Genetically edited potato plants having improved haplotypes

Abstract

The disclosure herein discloses potato varieties having unique haplotypes that allow for the improvement of cold storage properties of potatoes by potatoes having one or more of the unique haplotypes such that the ratio of reducing sugars to non-reducing sugars and / or acrylamide in processed potato products is improved compared to the ratio of reducing sugars to non-reducing sugars and / or acrylamide obtained from control plants. This improved cold storage property results in improved chip lightness scores for potatoes having these unique haplotypes.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Application No. 63 / 608,058, filed December 8, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0003] For all purposes, all references, articles, publications, patents, patent publications, and patent applications cited in the foregoing text and / or cited below are incorporated herein by reference in their entirety. However, any mention of any reference, article, publication, patent, patent publication, or patent application cited herein shall not constitute, nor should be construed as, an admission or implication of any kind that it constitutes valid prior art or is part of common general knowledge in any country of the world. Technical Field

[0004] This invention generally relates to the field of agricultural science, and more specifically to crop improvement. It also relates to potato cultivars with improved refrigeration properties, an improved ratio of reducing to non-reducing sugars, and an increased specific gravity. Furthermore, it relates to potato plant products obtained by this method. Background Technology

[0005] In terms of human consumption, the potato (Solanum tuberosum) is the world's third most important food crop, after rice and wheat. More than one billion people worldwide consume potatoes, and the global crop production exceeds 300 million metric tons (Clasen et al. 2016).

[0006] Most potatoes grown by American farmers for consumption are used by food processors to make potato chips, French fries, and other processed products. However, because potatoes are harvested only once a year, refrigerated tubers are necessary to ensure a year-round supply of high-quality potatoes for processing. Without refrigeration, potatoes have a shelf life of approximately six months, after which their quality deteriorates rapidly (Sowokinos 2001). In addition to extending shelf life, low temperatures reduce sprouting, losses due to water loss, and the spread of disease.

[0007] Unfortunately, refrigeration also has adverse effects on potato quality. One of these adverse effects is known in the industry as “low-temperature induced saccharification” (“CIS”). CIS involves the accumulation of reducing sugars glucose and fructose during starch breakdown. When processed at high temperatures, these reducing sugars form dark-colored products that are bitter and unacceptable to consumers (Sowokinos 2001). In the United States, CIS results in up to 15% of potatoes being rejected by processing plants annually (Bhaskar et al. 2010).

[0008] Besides producing bitter-tasting products, heat processing also causes reducing sugars to react with free amino acids (such as asparagine) to form acrylamide, a potential carcinogen. In products such as potato chips and French fries, acrylamide formation has been shown to occur via a non-enzymatic Maillard reaction (Mottram et al. 2002). Acrylamide is particularly prevalent in heat-processed potatoes that have undergone CIS (Chemical Induced Sugar Reduction) because these potatoes have high levels of reducing sugars. Therefore, potato growers are seeking ways to reduce acrylamide levels.

[0009] One approach to reducing CIS in heat-processed potatoes and thus lowering acrylamide content is to reduce the accumulation of reducing sugars formed during refrigeration. The accumulation of reducing sugars during refrigeration is influenced by several metabolic processes, including starch synthesis and starch degradation. The potato vacuolar invertase gene (“VINV”) plays a particularly important role in the conversion of starch into reducing sugars during storage (Sowokinos 2001). Therefore, there is a need to develop methods to rapidly and effectively reduce VINV activity in potatoes in a consumer- and regulatory-friendly manner.

[0010] Another potato trait is specific gravity (SpGr), which is used to estimate the processing and cooking quality of tubers. Typically, the SpGr of a larger population is estimated by measuring the SpGr of many tuber samples, and it is usually determined by the water displacement method, which is the weight of the tuber in air divided by the difference between the weight of the tuber in air and the weight of the tuber in water. The target SpGr phenotype for tubers used for processing is typically at least 1.080 (see Greenwood, ML, MH McKendrick, and A. Hawkins. 1952. The relationship of the specific gravity of six genotypes of potatoes to their mealiness as assessed by sensory methods. Am. Potato J. 29:192–196; Young, DA, PW Voisey, and N. Dixon. 1964. A specific gravity calculator for potatoes. Am. Potato J. 41:401–405; Wang, Y., PC Bethke, AJ Bussan, MTGlynn, DG Holm, FM Navarro, et al. 2015. Acrylamide-forming potential and agronomic properties of elite US potato germplasm from the National FryProcessing Trial. Crop Sci. 56:1–10).

[0011] Submission of sequence list

[0012] The sequence listing relating to this application was submitted via PatentCenter in xlm electronic format (OHLO.23WOU1; file size 200kb, created on December 6, 2024), and is incorporated herein by reference in its entirety.

[0013] SEQ ID NO: 1 discloses the DNA sequence of Atlantic E-PED165-7182 Hap1 with a phase shift of 7 bp.

[0014] SEQ ID NO: 2 discloses the DNA sequence of Atlantic E-PED165-7182 with a Hap2 phase shift of 8 bp.

[0015] SEQ ID NO: 3 discloses the DNA sequence of Atlantic E-PED165-7182 with a Hap3 phase shift of 13 bp.

[0016] SEQ ID NO: 4 discloses the DNA sequence of Atlantic E-PED165-7182 Hap4 phase shift = 7 bp.

[0017] SEQ ID NO: 5 discloses the DNA sequence of Atlantic E-PED165-7186 Hap1 with a phase shift of 10 bp.

[0018] SEQ ID NO: 6 discloses the DNA sequence of Atlantic E-PED165-7186 with a Hap2 phase shift of 9 bp.

[0019] SEQ ID NO: 7 discloses the DNA sequence of Atlantic E-PED165-7186 with a Hap3 phase shift of 9 bp.

[0020] SEQ ID NO: 8 discloses the DNA sequence of Atlantic E-PED165-7186 with a Hap4 phase shift of 8 bp.

[0021] SEQ ID NO: 9 discloses the DNA sequence of Atlantic E-PED165-7188 Hap1 phase shift = 7 bp.

[0022] SEQ ID NO: 10 discloses the DNA sequence of Atlantic E-PED165-7188 with a Hap2 phase shift of 16 bp.

[0023] SEQ ID NO: 11 discloses the DNA sequence of Atlantic E-PED165-7188 with a Hap3 phase shift of 14 bp.

[0024] SEQ ID NO: 12 discloses the DNA sequence of Atlantic E-PED165-7188 with a Hap4 phase shift of 7 bp.

[0025] SEQ ID NO: 13 discloses the DNA sequence of Atlantic E-PED165-7242 Hap1 with a phase shift of 9 bp.

[0026] SEQ ID NO: 14 discloses the DNA sequence of Atlantic E-PED165-7242 with a Hap2 phase shift of 33 bp.

[0027] SEQ ID NO: 15 discloses the DNA sequence of Atlantic E-PED165-7242 with a Hap3 phase shift of 9 bp.

[0028] SEQ ID NO: 16 discloses the DNA sequence of Atlantic E-PED165-7242 with a Hap4 phase shift of 8 bp.

[0029] SEQ ID NO: 17 discloses the DNA sequence of Atlantic E-PED165-7287 Hap1 with a phase shift of 13 bp.

[0030] SEQ ID NO: 18 discloses the DNA sequence of Atlantic E-PED165-7287 with a Hap2 phase shift of 7 bp.

[0031] SEQ ID NO: 19 discloses the DNA sequence of Atlantic E-PED165-7287 with a Hap3 phase shift of 10 bp.

[0032] SEQ ID NO: 20 discloses the DNA sequence of Atlantic E-PED165-7287 with a Hap4 phase shift of 6 bp.

[0033] SEQ ID NO: 21 discloses the DNA sequence of Atlantic E-PED165-7302 with a Hap1 phase shift of 7 bp.

[0034] SEQ ID NO: 22 discloses the DNA sequence of Atlantic E-PED165-7302 with a Hap2 phase shift of 9 bp.

[0035] SEQ ID NO: 23 discloses the DNA sequence of Atlantic E-PED165-7302 with a Hap3 phase shift of 6 bp.

[0036] SEQ ID NO: 24 discloses the DNA sequence of Atlantic E-PED165-7302 with a Hap4 phase shift of 5 bp.

[0037] SEQ ID NO: 25 discloses the DNA sequence of Atlantic E-PED165-7318 with a Hap1 phase shift of 9 bp.

[0038] SEQ ID NO: 26 discloses the DNA sequence of Atlantic E-PED165-7318 with a Hap2 phase shift of 9 bp.

[0039] SEQ ID NO: 27 discloses the DNA sequence of Atlantic E-PED165-7318 with a Hap3 phase shift of 12 bp.

[0040] SEQ ID NO: 28 discloses the DNA sequence of Atlantic E-PED165-7318 with a Hap4 phase shift of 6 bp.

[0041] SEQ ID NO: 29 discloses the DNA sequence of Atlantic E-PED165-7324 with a Hap1 phase shift of 9 bp.

[0042] SEQ ID NO: 30 discloses the DNA sequence of Atlantic E-PED165-7324 with a Hap2 phase shift of 15 bp.

[0043] SEQ ID NO: 31 discloses the DNA sequence of Atlantic E-PED165-7324 with a Hap3 phase shift of 15 bp.

[0044] SEQ ID NO: 32 discloses the DNA sequence of Atlantic E-PED165-7324 with a Hap4 phase shift of 10 bp.

[0045] SEQ ID NO: 33 discloses the DNA sequence of Atlantic E-PED165-7326 Hap1 with a phase shift of 5 bp.

[0046] SEQ ID NO: 34 discloses the DNA sequence of Atlantic E-PED165-7326 with a Hap2 phase shift of 7 bp.

[0047] SEQ ID NO: 35 discloses the DNA sequence of Atlantic E-PED165-7326 with a Hap3 phase shift of 11 bp.

[0048] SEQ ID NO: 36 discloses the DNA sequence of Atlantic E-PED165-7326 with a Hap4 phase shift of 11 bp.

[0049] SEQ ID NO: 37 discloses the DNA sequence of Atlantic E-PED165-7340 Hap1 with a phase shift of 13 bp.

[0050] SEQ ID NO: 38 discloses the DNA sequence of Atlantic E-PED165-7340 with a Hap2 phase shift of 6 bp.

[0051] SEQ ID NO: 39 discloses the DNA sequence of Atlantic E-PED165-7340 with a Hap3 phase shift of 6 bp.

[0052] SEQ ID NO: 40 discloses the DNA sequence of Atlantic E-PED165-7340 with a Hap4 phase shift of 7 bp.

[0053] SEQ ID NO: 41 discloses the DNA sequence of Atlantic E-PED165-7347 with a Hap1 phase shift of 7 bp.

[0054] SEQ ID NO: 42 discloses the DNA sequence of Atlantic E-PED165-7347 with a Hap2 phase shift of 8 bp.

[0055] SEQ ID NO: 43 discloses the DNA sequence of Atlantic E-PED165-7347 with a Hap3 phase shift of 11 bp.

[0056] SEQ ID NO: 44 discloses the DNA sequence of Atlantic E-PED165-7347 with a Hap4 phase shift of 7 bp.

[0057] SEQ ID NO: 45 discloses the DNA sequence of Atlantic E-PED165-7373 Hap1 phase shift = 9 bp.

[0058] SEQ ID NO: 46 discloses the DNA sequence of Atlantic E-PED165-7373 with a Hap2 phase shift of 0 bp (wild type).

[0059] SEQ ID NO: 47 discloses the DNA sequence of Atlantic E-PED165-7373 with a Hap3 phase shift of 13 bp.

[0060] SEQ ID NO: 48 discloses the DNA sequence of Atlantic E-PED165-7373 with a Hap4 phase shift of 10 bp.

[0061] SEQ ID NO: 49 discloses the DNA sequence of Atlantic E-PED165-7385 Hap1 with a phase shift of 15 bp.

[0062] SEQ ID NO: 50 discloses the DNA sequence of Atlantic E-PED165-7385 with a Hap2 phase shift of 4 bp.

[0063] SEQ ID NO: 51 discloses the DNA sequence of Atlantic E-PED165-7385 with a Hap3 phase shift of 8 bp.

[0064] SEQ ID NO: 52 discloses the DNA sequence of Atlantic E-PED165-7385 with a Hap4 phase shift of 13 bp.

[0065] SEQ ID NO: 53 discloses the DNA sequence of Atlantic E-PED165-7398 with a Hap1 phase shift of 8 bp.

[0066] SEQ ID NO: 54 discloses the DNA sequence of Atlantic E-PED165-7398 with a Hap2 phase shift of 13 bp.

[0067] SEQ ID NO: 55 discloses the DNA sequence of Atlantic E-PED165-7398 with a Hap3 phase shift of 8 bp.

[0068] SEQ ID NO: 56 discloses the DNA sequence of Atlantic E-PED165-7398 with a Hap4 phase shift of 10 bp.

[0069] SEQ ID NO: 57 discloses the DNA sequence of Atlantic E-PED165-7400 Hap1 with a phase shift of 8 bp.

[0070] SEQ ID NO: 58 discloses the DNA sequence of Atlantic E-PED165-7400 with a Hap2 phase shift of 6 bp.

[0071] SEQ ID NO: 59 discloses the DNA sequence of Atlantic E-PED165-7400 with a Hap3 phase shift of 6 bp.

[0072] SEQ ID NO: 60 discloses the DNA sequence of Atlantic E-PED165-7400 with a Hap4 phase shift of 13 bp.

[0073] SEQ ID NO: 61 discloses the DNA sequence of Atlantic E-PED165-7413 with a phase shift of 11 bp at Hap1.

[0074] SEQ ID NO: 62 discloses the DNA sequence of Atlantic E-PED165-7413 with a Hap2 phase shift of 17 bp.

[0075] SEQ ID NO: 63 discloses the DNA sequence of Atlantic E-PED165-7413 with a Hap3 phase shift of 13 bp.

[0076] SEQ ID NO: 64 discloses the DNA sequence of Atlantic E-PED165-7413 with a Hap4 phase shift of 7 bp.

[0077] SEQ ID NO: 65 discloses the DNA sequence of Atlantic E-PED165-7421 Hap1 with a phase shift of 10 bp.

[0078] SEQ ID NO: 66 discloses the DNA sequence of Atlantic E-PED165-7421 with a Hap2 phase shift of 13 bp.

[0079] SEQ ID NO: 67 discloses the DNA sequence of Atlantic E-PED165-7421 with a Hap3 phase shift of 11 bp.

[0080] SEQ ID NO: 68 discloses the DNA sequence of Atlantic E-PED165-7421 with a Hap4 phase shift of 6 bp.

[0081] SEQ ID NO: 69 discloses the DNA sequence of Atlantic E-PED165-7426 Hap1 with a phase shift of 11 bp.

[0082] SEQ ID NO: 70 discloses the DNA sequence of Atlantic E-PED165-7426 with a Hap2 phase shift of 7 bp.

[0083] SEQ ID NO: 71 discloses the DNA sequence of Atlantic E-PED165-7426 with a Hap3 phase shift of 5 bp.

[0084] SEQ ID NO: 72 discloses the DNA sequence of Atlantic E-PED165-7426 with a Hap4 phase shift of 9 bp.

[0085] SEQ ID NO: 73 discloses the DNA sequence of Atlantic E-PED165-7432 with a phase shift of 9 bp at Hap1.

[0086] SEQ ID NO: 74 discloses the DNA sequence of Atlantic E-PED165-7432 with a Hap2 phase shift of 14 bp.

[0087] SEQ ID NO: 75 discloses the DNA sequence of Atlantic E-PED165-7432 with a Hap3 phase shift of 16 bp.

[0088] SEQ ID NO: 76 discloses the DNA sequence of Atlantic E-PED165-7432 with a Hap4 phase shift of 7 bp.

[0089] SEQ ID NO: 77 discloses the DNA sequence of Atlantic E-PED165-7436 Hap1 phase shift = 7 bp.

[0090] SEQ ID NO: 78 discloses the DNA sequence of Atlantic E-PED165-7436 with a Hap2 phase shift of 9 bp.

[0091] SEQ ID NO: 79 discloses the DNA sequence of Atlantic E-PED165-7436 with a Hap3 phase shift of 6 bp.

[0092] SEQ ID NO: 80 discloses the DNA sequence of Atlantic E-PED165-7436 with a Hap4 phase shift of 6 bp.

[0093] SEQ ID NO: 81 discloses the DNA sequence of Atlantic E-PED165-7437 Hap1 phase shift = 7 bp.

[0094] SEQ ID NO: 82 discloses the DNA sequence of Atlantic E-PED165-7437 with a Hap2 phase shift of 8 bp.

[0095] SEQ ID NO: 83 discloses the DNA sequence of Atlantic E-PED165-7437 with a Hap3 phase shift of 7 bp.

[0096] SEQ ID NO: 84 discloses the DNA sequence of Atlantic E-PED165-7437 with a Hap4 phase shift of 7 bp.

[0097] SEQ ID NO: 85 discloses the DNA sequence of Atlantic E-PED165-7458 Hap1 with a phase shift of 8 bp.

[0098] SEQ ID NO: 86 discloses the DNA sequence of Atlantic E-PED165-7458 with a Hap2 phase shift of 13 bp.

[0099] SEQ ID NO: 87 discloses the DNA sequence of Atlantic E-PED165-7458 with a Hap3 phase shift of 10 bp.

[0100] SEQ ID NO: 88 discloses the DNA sequence of Atlantic E-PED165-7458 with a Hap4 phase shift of 7 bp.

[0101] SEQ ID NO: 89 discloses the DNA sequence of Atlantic E-PED165-7459 Hap1 with a phase shift of 11 bp.

[0102] SEQ ID NO: 90 discloses the DNA sequence of Atlantic E-PED165-7459 with a Hap2 phase shift of 11 bp.

[0103] SEQ ID NO: 91 discloses the DNA sequence of Atlantic E-PED165-7459 with a Hap3 phase shift of 11 bp.

[0104] SEQ ID NO: 92 discloses the DNA sequence of Atlantic E-PED165-7459 with a Hap4 phase shift of 7 bp.

[0105] SEQ ID NO: 93 discloses the DNA sequence of Atlantic E-PED165-7471 Hap1 with a phase shift of 8 bp.

[0106] SEQ ID NO: 94 discloses the DNA sequence of Atlantic E-PED165-7471 with a Hap2 phase shift of 6 bp.

[0107] SEQ ID NO: 95 discloses the DNA sequence of Atlantic E-PED165-7471 with a Hap3 phase shift of 6 bp.

[0108] SEQ ID NO: 96 discloses the DNA sequence of Atlantic E-PED165-7471 with a Hap4 phase shift of 13 bp.

[0109] SEQ ID NO: 97 discloses the DNA sequence of Atlantic E-PED165-7475 Hap1 with a phase shift of 10 bp.

[0110] SEQ ID NO: 98 discloses the DNA sequence of Atlantic E-PED165-7475 with a Hap2 phase shift of 10 bp.

[0111] SEQ ID NO: 99 discloses the DNA sequence of Atlantic E-PED165-7475 with a Hap3 phase shift of 8 bp.

[0112] SEQ ID NO: 100 discloses the DNA sequence of Atlantic E-PED165-7475 with a Hap4 phase shift of 8 bp.

[0113] SEQ ID NO: 101 discloses the DNA sequence of Atlantic E-PED165-7621 Hap1 with a phase shift of 8 bp, and SEQ ID NO: 102 discloses the DNA sequence of Atlantic E-PED165-7621 Hap2 with a phase shift of 7 bp.

[0114] SEQ ID NO: 103 discloses the DNA sequence of Atlantic E-PED165-7621 with a Hap3 phase shift of 8 bp.

[0115] SEQ ID NO: 104 discloses the DNA sequence of Atlantic E-PED165-7621 with a Hap4 phase shift of 9 bp.

[0116] SEQ ID NO: 105 discloses the DNA sequence of Atlantic E-PED165-7632 Hap1 with a phase shift of 5 bp.

[0117] SEQ ID NO: 106 discloses the DNA sequence of Atlantic E-PED165-7632 with a Hap2 phase shift of 5 bp.

[0118] SEQ ID NO: 107 discloses the DNA sequence of Atlantic E-PED165-7632 with a Hap3 phase shift of 10 bp.

[0119] SEQ ID NO: 108 discloses the DNA sequence of Atlantic E-PED165-7632 with a Hap4 phase shift of 7 bp.

[0120] SEQ ID NO: 109 discloses the DNA sequence of Russet Burbank E-PED060-8903 with a Hap1 phase shift of 7 bp.

[0121] SEQ ID NO: 110 discloses the DNA sequence of Russet Burbank E-PED060-8903 with a Hap2 phase shift of 8 bp.

[0122] SEQ ID NO: 111 discloses the DNA sequence of Russet Burbank E-PED060-8903 with a Hap5 phase shift of 7 bp.

[0123] SEQ ID NO: 112 discloses the DNA sequence of Russet Burbank E-PED060-8903 with a Hap5 phase shift of 8 bp.

[0124] SEQ ID NO: 113 discloses the DNA sequence of Russet Burbank E-PED060-8916 with a Hap1 phase shift of 7 bp.

[0125] SEQ ID NO: 114 discloses the DNA sequence of Russet Burbank E-PED060-8916 with a Hap2 phase shift of 9 bp.

[0126] SEQ ID NO: 115 discloses the DNA sequence of Russet Burbank E-PED060-8916 with a Hap5 phase shift of 12 bp.

[0127] SEQ ID NO: 116 discloses the DNA sequence of Russet Burbank E-PED060-8916 with a Hap5 phase shift of 9 bp.

[0128] SEQ ID NO: 117 discloses the DNA sequence of Russet Burbank E-PED060-8942 with a Hap1 phase shift of 6 bp.

[0129] SEQ ID NO: 118 discloses the DNA sequence of Russet Burbank E-PED060-8942 with a Hap2 phase shift of 1 bp.

[0130] SEQ ID NO: 119 discloses the DNA sequence of Russet Burbank E-PED060-8942 with a Hap5 phase shift of 7 bp.

[0131] SEQ ID NO: 120 discloses the DNA sequence of Russet Burbank E-PED060-8942 with a Hap5 phase shift of 14 bp.

[0132] SEQ ID NO: 121 discloses the DNA sequence of Russet Burbank E-PED060-9609 with a Hap1 phase shift of 6 bp.

[0133] SEQ ID NO: 122 discloses the DNA sequence of Russet Burbank E-PED060-9609 with a Hap2 phase shift of 10 bp.

[0134] SEQ ID NO: 123 discloses the DNA sequence of Russet Burbank E-PED060-9609 with a Hap5 phase shift of 10 bp.

[0135] SEQ ID NO: 124 discloses the DNA sequence of Russet Burbank E-PED060-9609 with a Hap5 phase shift of 10 bp.

[0136] SEQ ID NO: 125 discloses the DNA sequence of Russet Burbank E-PED060-9610 with a Hap1 phase shift of 11 bp.

[0137] SEQ ID NO: 126 discloses the DNA sequence of Russet Burbank E-PED060-9610 with a Hap2 phase shift of 11 bp.

[0138] SEQ ID NO: 127 discloses the DNA sequence of Russet Burbank E-PED060-9610 with a Hap5 phase shift of 7 bp.

[0139] SEQ ID NO: 128 discloses the DNA sequence of Russet Burbank E-PED060-9610 with a Hap5 phase shift of 16 bp.

[0140] SEQ ID NO: 129 discloses the DNA sequence of Russet Burbank E-PED060-9653 with a Hap1 phase shift of 11 bp.

[0141] SEQ ID NO: 130 discloses the DNA sequence of Russet Burbank E-PED060-9653 with a Hap2 phase shift of 10 bp.

[0142] SEQ ID NO: 131 discloses the DNA sequence of Russet Burbank E-PED060-9653 with a Hap5 phase shift of 11 bp.

[0143] SEQ ID NO: 132 discloses the DNA sequence of Russet Burbank E-PED060-9653 with a Hap5 phase shift of 8 bp.

[0144] SEQ ID NO: 133 discloses the DNA sequence of Russet Burbank E-PED060-9687 with a Hap1 phase shift of 9 bp.

[0145] SEQ ID NO: 134 discloses the DNA sequence of Russet Burbank E-PED060-9687 with a Hap2 phase shift of 8 bp.

[0146] SEQ ID NO: 135 discloses the DNA sequence of Russet Burbank E-PED060-9687 with a Hap5 phase shift of 8 bp.

[0147] SEQ ID NO: 136 discloses the DNA sequence of Russet Burbank E-PED060-9687 with a Hap5 phase shift of 13 bp.

[0148] SEQ ID NO: 137 discloses the DNA sequence of Russet Burbank E-PED060-9765 with a Hap1 phase shift of 9 bp.

[0149] SEQ ID NO: 138 discloses the DNA sequence of Russet Burbank E-PED060-9765 with a Hap2 phase shift of 10 bp.

[0150] SEQ ID NO: 139 discloses the DNA sequence of Russet Burbank E-PED060-9765 with a Hap5 phase shift of 8 bp.

[0151] SEQ ID NO: 140 discloses the DNA sequence of Russet Burbank E-PED060-9765 with a Hap5 phase shift of 7 bp.

[0152] SEQ ID NO: 141 discloses the DNA sequence of Russet Burbank E-PED060-9791vHap1.

[0153] SEQ ID NO: 142 discloses the DNA sequence of Russet Burbank E-PED060-9791 Hap2.

[0154] SEQ ID NO: 143 discloses the DNA sequence of Russet Burbank E-PED060-9791 Hap5.

[0155] SEQ ID NO: 144 discloses the DNA sequence of Russet Burbank E-PED060-9791 Hap5.

[0156] SEQ ID NO: 145 discloses the DNA sequence of Russet Burbank E-PED060-9808 with a Hap1 phase shift of 9 bp.

[0157] SEQ ID NO: 146 discloses the DNA sequence of Russet Burbank E-PED060-9808 with a Hap2 phase shift of 7 bp.

[0158] SEQ ID NO: 147 discloses the DNA sequence of Russet Burbank E-PED060-9808 with a Hap5 phase shift of 7 bp.

[0159] SEQ ID NO: 148 discloses the DNA sequence of Russet Burbank E-PED060-9808 with a Hap5 phase shift of 5 bp.

[0160] SEQ ID NO: 149 discloses the DNA sequence of the protospacer PRS155.

[0161] SEQ ID NO: 150 discloses the DNA sequence of the protospacer PRS156.

[0162] SEQ ID NO: 151 discloses the DNA sequence of the protospacer PRS157.

[0163] SEQ ID NO: 152 discloses the DNA sequence of the protospacer PRS158.

[0164] SEQ ID NO: 153 discloses the DNA sequence of the protospacer PRS159.

[0165] SEQ ID NO: 154 discloses the DNA sequence of the protospacer PRS160.

[0166] SEQ ID NO: 155 discloses the DNA sequence of the protospacer PRS161.

[0167] SEQ ID NO: 156 discloses the DNA sequence of the original spacer PRS162.

[0168] SEQ ID NO: 157 discloses the DNA sequence of the protospacer PRS163.

[0169] SEQ ID NO: 158 discloses the DNA sequence of the protospacer PRS164.

[0170] SEQ ID NO: 159 discloses the RNA sequence of guide RNA GR155.

[0171] SEQ ID NO: 160 discloses the RNA sequence of guide RNA GR156.

[0172] SEQ ID NO: 161 discloses the RNA sequence of guide RNA GR157.

[0173] SEQ ID NO: 162 discloses the RNA sequence of guide RNA GR158.

[0174] SEQ ID NO: 163 discloses the RNA sequence of guide RNA GR159.

[0175] SEQ ID NO: 164 discloses the RNA sequence of guide RNA GR160.

[0176] SEQ ID NO: 165 discloses the RNA sequence of guide RNA GR161.

[0177] SEQ ID NO: 166 discloses the RNA sequence of guide RNA GR162.

[0178] SEQ ID NO: 167 discloses the RNA sequence of guide RNA GR163.

[0179] SEQ ID NO: 168 discloses the RNA sequence of guide RNA GR164.

[0180] SEQ ID NO: 169 discloses the DNA sequence of the editing window.

[0181] SEQ ID NO: 170 discloses the DNA sequence of Atlantic E-PED165-7240 Hap1 with a phase shift of 12 bp.

[0182] SEQ ID NO: 171 discloses the DNA sequence of Atlantic E-PED165-7240 with a Hap2 phase shift of 8 bp.

[0183] SEQ ID NO: 172 discloses the DNA sequence of Atlantic E-PED165-7240 with a Hap3 phase shift of 10 bp.

[0184] SEQ ID NO: 173 discloses the DNA sequence of Atlantic E-PED165-7240 with a Hap4 phase shift of 12 bp.

[0185] SEQ ID NO: 174 discloses the DNA sequence of Atlantic E-PED165-7300 Hap1 with a phase shift of 11 bp.

[0186] SEQ ID NO: 175 discloses the DNA sequence of Atlantic E-PED165-7300 with a Hap2 phase shift of 8 bp.

[0187] SEQ ID NO: 176 discloses the DNA sequence of Atlantic E-PED165-7300 with a Hap3 phase shift of 0 bp (wild type).

[0188] SEQ ID NO: 177 discloses the DNA sequence of Atlantic E-PED165-7300 with a Hap4 phase shift of 7 bp.

[0189] SEQ ID NO: 178 discloses the DNA sequence of Atlantic E-PED165-7419 Hap1 phase shift = 0 bp (wild type).

[0190] SEQ ID NO: 179 discloses the DNA sequence of Atlantic E-PED165-7419 with a Hap2 phase shift of 7 bp.

[0191] SEQ ID NO: 180 discloses the DNA sequence of Atlantic E-PED165-7419 with a Hap3 phase shift of 11 bp.

[0192] SEQ ID NO: 181 discloses the DNA sequence of Atlantic E-PED165-7419 with a Hap4 phase shift of 11 bp.

[0193] SEQ ID NO: 182 discloses the DNA sequence of Atlantic E-PED165-7477 Hap1 with a phase shift of 2 bp.

[0194] SEQ ID NO: 183 discloses the DNA sequence of Atlantic E-PED165-7477 with a Hap2 phase shift of 0 bp (wild type).

[0195] SEQ ID NO: 184 discloses the DNA sequence of Atlantic E-PED165-7477 with a Hap3 phase shift of 8 bp.

[0196] SEQ ID NO: 185 discloses the DNA sequence of Atlantic E-PED165-7477 with a Hap4 phase shift of 15 bp.

[0197] SEQ ID NO: 186 discloses the DNA sequence of Atlantic E-PED165-7487 Hap1 phase shift = 0 bp (wild type).

[0198] SEQ ID NO: 187 discloses the DNA sequence of Atlantic E-PED165-7487 with a Hap2 phase shift of 0 bp (wild type).

[0199] SEQ ID NO: 188 discloses the DNA sequence of Atlantic E-PED165-7487 with a Hap3 phase shift of 8 bp.

[0200] SEQ ID NO: 189 discloses the DNA sequence of Atlantic E-PED165-7487 with a Hap4 phase shift of 0 bp (wild type).

[0201] SEQ ID NO: 190 discloses the DNA sequence of Atlantic E-PED165-7590 with a phase shift of 19 bp at Hap1.

[0202] SEQ ID NO: 191 discloses the DNA sequence of Atlantic E-PED165-7590 with a Hap2 phase shift of 0 bp (wild type).

[0203] SEQ ID NO: 192 discloses the DNA sequence of Atlantic E-PED165-7590 with a Hap3 phase shift of 8 bp.

[0204] SEQ ID NO: 193 discloses the DNA sequence of Atlantic E-PED165-7590 with a Hap4 phase shift of 7 bp.

[0205] SEQ ID NO: 194 discloses the DNA sequence of Atlantic E-PED165-7651 Hap1 phase shift = 0 bp (wild type).

[0206] SEQ ID NO: 195 discloses the DNA sequence of Atlantic E-PED165-7651 with Hap2 phase shift = 0b (wild type).

[0207] SEQ ID NO: 196 discloses the DNA sequence of Atlantic E-PED165-7651 with a Hap3 phase shift of 13 bp.

[0208] SEQ ID NO: 197 discloses the DNA sequence of Atlantic E-PED165-7651 with a Hap4 phase shift of 0 bp (wild type).

[0209] SEQ ID NO: 198 discloses the DNA sequence of Russet Burbank E-PED060-8917 with a Hap1 phase shift of 10 bp.

[0210] SEQ ID NO: 199 discloses the DNA sequence of Russet Burbank E-PED060-8917 with a Hap2 phase shift of 13 bp.

[0211] SEQ ID NO: 200 discloses the DNA sequence of Russet Burbank E-PED060-8917 with a Hap5 phase shift of 0 bp (wild type).

[0212] SEQ ID NO: 201 discloses the DNA sequence of Russet Burbank E-PED060-8917 with a Hap5 phase shift of 8 bp.

[0213] SEQ ID NO: 202 discloses the DNA sequence of Russet Burbank E-PED060-9590 Hap1 phase shift = 0 bp (wild type).

[0214] SEQ ID NO: 203 discloses the DNA sequence of Russet Burbank E-PED060-9590 with a Hap2 phase shift of 11 bp.

[0215] SEQ ID NO: 204 discloses the DNA sequence of Russet Burbank E-PED060-9590 with a Hap5 phase shift of 0 bp (wild type).

[0216] SEQ ID NO: 205 discloses the DNA sequence of Russet Burbank E-PED060-9590 with a Hap5 phase shift of 8 bp.

[0217] SEQ ID NO: 206 discloses the DNA sequence of Russet Burbank E-PED060-9711 with a Hap1 phase shift of 8 bp.

[0218] SEQ ID NO: 207 discloses the DNA sequence of Russet Burbank E-PED060-9711 with a Hap2 phase shift of 11 bp.

[0219] SEQ ID NO: 208 discloses the DNA sequence of Russet Burbank E-PED060-9711 with a Hap5 phase shift of 0 bp (wild type).

[0220] SEQ ID NO: 209 discloses the DNA sequence of Russet Burbank E-PED060-9711 with a Hap5 phase shift of 8 bp.

[0221] SEQ ID NO: 210 discloses the DNA sequence of Russet Burbank E-PED060-9846 with a Hap1 phase shift of 9 bp.

[0222] SEQ ID NO: 211 discloses the DNA sequence of Russet Burbank E-PED060-9846 with a Hap2 phase shift of 0 bp (wild type).

[0223] SEQ ID NO: 212 discloses the DNA sequence of Russet Burbank E-PED060-9846 with a Hap5 phase shift of 7 bp.

[0224] SEQ ID NO: 213 discloses the DNA sequence of Russet Burbank E-PED060-9846 with a Hap5 phase shift of 16 bp.

[0225] SEQ ID NO: 214 discloses the DNA sequence of Atlantic E-PED165-7393 with a Hap1 phase shift of 0 bp (wild type).

[0226] SEQ ID NO: 215 discloses the DNA sequence of Atlantic E-PED165-7393 with a Hap2 phase shift of 7 bp.

[0227] SEQ ID NO: 216 discloses the DNA sequence of Atlantic E-PED165-7393 with a Hap3 phase shift of 0 bp (wild type).

[0228] SEQ ID NO: 217 discloses the DNA sequence of Atlantic E-PED165-7393 with a Hap4 phase shift of 0 bp (wild type).

[0229] SEQ ID NO: 218 discloses the DNA sequence of Atlantic E-PED165-7594 Hap1 phase shift = 0 bp (wild type).

[0230] SEQ ID NO: 219 discloses the DNA sequence of Atlantic E-PED165-7594 with a Hap2 phase shift of 0 bp (wild type).

[0231] SEQ ID NO: 220 discloses the DNA sequence of Atlantic E-PED165-7594 with a Hap3 phase shift of 0 bp (wild type).

[0232] SEQ ID NO: 221 discloses the DNA sequence of Atlantic E-PED165-7594 with a Hap4 phase shift of 0 bp (wild type).

[0233] SEQ ID NO: 222 discloses the DNA sequence of Atlantic E-PED165-7286 with a Hap1 phase shift of 0 bp (wild type).

[0234] SEQ ID NO: 223 discloses the DNA sequence of Atlantic E-PED165-7286 with a Hap2 phase shift of 0 bp (wild type).

[0235] SEQ ID NO: 224 discloses the DNA sequence of Atlantic E-PED165-7286 with a Hap3 phase shift of 0 bp (wild type).

[0236] SEQ ID NO: 225 discloses the DNA sequence of Atlantic E-PED165-7286 with a Hap4 phase shift of 0 bp (wild type).

[0237] SEQ ID NO: 226 discloses the DNA sequence of Atlantic E-PED165-7384 Hap1 phase shift = 0 bp (wild type).

[0238] SEQ ID NO: 227 discloses the DNA sequence of Atlantic E-PED165-7384 with a Hap2 phase shift of 0 bp (wild type).

[0239] SEQ ID NO: 228 discloses the DNA sequence of Atlantic E-PED165-7384 with a Hap3 phase shift of 0 bp (wild type).

[0240] SEQ ID NO: 229 discloses the DNA sequence of Atlantic E-PED165-7384 with a Hap4 phase shift of 0 bp (wild type). Summary of the Invention

[0241] This article provides compositions, methods, kits, and genomes relating to the production of modified potato plants and products exhibiting lighter color, reduced low-temperature induced saccharification, and acrylamide accumulation.

[0242] The following implementation schemes and aspects thereof are described and illustrated in conjunction with systems, tools and methods, which are intended to be exemplary and illustrative, and not limiting in scope.

[0243] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains a genetic modification in at least one VINV allele, wherein the potato chip brightness score of a potato food product derived from said potato plant is at least 10%, 25%, 50% or higher than that of a potato food product derived from a control potato plant.

[0244] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains a genetic modification in at least one VINV allele, wherein potato food products derived from said potato plant have a chip brightness score higher than 63.

[0245] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains a genetic modification in at least one VINV allele, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than the control plant, or both.

[0246] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modifications in at least two VINV alleles, wherein potato food products derived from said potato plant have a chip brightness score at least 10% higher than potato food products derived from control potato plants.

[0247] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided, wherein the potato chip brightness score of the potato food product derived from said potato plant is between 10% and 25% higher than that of the potato food product derived from the control potato plant.

[0248] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modifications in at least two VINV alleles, wherein potato food products derived from said potato plant have a chip brightness score higher than 63.

[0249] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modification in at least two VINV alleles, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than the control plant, or both.

[0250] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modifications in at least three VINV alleles, wherein potato food products derived from said potato plant have a chip brightness score at least 10% higher than potato food products derived from control potato plants.

[0251] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modifications in at least three VINV alleles, wherein potato food products derived from said potato plant have a chip brightness score higher than 63.

[0252] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modification in at least three VINV alleles, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than the control plant, or both.

[0253] In some aspects of the invention, a modified potato plant, plant part or plant cell is provided that contains genetic modifications in four VINV alleles, wherein potato food products derived from said potato plant have a chip brightness score at least 10% higher than potato food products derived from control potato plants.

[0254] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modifications in four VINV alleles, wherein potato food products derived from said potato plant have a chip brightness score higher than 63.

[0255] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains genetic modifications in four VINV alleles, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than the control plant, or both.

[0256] In some aspects of the invention provided, a modified potato plant, plant part or plant cell is provided that contains a genetic modification or mutation, such as but not limited to deletion, editing, phase shift, inversion or duplication, in at least one VINV allele, wherein the VINV allele is selected from the group consisting of Hap1 VINV allele, Hap2 VINV allele, Hap3 VINV allele, Hap4 VINV and Hap5 VINV allele.

[0257] In some aspects of this disclosure, a modified potato plant, plant part, or plant cell is provided that contains modifications or mutations, such as but not limited to deletions, edits, phase shifts, inversions, or duplications, in at least one of the Hap1, Hap2, Hap3, Hap4, and Hap5 VINV alleles, wherein the deletions, edits, phase shifts, inversions, or duplications are generated by a guiding endonuclease, and wherein the endonuclease binds a protospacer sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or a sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 158. 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158 are sequences having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity.

[0258] In some aspects of the invention, a modified potato plant, plant part, or plant cell is provided that contains modifications, such as but not limited to deletions, edits, phase shifts, inversions, or duplications, in at least one or more of the Hap1, Hap2, Hap3, Hap4, and Hap5 VINV alleles, wherein each deletion, edit, phase shift, inversion, or duplication is generated by a guided endonuclease, and wherein each deletion, edit, phase shift, inversion, or duplication contains one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is aligned with SEQ ID NO: 169.

[0259] In some aspects of the invention, a modified potato plant, plant part, or plant cell is provided, wherein the Hap1 VINV allele comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, ... NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 1121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 170. SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, SEQ ID NO: 202, SEQ ID NO: 206, SEQ ID NO: 210, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 222 and SEQ ID NO: 226.

[0260] In some aspects of the invention, a modified potato plant, plant part, or plant cell is provided, wherein the Hap2 VINV allele comprises a sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: ...70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 171. SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223 and SEQ ID NO: 227.

[0261] In some aspects of the invention, a modified potato plant, plant part, or plant cell is provided, wherein the Hap3 VINV allele comprises a sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103 ... NO: 107, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 216, SEQ ID NO: 220, SEQ ID NO: 224 and SEQ ID NO: 228.

[0262] In some aspects of the invention, a modified potato plant, plant part, or plant cell is provided, wherein the Hap4 VINV allele comprises a sequence selected from the group consisting of the following SEQ ID NOs: SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104. SEQ ID NO: 108, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225 and SEQ ID NO: 229.

[0263] In some aspects of the invention, a modified potato plant, plant part, or plant cell is provided, wherein the Hap5 VINV allele comprises a sequence selected from the group consisting of the following SEQ ID NOs: SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 147, and SEQ ID NO: 148.

[0264] In some aspects of the invention, a modified potato plant, plant part, or plant cell is provided, which includes editing in four VINV alleles, wherein the editing is generated by a guided endonuclease, such that the VINV alleles of the potato plant, plant part, or plant cell contain four sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0265] In some aspects of this disclosure, a modified potato plant, plant part, or plant cell is provided, comprising a mutation, deletion, editing, phase shift, inversion, or duplication in at least one VINV allele, wherein the mutation, deletion, editing, phase shift, inversion, or duplication is generated by a guiding endonuclease, and wherein the endonuclease binds a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or a sequence thereof. 156, SEQ ID NO: 157 or SEQ ID NO: 158, having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity.

[0266] In some aspects of the invention provided, a modified potato plant, plant part, or plant cell is provided, wherein the potato plant, plant part, or plant cell contains mutations, deletions, edits, phase shifts, inversions, or duplications in two, three, or four VINV alleles, wherein a nuclease is complexed with a guide RNA comprising a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168. The group consisting of SEQ ID NO: 166, SEQ ID NO: 167, or SEQ ID NO: 168 has a sequence with at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity, and wherein each edit contains an edit of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO: 169.

[0267] In some aspects of the invention provided, a modified potato plant, plant part, or plant cell is provided, wherein the potato plant, plant part, or plant cell contains mutations, deletions, edits, phase shifts, inversions, or duplications in two, three, or four VINV alleles, wherein a nuclease is complexed with a guide RNA comprising a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168. 166, the group consisting of SEQ ID NO: 167 or SEQ ID NO: 168 having sequences with at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity, wherein each edit contains an edit of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO: 169, and wherein the guiding endonuclease is a Cas protein.

[0268] In some aspects of the invention, a modified potato plant, plant part or plant cell as provided herein is provided, wherein the potato plant, plant part or plant cell is derived from a breeding line selected from the group consisting of Russet Burbank or Atlantic.

[0269] In some aspects of the invention, a modified potato plant, plant part or plant cell as provided herein is provided, wherein the tuber sugar profile obtained from said plant contains lower levels of glucose, fructose or both compared to the tuber sugar profile obtained from a control plant.

[0270] In some aspects of the invention, a modified potato plant, plant part or plant cell as provided herein is provided, wherein the tuber sugar profile obtained from said plant contains a lower level of glucose compared to the tuber sugar profile obtained from a control plant.

[0271] In some aspects of the invention, a modified potato plant, plant part or plant cell as provided herein is provided, wherein the tuber sugar profile obtained from said plant contains a lower level of fructose compared to the tuber sugar profile obtained from a control plant.

[0272] In some aspects of this disclosure, a modified potato plant, plant part, or plant cell as provided herein is provided, wherein the specific gravity spectrum obtained from said plant contains a higher specific gravity than that obtained from a control plant.

[0273] In some aspects of the invention, a modified potato plant, plant part or plant cell as provided herein is provided, wherein the acrylamide content after refrigeration is at least 85% lower than that of a control potato plant, plant part or plant cell after refrigeration.

[0274] In some aspects of the invention, a processed potato product as provided herein is provided, wherein the processed potato product is selected from the group consisting of: biomass, oil, meal, edible starch, syrup, sugar, animal feed, flour, flakes, potato chips, French fries, potato wedges, potato cakes, potato balls, baked potatoes, mashed potatoes, dehydrated potatoes, granules, peels, cooked skins, potato pulp, mashed potatoes, filter cake, sieve residue, potato residue, potato protein isolate or concentrate, discarded French fries, discarded potato chips, scraps, batter, crumbs, defective pieces, or alcoholic beverages.

[0275] In some aspects of the invention, a processed potato product as provided herein is provided, wherein the processed potato product is non-renewable.

[0276] In some aspects of the invention provided, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease together binding with a protospacer sequence of at least one VINV allele; and regenerating the modified potato plant, plant, part, or plant cell from the potato cell, wherein the modified potato plant, plant, part, or plant cell contains editing in at least one VINV allele such that at least one VINV allele of the modified potato plant, plant part, or plant cell contains at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0277] In some aspects of the invention provided, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease together binding with protospacer sequences of at least two VINV alleles; and regenerating modified potato plants, plants, parts, or plant cells from potato cells, wherein the modified potato plants, plants, parts, or plant cells contain editing in at least two VINV alleles such that the at least two VINV alleles of the modified potato plants, plant parts, or plant cells contain at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0278] In some aspects of the invention provided, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease together binding with protospacer sequences of at least three VINV alleles; and regenerating modified potato plants, plants, parts, or plant cells from potato cells, wherein the modified potato plants, plants, parts, or plant cells contain edits in at least three VINV alleles such that the at least three VINV alleles of the modified potato plants, plant parts, or plant cells contain at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0279] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease together binding with the protospacer sequences of four VINV alleles; and regenerating the modified potato plants, plants, parts, or plant cells from the potato cells, wherein the modified potato plants, plants, parts, or plant cells contain edits in the four VINV alleles such that the four VINV alleles of the modified potato plants, plant parts, or plant cells contain at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0280] In some aspects of the invention, a method for producing modified potato plants is provided, wherein the method comprises: introducing at least one guide endonuclease into a plurality of potato cells; regenerating a plurality of potato plants or plant parts from the plurality of potato cells; and selecting from the plurality of regenerated potato plants or plant parts that have editing in at least one VINV allele.

[0281] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, wherein the method comprises: introducing a guiding endonuclease into potato cells, the endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or with a protospacer sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO: 158. The group consisting of 158 has a sequence with at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity; and the regeneration of modified potato plants, plants, parts, or plant cells from potato cells, wherein the modified potato plants, plants, parts, or plant cells contain deletions, edits, phase shifts, inversions, or duplications in at least one VINV allele.

[0282] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, wherein the method comprises: introducing a guiding endonuclease into potato cells, the endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or with a protospacer sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO: 158. The group consisting of 158 has a sequence with at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity; and the modified potato plant, plant, part, or plant cell is regenerated from potato cells, wherein the modified potato plant, plant, part, or plant cell contains deletions, edits, phase shifts, inversions, or duplications in at least two VINV alleles.

[0283] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, wherein the method comprises: introducing a guiding endonuclease into potato cells, the endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or with a protospacer sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO: 158. The group consisting of 158 has a sequence with at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity; and the modified potato plant, plant, part, or plant cell is regenerated from potato cells, wherein the modified potato plant, plant, part, or plant cell contains deletions, edits, phase shifts, inversions, or duplications in at least three VINV alleles.

[0284] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, wherein the method comprises: introducing a guiding endonuclease into potato cells, the endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or with a protospacer sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO: 158. The group consisting of 158 has a sequence with at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity; and the modified potato plant, plant, part, or plant cell is regenerated from potato cells, wherein the modified potato plant, plant, part, or plant cell contains deletions, edits, phase shifts, inversions, or duplications in four VINV alleles.

[0285] In some aspects of this disclosure, methods as discussed herein are provided, wherein a nuclease is complexed with a guide RNA selected from the group consisting of: SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, or sequences having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168.

[0286] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease binding to a protospacer sequence of at least one VINV allele; and regenerating the modified potato plants, plants, parts, or plant cells from the potato cells, wherein the modified potato plants, plants, parts, or plant cells contain mutations, such as, but not limited to, deletions, edits, phase shifts, inversions, or duplications, in at least one VINV allele, such that when the modified VINV allele is compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169, such as, but not limited to, deletions, edits, phase shifts, inversions, or duplications.

[0287] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease binding to the protospacer sequences of at least two VINV alleles; and regenerating the modified potato plants, plants, parts, or plant cells from the potato cells, wherein the modified potato plants, plants, parts, or plant cells contain mutations, such as but not limited to deletions, edits, phase shifts, inversions, or duplications, in at least two VINV alleles, such that when the modified VINV alleles are compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169, such as but not limited to deletions, edits, phase shifts, inversions, or duplications.

[0288] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease binding to the protospacer sequences of at least three VINV alleles; and regenerating the modified potato plants, plants, parts, or plant cells from the potato cells, wherein the modified potato plants, plants, parts, or plant cells contain mutations, such as but not limited to deletions, edits, phase shifts, inversions, or duplications, in at least three VINV alleles, such that when the modified VINV alleles are compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169, such as but not limited to deletions, edits, phase shifts, inversions, or duplications.

[0289] In some aspects of the invention, a method for producing modified potato plants, plant parts, or plant cells is provided, the method comprising: introducing at least one guide endonuclease into potato cells, the endonuclease binding together with the protospacer sequences of four VINV alleles; and regenerating the modified potato plants, plants, parts, or plant cells from the potato cells, wherein the modified potato plants, plants, parts, or plant cells contain mutations in the four VINV alleles, such as, but not limited to, deletions, edits, phase shifts, inversions, or duplications, such that when the modified VINV alleles are compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169, such as, but not limited to, deletions, edits, phase shifts, inversions, or duplications.

[0290] In some aspects of the invention, a method is provided for producing modified potatoes, potato plants, portions, or plant cells as provided herein, wherein a nuclease binds to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or a sequence with SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO: 158. 158 sequences having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity.

[0291] In some aspects of the invention, a method is provided for producing modified potatoes, potato plants, portions, or plant cells as provided herein, wherein a nuclease is complexed with a guide RNA comprising a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or a sequence consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, or SEQ ID NO: 168. 168 sequences having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity.

[0292] In some aspects of the invention, a potato genome is provided, characterized in that it contains a mutation in at least one VINV allele, the at least one VINV allele comprising at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0293] In some aspects of the invention, a potato genome is provided, characterized in that it contains mutations in at least two VINV alleles, the at least two VINV alleles comprising at least two sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0294] In some aspects of the invention, a potato genome is provided, characterized in that it contains mutations in at least three VINV alleles, the at least three VINV alleles comprising at least three sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0295] In some aspects of the invention, a potato genome is provided, characterized by containing mutations in four VINV alleles, the four VINV alleles comprising four sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0296] In some aspects of the invention, a potato genome is provided, characterized in that at least one VINV allele contains a mutation, each mutation comprising one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO: 169.

[0297] In some aspects of the invention, a potato genome is provided, characterized in that it contains mutations in at least two VINV alleles, each mutation containing one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are compared with SEQ ID NO: 169.

[0298] In some aspects of the invention, a potato genome is provided, characterized in that it contains mutations in at least three VINV alleles, each mutation containing one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are compared with SEQ ID NO: 169.

[0299] In some aspects of the invention, a potato genome is provided, characterized in that it contains mutations in four VINV alleles, each mutation containing one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are compared with SEQ ID NO: 169.

[0300] In another aspect, this document provides a recombinant DNA construct comprising a first expression cassette containing a first DNA sequence encoding any of the aforementioned guide RNAs. In some embodiments, the first DNA sequence is operatively linked to a first plant-expressible promoter. In some embodiments, the recombinant DNA construct further comprises an expression cassette containing a second DNA sequence encoding a guide endonuclease, wherein the second DNA sequence is operatively linked to a second plant-expressible promoter. In some embodiments, the guide endonuclease is a Cas protein.

[0301] In another respect, this article provides a vector that contains any of the aforementioned recombinant DNA constructs.

[0302] In another aspect, this document provides a host cell comprising the aforementioned vector construct. In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is an Agrobacterium cell. In some embodiments, the host cell is a plant cell.

[0303] In another aspect, this document provides a composition comprising any of the aforementioned guide RNAs complexed with a guiding endonuclease. In some embodiments, the guiding endonuclease is a Cas protein.

[0304] In another aspect, this document provides a kit for producing modified potato plants, plant parts, or plant cells comprising any one or any combination of the aforementioned guide RNA, recombinant DNA construct, vector, host cell, composition, or combination thereof. In some embodiments, the kit also includes instructions for using the guide RNA, recombinant DNA construct, vector, host cell, composition, or combination thereof to introduce one or more guide endonucleases into potato cells, which together bind to the original spacer sequence of one or more VINV alleles, two or more VINV alleles, three or more VINV alleles, or each of four VINV alleles.

[0305] In some respects, this article provides a modified potato plant, plant part, or plant cell containing a mutation, such as but not limited to deletion, editing, phase shift, inversion, or duplication, in one, two, three, or four VINV alleles, wherein the mutation is generated by a guide endonuclease, and wherein the endonuclease binds a protospacer sequence comprising the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or the original spacer sequence of the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, ... consisting of SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: Sequences NO:157 or SEQ ID NO:158 having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity.

[0306] In some embodiments, the potato plant, plant part, or plant cell contains a mutation in one, two, three, or four VINV alleles. In some embodiments, the endonuclease is complexed with a guide RNA selected from the group consisting of: SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or sequences having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 159.

[0307] In some respects, this article provides a modified potato plant, plant part, or plant cell containing a mutation in one, two, three, or four VINV alleles, wherein each mutation is generated by a guided endonuclease, and wherein each mutation contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is aligned with SEQ ID NO: 169.

[0308] In some embodiments of the foregoing aspects, the tuber sugar profile obtained from said plant contains lower levels of glucose, fructose, or both compared to the tuber sugar profile obtained from the control plant. In some embodiments, the tuber sugar profile obtained from said plant contains lower levels of glucose compared to the tuber sugar profile obtained from the control plant. In some embodiments, the tuber sugar profile obtained from said plant contains lower levels of fructose compared to the tuber sugar profile obtained from the control plant. In some embodiments of the foregoing aspects, the tuber sugar profile obtained from said plant contains higher levels of sucrose compared to the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 200% compared to the tuber sugar profile obtained from the control plant. In some embodiments, the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 100%, 50%, or 25% compared to the tuber sugar profile obtained from the control plant. In some embodiments, the tuber sugar profile is obtained using colorimetric determination. In other embodiments, the tuber sugar profile is obtained using high-performance liquid chromatography (HPLC). In some embodiments, the tuber sugar profile is obtained at harvest. In other embodiments, the tuber sugar profile is obtained after refrigeration.

[0309] In some embodiments of the foregoing, the refrigerated acrylamide content is lower than that of control potato plants, plant parts, or plant cells. In some embodiments, the refrigerated acrylamide content is at least 50%, at least 75%, at least 85%, at least 95%, or at least 99% lower than that of control potato plants, plant parts, or plant cells. In some embodiments, the refrigerated acrylamide content is obtained from potato food products.

[0310] In some embodiments of the foregoing, the specific gravity of potatoes is higher than that of control potato plants, plant parts, or plant cells. In some embodiments, the specific gravity level is obtained from potato food products.

[0311] In some embodiments of the foregoing aspects, the potato chip brightness score of potato food products produced from said plant is higher than that of potato chip brightness scores of potato food products produced from control plants. In some embodiments, the potato chip brightness score is determined by colorimetric readings. In some embodiments, the potato chip brightness score is between 25% and 100% higher than that of processed potato products produced from control plants.

[0312] In some variations of the foregoing aspects and implementation schemes, the control potato plant, plant part, or plant cell lacks one or more or all of the deletions, edits, inversions, or duplications in the VINV alleles. In some variations of the foregoing aspects and implementation schemes, the control plant is unedited. In some variations of the foregoing aspects and implementation schemes, the control plant is wild-type. In some variations of the foregoing aspects and implementation schemes, the control plant is a null isolate. In some variations of the foregoing aspects and implementation schemes, the control potato plant, plant part, or plant cell belongs to the same breeding line as the modified potato plant, plant, plant part, or plant cell.

[0313] In other respects, this document provides a processed potato product derived from any of the aforementioned modified potato plants, plant parts, or plant cells, wherein the processed potato product contains a detectable amount of one, two, three, or four VINV alleles from the modified plant, plant part, or plant cell. In some embodiments, the processed potato product is selected from the group consisting of: biomass, oil, meal, edible starch, syrup, sugar, animal feed, flour, flakes, potato chips, French fries, potato wedges, potato cakes, potato balls, baked potatoes, mashed potatoes, dehydrated potatoes, granules, peels, cooked skins, potato pulp, mashed potatoes, filter cake, sieve residue, potato residue, potato protein isolate or concentrate, discarded French fries, discarded potato chips, scraps, batter, crumbs, defective chunks, or alcoholic beverages. In some embodiments, the processed potato product is potato chips. In some embodiments, the processed potato product is non-renewable.

[0314] In some embodiments of the aforementioned method, the VINV activity in potato plants, plant parts, or plant cells is reduced by at least 50% compared to control potato plants, plant parts, or plant cells. In some embodiments, the VINV activity in potato plants, plant parts, or plant cells is reduced by at least 85%, at least 95%, or at least 99% compared to control potato plants, plant parts, or plant cells.

[0315] Preferably, in one embodiment, the method as described herein includes: the at least one plant cell, tissue, organ, plant, or seed is not obtained by a method that is inherently biological. Instead, the at least one plant cell, tissue, organ, plant, or seed is obtained through at least one step of artificial intervention that would not occur naturally, which affects the plant cell by modifying and / or introducing steps that influence the technical properties of sexual hybridization and selection. Such steps may include genome editing steps (e.g., exchanging target bases or nucleotides), chemical treatment steps (e.g., for chromosome doubling), reagents or genes or gene products including chromosome elimination, introducing foreign genes or genetic material into the plant genome (nuclear genome, mitochondrial genome, or plastid genome), etc., or any combination thereof. Attached Figure Description

[0316] This application can be understood by referring to the following description in conjunction with the accompanying drawings.

[0317] The accompanying drawings, which are incorporated herein and form part of this specification, illustrate some, but not unique or exclusive, exemplary embodiments and / or features. The embodiments and drawings disclosed herein are intended to be illustrative rather than restrictive.

[0318] Figure 1 The present disclosure of Atlantic shows the haplotypes of Hap1, Hap2, Hap3 and Hap4, and the haplotypes of Russet Burban show Hap1, Hap2 and two copies of Hap5.

[0319] Figure 2 The target sites within the editing window on chromosome 3 of the potato genome are shown.

[0320] Figure 3 A diagram showing the visualization of the six fully edited potato allele sequences, highlighting the location of VINV on chromosome 3, as well as exons and introns.

[0321] Figure 4 The diagram shows the amino acid sequences of the edited and wild-type alleles among the six samples.

[0322] Figure 5 The editing efficiency of guide endonucleases targeting protospacers based on endonucleases was described.

[0323] Figure 6 The tuberous sugar profiles of Russet Burbank potatoes were depicted before and after VINV editing.

[0324] Figure 7 The tuberous sugar profiles of Atlantic potatoes were depicted before and after VINV editing.

[0325] Figure 8 The color changes are depicted when unedited Russet Burbank potato chips are fried.

[0326] Figure 9 The edited Russet Burbank fries were shown to be lighter in color after frying compared to the unedited control.

[0327] Figure 10 The edited Russet Burbank tubers were depicted as having a similar color before frying compared to the unedited control.

[0328] Figure 11 The study showed that, compared to the unedited control, the edited Atlantic potato chips were lighter in color after frying.

[0329] Figure 12 The edited Atlantic tubers were shown to have a similar color before frying compared to the unedited control.

[0330] Figure 13 The chip brightness ratings were depicted for various combinations of VINV haplotype edits.

[0331] Figure 14 The total field yield of eight samples with the edited VINV allele was depicted when compared with the unedited sample and the wild type.

[0332] Figure 15 The chip brightness scores of five samples with the edited VINV allele were depicted when compared to unedited samples and wild type.

[0333] Figure 16 Describe reducing sugars and non-reducing sugars in wild-type potatoes.

[0334] Figure 17 The reducing and non-reducing sugars in potatoes edited by the triple mutant VINV were depicted.

[0335] Figure 18The reducing and non-reducing sugars in potatoes with complete knockout of VINV (four alleles) were depicted.

[0336] Figure 19 A comparison of total sugar in wild-type (WT), triple-mutant, and completely knocked-out VINV edited potatoes was depicted.

[0337] Figure 20 The total tuber yield of ten greenhouse-grown samples with the edited VINV allele was depicted compared to unedited samples and wild type.

[0338] Figure 21 The weight scores of various combinations of VINV haplotypes were described.

[0339] Figure 22 The study depicted a comparison of edited Atlantic E-PED165-7398 tubers with wild-type and unedited potatoes at harvest time.

[0340] Figure 23 Edited Atlantic E-PED165-7398 tubers were compared with wild-type and unedited potatoes after one month of refrigeration (FRY1) and three months of refrigeration (FRY2).

[0341] Figure 24 The edited Atlantic E-PED165-7398 tubers at sites BG and MR, after one month of refrigeration, were compared with wild-type and unedited potatoes.

[0342] Figure 25 The edited Atlantic E-PED165-7398 tubers were compared with wild-type and unedited potatoes at sites BG and MR after being refrigerated for two to three months at FRY2. Detailed Implementation

[0343] The following description is provided to enable those skilled in the art to make and use the various embodiments. The descriptions of specific apparatuses, techniques, and applications are provided by way of example only. Various modifications to the examples described herein will be apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and shown herein, but should be given the scope consistent with the claims.

[0344] As will be discussed in further detail herein, this specification discloses potato varieties with unique edited haplotypes, designated Hap1, Hap2, Hap3, Hap4, and Hap5. These edited haplotypes result in improved refrigeration properties in the potatoes; specifically, potatoes with one or more unique edited haplotypes exhibit lower levels of glucose, fructose, and / or acrylamide in their tuber sugar profiles compared to control plants, leading to improved refrigeration characteristics. This improved refrigeration results in higher brightness scores for potato chips from potatoes with these unique edited haplotypes.

[0345] As will be discussed in more detail in this article, a total of five haplotypes have been identified in Atlantic and Russet Burbank type potatoes, such as Figure 1 As shown in the diagram. Atlantic contains Hap1, Hap2, Hap3, and Hap4, which are edited to produce a modified sucrose to fructose ratio and reduced acrylamide in processed potato products. Also as... Figure 1 As shown, the Rustet Burbank type potato has Hap1, Hap2 and two copies of Hap5, which can be edited to achieve a modified sucrose to fructose ratio and reduced acrylamide in processed potato products.

[0346] In one aspect of the invention, a potato plant, plant part, or plant cell is described herein comprising one or more VINV alleles, two or more VINV alleles, three or more VINV alleles, or mutations in four VINV alleles, such as, but not limited to, deletion, editing, phase shift, inversion, or duplication, wherein the VINV alleles are identified as Hap1, Hap2, Hap3, Hap4, and Hap5, wherein the mutation is generated by a guide endonuclease such that the VINV alleles of the potato plant, plant part, or plant cell comprise one or more sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0347] On the other hand, this document provides a potato plant, plant part, or plant cell containing mutations in one or more, two or more, three or more, or four VINV alleles, such as, but not limited to, deletions, edits, phase shifts, inversions, or duplications, wherein the mutations are generated by a guide endonuclease, and wherein the endonuclease binds to the original spacer sequence comprising SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or with SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, and SEQ ID NO: 158. 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158 are sequences having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity.

[0348] On the other hand, this article describes a potato plant, plant part, or plant cell containing mutations, such as but not limited to deletions, edits, phase shifts, inversions, or duplications, in one or more, two or more, three or more, or four VINV alleles, generated by a guided endonuclease, and wherein each mutation contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO: 169. These potato plants, plant parts, or plant cells address a long-standing need for rapidly and effectively mitigating the effects of low-temperature induced saccharification, including the accumulation of reducing sugars and acrylamide, and the formation of dark spots.

[0349] On the other hand, this document describes a method for producing potato plants, plant parts, or plant cells, wherein the method comprises: introducing one or more guide endonucleases into potato cells, the endonucleases together binding to the original spacer sequence of each of one or more, two or more, three or more, or four VINV alleles; and regenerating potato plants, plants, parts, or plant cells from potato cells, wherein the potato plants, plants, parts, or plant cells contain mutations in one or more, two or more, three or more, or four VINV alleles, such as, but not limited to, deletions, edits, phase shifts, inversions, or duplications, such that the VINV alleles of the potato plants, plant parts, or plant cells comprise one or more, two or more, three or more, or four VINV alleles, four selected from SEQ ID NO: 1-148 and SEQ ID NO: The sequence of groups consisting of 170-229.

[0350] In another aspect, this document describes a method for producing potato plants, plant parts, or plant cells, wherein the method comprises introducing a guiding endonuclease into potato cells, the endonuclease binding to a protospacer sequence comprising SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, and SEQ ID NO:158, or with SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, or SEQ ID NO:158. NO:158 has a sequence with at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity; and the regeneration of potato plants, plants, parts, or plant cells from potato cells, wherein the potato plants, plants, parts, or plant cells contain deletions, edits, phase shifts, inversions, or duplications in one or more, two or more, three or more, or four VINV alleles.

[0351] On the other hand, this article describes a method for producing potato plants, plant parts, or plant cells, wherein the method includes introducing one or more guide endonucleases into potato cells, the endonucleases together binding to the original spacer sequence of each of one or more, two or more, three or more, or four VINV alleles; and regenerating potato plants, plants, parts, or plant cells from potato cells, wherein the potato plants, plants, parts, or plant cells contain mutations in one or more, two or more, three or more, or four VINV alleles such that when the modified VINV alleles are compared with SEQ ID NO: 169, each mutation contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169.

[0352] In another aspect, this article provides a potato genome characterized by containing mutations in one or more, two or more, three or more, or four VINV alleles, of which the one or more, two or more, three or more, or four VINV alleles are selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

[0353] On the other hand, this article describes the potato genome, characterized in that when the modified VINV allele is compared with SEQ ID NO: 169, a mutation is contained in one or more, two or more, three or more, or four VINV alleles corresponding to SEQ ID NO: 169.

[0354] On the other hand, this article describes guide RNAs, recombinant DNA constructs, host cells, and kits associated with the production of potato plants, plant parts, or plant cells, and their use in their manufacturing methods.

[0355] definition

[0356] The term "a" or "an" refers to one or more of the entities, i.e., it refers to a plural entity. Therefore, the terms "a" or "an," "one / a kind or multiple / kinds," and "at least one / kind" are used interchangeably herein. Furthermore, the reference to "component" by the indefinite article "a" or "an" does not preclude the possibility of more than one of the components, unless the context explicitly requires the existence of exactly one of the components.

[0357] As used in this specification, unless otherwise stated, the term "and / or" is used in this disclosure to mean "and" or "or".

[0358] As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerical value used in this application, whether or not it is used with “about” or “approximately”, is intended to cover any normal fluctuations as understood by one of ordinary skill in the art. In some embodiments, the terms “about” or “about” refer to a range of values ​​that fluctuate (greater or less than) by 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower in either direction of the specified reference value, unless otherwise stated or clearly apparent from the context (but such value should not exceed 100% of the possible value).

[0359] As used herein, the term "allele" refers to any one or more alternative forms of a gene located at a specific locus. In the diploid (or double diploid) cells of an organism, the alleles of a given gene are located at a specific location or locus on a chromosome, with one allele present on each chromosome of a homologous chromosome pair. Similarly, in the tetraploid cells of an organism, one allele is present on each of the four homologous chromosomes in a set. A "heterozygous" allele is a different allele located at a specific locus, which is located alone on the corresponding homologous chromosome. A "homozygous" allele is the same allele located at a specific locus, which is located alone on the corresponding homologous chromosome in the cell.

[0360] As used herein, the terms “at least a portion” or “fragment” for nucleic acid or polypeptide mean a portion having the smallest size characteristic of such a sequence, or any larger fragment (at most and including the full-length molecule). Fragments of polynucleotides disclosed herein may encode the biologically active portion of a gene regulatory element. The biologically active portion of a gene regulatory element may be prepared by isolating a portion of one of the polynucleotides disclosed herein that contains the gene regulatory element and evaluating its activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, etc., up to a full-length polypeptide. The length of the portion to be used will depend on the specific application. A portion of a nucleic acid that can be used as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide that can be used as an epitope may be as short as 4 amino acids. A portion of a polypeptide that performs the function of a full-length polypeptide will typically be longer than 4 amino acids. In some embodiments, the peptide or polynucleotide fragment comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire length of the reference peptide or polynucleotide. In some embodiments, the peptide or polynucleotide fragment may contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 or more nucleotides or amino acids.

[0361] As used herein, a “base editor” refers to a protein or fragment thereof having the same catalytic activity as the protein from which it originates, and which, when provided alone or as a molecular complex (referred to herein as a base editing complex), has the ability to mediate targeted base modification, i.e., the conversion of a target base, thereby resulting in a target point mutation; if the base conversion does not cause a silent mutation but results in a conversion of the amino acid encoded by the codon containing the position to be converted by the base editor to be used, then the point mutation may in turn lead to a targeted mutation. At least one base editor according to this disclosure is temporarily or permanently linked to at least one CRISPR-related effector, or optionally linked to a component of at least one CRISPR-related effector complex.

[0362] As used herein, the terms “Cas9 nuclease” and “Cas9” are used interchangeably to refer to an RNA-guided DNA endonuclease associated with CRISPR (clustered regularly spaced short palindromic repeats), including the Cas9 protein or fragments thereof (such as proteins containing the active DNA-cutting domain of Cas9 and / or the gRNA-binding domain of Cas9). Cas9 is an integral part of the CRISPR / Cas genome editing system, which, guided by guide RNA, targets and cuts DNA target sequences, creating DNA double-strand breaks (DSBs).

[0363] The terms “Cas12 nuclease” and “Cas12” are used interchangeably in this document and refer to RNA-guided DNA endonucleases associated with CRISPR (clustered regularly spaced short palindromic repeats), including the Cas12 protein or fragments thereof (such as proteins containing the active DNA-cutting domain of Cas12 and / or the gRNA-binding domain of Cas12). Cas12 is an integral part of the CRISPR / Cas genome editing system, which, guided by guide RNA, targets and cuts DNA target sequences, creating DNA double-strand breaks (DSBs).

[0364] As used in this article, “centimolecular weight” (cM) is a unit of measurement for recombination frequency and genetic distance between two loci. One cM is equal to the 1% probability that a marker at one locus will segregate from a marker at a second locus due to crossing over in a single generation.

[0365] As used in this article, the term "cloning" or "performing cloning" in the context of plants refers to the production of "suckers" that are genetically identical to the original plant. These suckers can be cut from the original plant, planted in the ground, and will also grow into fruit-bearing plants.

[0366] A cloning vector typically contains one or a few restriction endonuclease recognition sites, where foreign DNA sequences can be inserted in a deterministic manner without losing the vector's basic biological function; and marker genes for identifying and selecting cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance, or ampicillin resistance.

[0367] As used herein, “closely linked” means that a marker or locus is located within approximately 20 cM, 15 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of another marker or locus. For example, 20 cM means that recombination occurs between the marker and the locus at a frequency equal to or less than approximately 20%.

[0368] As used herein, the term "codon optimization" means adapting the codon usage of DNA or RNA to the codon usage of a target cell or organism to improve the transcription rate of the recombinant nucleic acid in that target cell or organism. It is well known to those skilled in the art that, due to codon degeneracy, a target nucleic acid can be modified at a position that, after translation, still results in the same amino acid sequence at that position; this is achieved through codon optimization, taking into account the species-specific codon usage of the target cell or organism.

[0369] As used herein, “refrigeration” means storing potatoes at 12°C or lower. Alternatively, “refrigeration” refers to a temperature range of 2°C to 12°C. Examples of “refrigeration” temperatures for potatoes are 2°C to 4°C or 8°C to 10°C. Refrigeration can be carried out for a period of at least 2 hours. More specifically, refrigeration can be carried out for periods of at least three hours, at least four hours, at least five hours, at least six hours, at least eight hours, at least ten hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, or longer.

[0370] As used herein, “complementarity” refers to the ability to pair between two sequences containing naturally occurring or non-naturally occurring bases or their analogues through base stacking and specific hydrogen bonding. For example, if a base at one position of a nucleic acid can form a hydrogen bond with a base at a corresponding position of a target, those bases are considered complementary to each other at that position. Nucleic acids may contain universal bases or inert, base-free spacers that do not contribute positively or negatively to hydrogen bonding. Base pairing can include canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., wobbling base pairing and Hogstein base pairing). It should be understood that for complementary base pairing, an adenosine base (A) is complementary to a thymidine base (T) or a uracil base (U), a cytosine base (C) is complementary to a guanosine base (G), and universal bases (such as 3-nitropyrrole or 5-nitroindole) can hybridize with any A, C, U, or T and are considered complementary to them. Nichols et al., Nature, 1994;369:492-493; and Loakes et al., Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) is also considered a universal base in the field and is thought to be complementary to any A, C, U, or T. See Watkins and Santa Lucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.

[0371] As mentioned in this article, a "complementary nucleic acid sequence" is a nucleic acid sequence containing a nucleotide sequence that enables it to nonvalently bind to another nucleic acid in a sequence-specific, antiparallel manner under appropriate in vitro and / or in vivo temperature and solution ionic strength conditions (i.e., nucleic acid-specific binding of complementary nucleic acids).

[0372] Sequence alignment methods for comparing and determining the percentage of sequence identity and complementarity are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the following methods: the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI); manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology); using algorithms known in the art, including BLAST and BLAST2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990). J. Mol. Biol. 215:403-410. Software used for BLAST analysis is publicly available from the National Center for Biotechnology Information. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK), ALIGN Plus (Scientific and Educational Software, Pennsylvania), and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Other alignment programs are Sequencher (Gene Codes, Ann Arbor, Michigan) using default parameters, and MUSCLE (multiple sequence alignment by log-expectation, a publicly available computer software).

[0373] As described herein, a “control potato plant” is a plant that does not possess at least one genetic modification of the VINV allele. This can be a plant that has undergone a process designed to produce the genetic modification, but for whatever reason the modification did not occur, i.e., an “unedited control.” In some cases, control potato plants can be “wild-type” potato plants, such as control potato plants that have not undergone the experimental treatments experienced by other potato plants. Examples of control potato plants can be plants used by potato breeders in planting trials as a “test variety” to which the performance of the experimental potato groups can be compared.

[0374] As used in this article, "control plant" generally refers to a potato plant, plant part, or plant cell that has not undergone gene editing. A control plant can be a "null isolate," which is an isogenetic plant that has undergone the same gene editing and regeneration process but lacks the gene-edited material. A control plant can be "unedited," referring to a plant that has undergone protoplast preparation and subsequent regeneration from the resulting protoplasts. A control plant can also be "wild-type," referring to a plant that has not undergone protoplast preparation and regeneration.

[0375] "Corresponding to" or "equal to" means that a polynucleotide (a) has a nucleotide sequence that is wholly or partially identical or complementary to that of a reference polynucleotide sequence, or (b) encodes an amino acid sequence that is identical to the amino acid sequence in a peptide or protein. Within its scope, this phrase also includes peptides or polypeptides that have an amino acid sequence that is substantially identical to the amino acid sequence in a reference peptide or protein.

[0376] The term "CRISPR RNA" or "crRNA" refers to an RNA strand responsible for hybridizing with a target DNA sequence and recruiting CRISPR endonucleases and / or CRISPR-related effectors. crRNA may be naturally occurring or synthesized using any known method for producing RNA.

[0377] Therefore, as used herein, “CRISPR-related effector” can be defined as any CRISPR (clustered regularly spaced short palindromic repeats)-related nuclease, nickase, or recombinase capable of introducing single-stranded or double-stranded cleavage into a genomic target site, or capable of introducing targeted modifications (including point mutations, insertions, or deletions) into a target genomic target site. At least one CRISPR-related effector may function alone or in combination with other molecules as part of a molecular complex. CRISPR-related effectors may exist as fusion molecules or as individual molecules that bind to or are bound to at least one of the covalent or non-covalent interactions with gRNA and / or target sites, such that the components of the CRISPR-related complex are physically close to each other.

[0378] As used herein, the term "CRISPR landing site" refers to a DNA sequence that can be targeted by the CRISPR-Cas complex. In some embodiments, the CRISPR landing site comprises a proximal spacer / spacer-adjacent motif combination sequence that can be cleaved by the CRISPR complex.

[0379] The terms “CRISPR complex,” “CRISPR endonuclease complex,” “CRISPR Cas complex,” or “CRISPR-gRNA complex” are used interchangeably herein. “CRISPR complex” refers to a Cas9 nuclease and / or a CRISPR-related effector complexed with a guide RNA (gRNA). Therefore, the term “CRISPR complex” refers to a combination of a CRISPR endonuclease and a guide RNA capable of inducing double-strand breaks at the CRISPR landing site. In some embodiments, the “CRISPR complex” of this disclosure refers to a combination of a catalytically inactivating Cas9 protein and a guide RNA capable of targeting a target sequence, but which is unable to induce double-strand breaks at the CRISPR landing site due to its loss of nuclease activity. In other embodiments, the “CRISPR complex” of this disclosure refers to a combination of a Cas9 cleavage enzyme and a guide RNA capable of introducing gRNA-targeted single-strand breaks into DNA, rather than double-strand breaks generated by wild-type Cas enzymes.

[0380] As used in this article, the terms “hybridization,” “cross-pollination,” or “hybrid breeding” refer to the process of applying pollen from one flower on one plant (by artificial or natural means) to the ovule (stigma) of another flower on another plant.

[0381] As used herein, the term "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments of this disclosure, the deaminase refers to cytidine deaminase, which catalyzes the deamination of cytidine or deoxycytidine to uracil or deoxyuridine, respectively. In other embodiments of this disclosure, the deaminase refers to adenosine deaminase, which catalyzes the deamination of adenine to form hypoxanthine (in the form of its nucleoside inosine), which is then read by DNA polymerase as guanine.

[0382] As used herein, the term “derived from” means origin or source and can include naturally occurring, recombinant, unpurified, or purified molecules. Nucleic acids or amino acids derived from a particular origin or source may have all kinds of nucleotide variations or protein modifications as defined elsewhere herein.

[0383] As used herein, the terms "dicotyledon" and "dicotyledonous" refer to flowering plants that have an embryo containing two seed halves or cotyledons. Examples include tobacco; tomatoes; legumes, including peas, alfalfa, clover, and soybeans; oaks; maples; roses; mint; pumpkins; daisies; walnuts; cacti; violets; and ranunculus.

[0384] As used herein, in the case of the CRISPR complex, the term "guide sequence-specific binding" refers to the ability of the guide RNA to recruit CRISPR endonucleases and / or CRISPR-related effectors to the CRISPR landing site.

[0385] As used in this article, a "double" mutation refers to a genetic modification that occurs in the two alleles of a given gene.

[0386] As used herein, the term "endogenous" or "endogenous gene" refers to a gene that is naturally present in a location within the host cell genome. As used herein, "endogenous gene" is synonymous with "natural gene." Endogenous genes as described herein may include alleles of naturally present genes that have been mutated according to any method of this disclosure, i.e., endogenous genes can be modified at some point by conventional plant breeding methods and / or next-generation plant breeding methods.

[0387] As used herein, the term "exogenous" refers to a substance that originates from a source other than its natural origin. For example, the terms "exogenous protein" or "exogenous gene" refer to a protein or gene that originates from a non-natural source and has been artificially supplied to a biological system. As used herein, the terms "exogenous" and "heterogeneous" are used interchangeably and refer to substances that originate from sources other than their natural origin.

[0388] As used herein, “expression” and “expression level” refer to the relative or absolute amount of functional gene products present in a cell. As used herein, “gene product” includes, but is not limited to, nucleic acids (e.g., RNA), post-transcriptionally modified nucleic acids (e.g., spliced ​​RNA, polyadenylated mRNA), proteins (e.g., enzymes, structural proteins, etc.), and post-translational modified proteins (e.g., glycoproteins, lipoproteins, etc.). The function of a gene product refers to its wild-type, unmodified, and unrepressed function. As used herein, “reduced expression” refers to a relative decrease in the amount of functional gene product present in a cell. Reduced expression can refer to a decrease in the total amount of gene product present in a cell (e.g., a decrease in the amount of protein), or a decrease in the amount of functional gene product present in a cell (e.g., a decrease in the percentage of proteins with wild-type function, e.g., altered protein activity), or a decrease in the function of a gene product present in a cell (e.g., decreased protein activity compared to proteins with wild-type function). Reduced expression can be a decrease in the expression of a gene product encoding a gene product at a specific genomic locus. Reduced expression also includes “non-expression.” As used in this article, "not expressed" means that there is no functional gene product in the cell, or the expression level is insufficient to detect the gene product in the cell, or the expression level is insufficient to produce the function of the gene product in the cell, or the activity level is insufficient to result in the detectable activity of the gene product in the cell.

[0389] As used herein, an "expression cassette" is a DNA sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell. It contains a promoter operatively linked to the target nucleotide sequence, which in turn is operatively linked to a termination signal. It typically also contains the sequence required for the correct translation of the nucleotide sequence. The coding region typically encodes the target protein, but may also encode a target functional RNA in a sense or antisense direction, such as antisense RNA or untranslated RNA. Expression cassettes containing the target nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. Expression cassettes can also be naturally occurring but obtained in a recombinant form suitable for heterologous expression. Expression of the nucleotide sequence in the expression cassette can be controlled by a constitutive or inducible promoter, which initiates transcription only when the host cell is exposed to certain specific external stimuli. In the case of multicellular organisms, the promoter may also be specific to specific tissues or organs or developmental stages in animals and / or plants (including potato species).

[0390] As used herein, the term "gene" refers to any segment of DNA that is associated with a biological function. Therefore, genes include, but are not limited to, coding sequences and / or regulatory sequences required for their expression. Genes may also include unexpressed segments of DNA that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a target source or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0391] As used herein, the term "gene-edited plant, part, or cell" refers to a plant, part, or cell containing one or more endogenous genes edited by a gene-editing system. The gene-editing system disclosed herein includes a targeting element and / or an editing element. The targeting element recognizes a target genomic sequence. The editing element can modify the target genomic sequence, for example by replacing or inserting one or more nucleotides in the genomic sequence, deleting one or more nucleotides in the genomic sequence, altering the genomic sequence to include regulatory sequences, inserting transgenes at safe harbor genomic sites or other specific locations in the genome, or any combination thereof. The targeting element and the editing element may be located on the same nucleic acid molecule or different nucleic acid molecules. In some embodiments, the editing element is capable of precise genome editing by replacing single nucleotides using a base editor (such as a cytosine base editor (CBE) and / or an adenine base editor (ABE)) that is directly or indirectly fused to a CRISPR-related effector protein.

[0392] As used herein, “genetic modification” or “modification” refers to any sequence or portion thereof within a nucleic acid molecule that differs from the sequence of its ancestral nucleic acid molecule. For example, seeds containing an insertion or deletion of a genomic sequence that is not present in one of its parent plants contain genetic modifications. Genetic modifications can be naturally occurring or introduced. Genetic modifications can be introduced, for example, through plant breeding to introduce a genetic modification naturally occurring in one plant line into another; transgenic methods; gene editing; chemical mutagenesis; etc.

[0393] As used herein, “genotype” is the genetic makeup of an individual (or group of individuals) at one or more gene loci, as opposed to an observable trait (phenotype). A genotype is defined by the alleles at one or more known loci inherited by an individual from its parents. The term “genotype” can be used to refer to the genetic makeup of an individual at a single locus, multiple loci, or more generally, to the genetic makeup of an individual across all the genes in its genome. The term “genotype” can also refer to the genetic makeup that identifies an individual (or group of individuals) at one or more gene loci.

[0394] As used herein, “germstone” refers to a living source of genetic material. Germstone can be part of an organism or cell, or it can be separated from an organism or cell. Typically, germstone provides genetic material with a specific molecular composition, providing the material basis for some or all of the genetic characteristics of an organism or cell culture. As used herein, germstone includes cells, seeds, or tissues from which new plants can grow, or plant parts such as leaves, stems, pollen, or cells that can be cultured into complete plants.

[0395] As used herein, the terms “growth” or “regeneration” refer to the growth of a complete, differentiated plant from plant cells, plant cell groups, plant parts (including seeds), or plant fragments (e.g., from protoplasts, callus, or tissue parts).

[0396] The term "guided endonuclease" refers to a polypeptide possessing RNA-binding, DNA-binding, and / or DNA-cutting activity. The RNA-guided endonuclease forms a complex with guide RNA containing a sequence capable of binding to a target sequence on double-stranded DNA. In some embodiments, the RNA-guided endonuclease cleaves the double-stranded target DNA.

[0397] As used herein, the term "guide RNA" or "gRNA" refers to an RNA sequence or combination of sequences capable of recruiting CRISPR endonucleases and / or CRISPR-related effectors to a target sequence. Typically, gRNAs consist of crRNA and tracrRNA molecules that form a complex through partial complementarity, wherein the crRNA contains a sequence fully complementary to the target sequence used for hybridization and directs the CRISPR complex (i.e., the Cas9-crRNA / tracrRNA hybrid) to specifically bind to the target sequence. Additionally, single guide RNAs (sgRNAs) can be designed that incorporate characteristics of both crRNA and tracrRNA. Therefore, as used herein, guide RNAs can be natural or synthetic crRNAs (e.g., for Cpf1), natural or synthetic crRNA / tracrRNA hybrids (e.g., for Cas9), or single guide RNAs (sgRNAs).

[0398] The term "guide sequence" or "spacer sequence" refers to the portion of crRNA or guide RNA (gRNA) responsible for hybridization with target DNA.

[0399] As used herein, a “haplotype” refers to a unique set of 1n chromosomes with a unique allele genome. As used herein, each haplotype is distinguished from other haplotypes by containing a set of alleles that confer a unique combination of characteristics, which other haplotypes do not. As used herein, as a feature of this disclosure, each unique haplotype does not necessarily inherit from different parents—the polyploid organisms of this disclosure may contain three or more haplotypes inherited from two parents. As used herein, a “monoeleptic plant” generally refers to a plant line containing a single haplotype, a “dioeleptic plant” generally refers to a plant line containing two haplotypes, and a “multieleptic plant” generally refers to a plant line containing three or more haplotypes. In the case of allopolyploid potato plants containing multiple subgenomes, there is very little or no recombination between these subgenomes. As used herein, the term "at least one haplotype" usually refers to one or more haplotypes of the same subgenome, "at least two haplotypes" usually refers to two or more haplotypes of the same subgenome, and "at least three haplotypes" usually refers to three or more haplotypes of the same subgenome.

[0400] As used herein, the term "heterologous" refers to a substance originating from a source or location other than its natural source or location. In some embodiments, the term "heterologous nucleic acid" refers to a nucleic acid sequence that is not naturally present in a particular organism. For example, the term "heterologous promoter" may refer to a promoter derived from one source organism and utilized in another organism where the promoter is not naturally present. However, the term "heterologous promoter" may also refer to a promoter from the same source organism, but moved to a new location where the promoter would not normally be located.

[0401] Heterogeneous gene sequences can be introduced into target cells using an "expression vector," which can be a eukaryotic expression vector, such as a plant expression vector. Methods for constructing vectors are well known to those skilled in the art and are described in various publications. In particular, techniques for constructing suitable vectors are reviewed in the prior art, including descriptions of functional components such as promoters, enhancers, termination signals and polyadenylation signals, selection markers, origins of replication, and splicing signals. Vectors can include, but are not limited to, plasmid vectors, phage particles, granules, artificial / miniature chromosomes (e.g., ACE), or viral vectors such as baculoviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, retroviruses, and bacteriophages. Eukaryotic expression vectors typically also contain prokaryotic sequences that promote vector replication in bacteria, such as origins of replication and antibiotic resistance genes for selection in bacteria. Various eukaryotic expression vectors containing cloning sites operably linked to polynucleotides are well known in the art, and some are commercially available from companies such as Stratagene, La Jolla, Calif.; Invitrogen, Carlsbad, Calif.; Promega, Madison, Wis.; or BD Biosciences Clontech, Palo Alto, Calif. In one embodiment, the expression vector comprises at least one nucleic acid sequence that is a regulatory sequence necessary for transcription and translation of a nucleotide sequence encoding a target peptide / polypeptide / protein.

[0402] As used in this article, the term "hemiszygote" refers to a cell, tissue, or organism in which a gene is present only once in the genotype, such as a gene in a haploid cell or organism, a sex-linked gene in a heterogametic sex, or a gene in a chromosomal segment in a diploid cell or organism in which the mate segment has been missing.

[0403] As used herein, the terms “homologous” or “homophore” are known in the art and refer to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homophore,” “substantially similar,” and “substantially corresponding” are used interchangeably herein. Homologous sequences typically control, mediate, or influence the same or similar biochemical pathways, but specific homologous sequences may produce different phenotypes. Therefore, it should be understood that, as those skilled in the art will appreciate, this disclosure covers more than just specific exemplary sequences. These terms describe the relationship between genes present in one species, subspecies, variety, cultivar, or strain and corresponding or equivalent genes in another species, subspecies, variety, cultivar, or strain. For the purposes of this disclosure, homologous sequences are compared.

[0404] As used herein, the term "heterozygote" refers to a diploid or polyploid single cell or plant having different alleles (in the form of a given gene) present at at least one locus.

[0405] As used in this article, the term "heterozygous" refers to the presence of different alleles (in the form of a given gene) at a particular locus.

[0406] As used in this article, the term "homozygote" refers to a single cell or plant that has the same allele at one or more loci.

[0407] As used in this article, the term "homozygous" means that the same alleles are present at one or more loci in a homologous chromosomal region.

[0408] The term “homologous” is sometimes used to refer to the relationship between genes separated by speciation events (see “orthologous”) or the relationship between genes separated by gene duplication events (see “paralogous”).

[0409] The term "partial homolog" refers to a partially homologous gene or chromosome resulting from a polyploidization or chromosome duplication event. This contrasts with the more common definition of "homolog" above.

[0410] "Homologous sequences" or "homologous" or "orthologous" sequences are considered, believed, or known to be functionally related. Functional relationship can be indicated in any of several ways, including but not limited to: (a) the degree of sequence identity and / or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. The degree of sequence identity can vary, but in one embodiment, it is at least 50% (when using standard sequence alignment procedures known in the art), at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987), Supplement 30, Section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK) and ALIGN Plus (Scientific and Educational Software, Pennsylvania). Other non-restrictive alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector NTI (Invitrogen, Carlsbad, CA).

[0411] The terms “host cell,” “genetically engineered host cell,” “recombinant host cell,” and “recombinant strain” are used interchangeably herein and refer to a host cell that has been genetically engineered using the methods of this disclosure. Therefore, the term includes host cells that have undergone genetic alteration, modification, or engineering (e.g., bacterial, yeast, fungal, CHO, human, plant, plant-derived protoplasts, callus, etc.) such that they exhibit altered, modified, or different genotypes and / or phenotypes compared to their naturally occurring host cell origin (e.g., when genetic modifications affect the coding nucleic acid sequence). It should be understood that the term refers not only to the specific recombinant host cell discussed but also to the offspring or potential offspring of such host cells.

[0412] As used herein, the term "hybridization" refers to the pairing of complementary nucleotide bases (e.g., adenine (A) forming a base pair with thymine (T) in a DNA molecule and uracil (U) in an RNA molecule, and guanine (G) forming a base pair with cytosine (C) in both DNA and RNA molecules) to form a double-stranded nucleic acid molecule. (See, for example, Wahl and Berger (1987) Methods Enzymol. 152:399; Kimmel, (1987) Methods Enzymol. 152:507). Additionally, it is known in the art that for hybridization between two RNA molecules (e.g., dsRNA), the guanine (G) base pairs with uracil (U). For example, G / U base pairing is partly responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of base pairing between the anticodon of tRNA and the codon in mRNA. In the context of this disclosure, guanine (G) in the protein-binding region (dsRNA double strand) of a guide RNA molecule is considered complementary to uracil (U), and vice versa. Therefore, when a G / U base pair can be formed at a given nucleotide position in the protein-binding region (dsRNA double strand) of a guide RNA molecule, that position is not considered non-complementary, but rather complementary. It should be understood in the art that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid for specific hybridization. Furthermore, the polynucleotide can hybridize on one or more segments such that intermediate or adjacent segments do not participate in the hybridization event (e.g., loop structures or hairpin structures). The polynucleotide may contain at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity with the target region within the target nucleic acid sequence it targets.

[0413] As used herein, the terms “genetic introgression,” “introgressive,” and “introgression” refer to the process by which genes from one species, variety, or cultivar are transferred into the genome of another species, variety, or cultivar through hybridization. Hybridization can be natural or artificial. The process can optionally be accomplished by backcrossing with a recurrent parent, in which case genetic introgression refers to the transfer of genes from one species into the gene pool of another species through repeated backcrossing of an interspecific hybrid with one of its parents. Genetic introgression can also be described as heterologous genetic material stably integrated into the genome of the recipient plant.

[0414] This disclosure covers isolated or substantially purified nucleic acid or protein compositions. As used herein, “isolated” or “purified” nucleic acid molecules or proteins, or their biologically active portions, are substantially or essentially free of components that typically accompany or interact with nucleic acid molecules or proteins, as found in their natural environment. Therefore, isolated or purified polynucleotides or polypeptides are substantially free of other cellular material or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Suitably, “isolated” polynucleotides do not contain sequences naturally flanking the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) (especially protein-coding sequences). For example, in various embodiments, the isolated polynucleotide may contain nucleotide sequences shorter than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which are naturally flanking the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Peptides that are essentially free of cellular material include protein formulations containing less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating proteins. When recombinantly producing the proteins of this disclosure or their bioactive portions, the culture medium suitably represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-target protein chemicals.

[0415] As used herein, the term "strain" is used broadly to refer to, but not limited to, a group of plants asexually propagated from a single parent using tissue culture techniques, or a group of inbred plants that are genetically very similar due to their common parent. A plant is considered to "belong" to a particular strain if (a) it is a primary transformant (T0) plant regenerated from material of a strain; (b) it has a lineage consisting of T0 plants of that strain; or (c) it is genetically very similar due to a common ancestor (e.g., through inbreeding or self-pollination). In this case, the term "lineage" refers to the plant's pedigree, for example, based on the sexual hybridization performed, such that heterozygous (hemozygous) or homozygous genes or combinations of genes confer the desired trait on the plant.

[0416] As used herein, the term "locus" (plural loci) refers to any genetically defined site. A locus can be a gene, a part of a gene, or a DNA sequence that has some regulatory function, and can be occupied by different sequences.

[0417] As used herein, the term "population selection" refers to a form of selection in which individual plants are chosen to reproduce the next generation from their seed collection. Further details of population selection are described in this specification.

[0418] The term "modified" refers to a substance or compound that has been altered or changed compared to its unmodified counterpart (e.g., cells, polynucleotide sequences, and / or polypeptide sequences).

[0419] As used herein, the term "molecular marker" or "genetic marker" refers to an indicator used in methods for visualizing characteristic differences in nucleic acid sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), enzyme digestion amplified polymorphic sequences (CAPS) markers, or isoenzyme markers, or combinations of markers described herein, which define specific gene and chromosomal locations. The localization of molecular markers near alleles is a procedure readily performed by those skilled in the art of molecular biology, techniques described, for example, in Lefebvre and Chevre, 1995; Lorez and Wenzel, 2007; Srivastava and Narula, 2004; Meksem and Kahl, 2005; and Phillips and Vasil, 2001. General information about AFLP technology can be found in Vos et al. (1995, AFLP: a new technique for DNA fingerprinting, Nucleic Acids Res. 11 Nov 1995; 23(21): 4407-4414).

[0420] Probes contain identifiable, isolated nucleic acids that recognize target nucleic acid sequences. Probes include nucleic acids attached to addressable sites, detectable markers, or other reporter molecules and hybridizing with the target sequence. Typical markers include radioactive isotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidelines for selecting markers suitable for various purposes are discussed, for example, in Sambrook et al. (eds.), *Molecular Cloning: A Laboratory Manual*, 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., *Short Protocols in Molecular Biology*, 4th ed., John Wiley & Sons, Inc., 1999.

[0421] As used herein, “mutation” refers to the process of introducing a mutation into a selected DNA sequence. Mutations induced by nucleases are typically obtained through double-strand breaks, resulting in insertion / deletion mutations (“insertions”) that can be detected by deep sequencing analysis. Such mutations are typically the loss of several base pairs and have the effect of inactivating the mutated allele. In the methods described herein, mutagenesis occurs, for example, through double-strand DNA breaks produced by a TALE nuclease targeting a selected DNA sequence in plant cells. This mutagenesis results in a “TALE nuclease-induced mutation” (e.g., a TALE nuclease-induced knockout) and reduced expression of the target gene. Following mutagenesis, plants can be regenerated from the treated cells using known techniques (e.g., sown according to a standard planting procedure followed by self-pollination).

[0422] As used herein, the term "naturally occurring" applied to nucleic acids, peptides, cells, or organisms refers to nucleic acids, peptides, cells, or organisms found in nature. The term "naturally occurring" may refer to a gene or sequence derived from a naturally occurring source. Therefore, for the purposes of this disclosure, a "non-naturally occurring" sequence is a sequence that has been synthesized, mutated, engineered, edited, or otherwise modified to have a sequence different from a known natural sequence. In some embodiments, modifications may be made at the protein level (e.g., amino acid substitutions). In other embodiments, modifications may be made at the DNA level (e.g., nucleotide substitutions).

[0423] As used herein, the term “non-renewable” generally means that parts or cells of a potato plant, plant cells, processed potato products, or any of the foregoing cannot be induced to form a complete potato plant, or cannot be induced to form a complete potato plant capable of sexual and / or asexual reproduction.

[0424] As used herein, the terms "nucleotide change" or "nucleotide modification" refer to, for example, nucleotide substitutions, deletions, and / or insertions, as well as are well known in the art. For example, such nucleotide changes / modifications include mutations containing alterations that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded protein or the manner in which the protein is produced. As another example, such nucleotide changes / modifications include mutations containing alterations that produce substitutions, additions, or deletions that change the properties or activity of the encoded protein or the manner in which the protein is produced.

[0425] As used herein, the term "offspring" refers to any plant produced as a descendant from one or more parent plants or their offspring through vegetative or sexual reproduction. For example, offspring plants can be obtained by cloning or self-pollination of parent plants, or by hybridization of two parent plants, and include inbred lines and F1 or F2 or higher generations. F1 is the first generation of offspring produced by the parents, at least one of which is used as a donor for the trait for the first time, while the offspring of the second generation (F2) or subsequent generations (F3, F4, etc.) are samples produced by self-pollination of F1, F2, etc. Thus, F1 can be (and usually is) a hybrid produced by hybridization between two purebred parents (purebred being homozygous for a certain trait), while F2 can be (and usually is) an offspring produced by self-pollination of said F1 hybrid.

[0426] As used in this article, the term "open pollination" refers to a plant population being freely exposed to some gene flow, as opposed to "closed pollination," in which there is an effective barrier to gene flow.

[0427] As used herein, the terms "open-pollinated population" or "open-pollinated variety" refer to plants that are typically capable of at least some degree of cross-pollination and are selected according to standards. These plants may exhibit variation but also possess one or more genotypic or phenotypic characteristics that distinguish the population or variety from others. Hybrids do not have the barrier of cross-pollination and are therefore open-pollinated populations or open-pollinated varieties.

[0428] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one nucleic acid sequence is regulated by another. For example, a promoter is operably linked to a coding sequence when it is capable of regulating the expression of that sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in either a sense or antisense direction. In another instance, the complementary RNA region of this disclosure can be operably linked directly or indirectly to the 5′ or 3′ of a target mRNA, or to the interior of the target mRNA, or the first complementary region may be located at the 5′ of the target mRNA and its complementary sequence at the 3′ of the target mRNA.

[0429] The term "ortholog" refers to genes in different species that evolved from a common ancestor through speciation. Orthologs typically retain the same function during evolution. Identification of orthologs is crucial for reliably predicting gene functions in newly sequenced genomes.

[0430] As used in this article, when discussing plants, the term "ovule" refers to the female gametophyte, while the term "pollen" refers to the male gametophyte.

[0431] The term "paralog" refers to genes within the genome that are related through duplication. Orthologs typically retain the same function during evolution, while paralogs may evolve new functions, even if these functions are related to the original function.

[0432] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to an amino acid of any length in polymeric form, which may include coding and non-coding amino acids, amino acids that are chemically or biochemically modified or derived, and polypeptides having a modified peptide backbone.

[0433] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism (e.g., plant), or population of organisms, resulting from the interaction between an individual’s genetic makeup (i.e., genotype) and its environment.

[0434] The term "plant" refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, including any of the following: a whole plant, plant components or organs (e.g., leaves, stems, roots, tubers, etc.), plant tissues, tubers, microtubers, seeds, embryos, plant cells, protoplasts, and / or their offspring. Plant cells are the biological cells of a plant, taken from the plant or obtained by culturing cells taken from the plant.

[0435] The term "plant part" includes differentiated and undifferentiated tissues, including but not limited to tubers, microtubers, plant organs, plant tissues, roots, stems, branches, rootstocks, scions, stipules, petals, leaves, flowers, ovules, pollen, bracts, petioles, internodes, bark, pubescence, tillers, rhizomes, thallus, leaves, stamens, fruits, seeds, tumor tissues, and plant cells (e.g., single cells, protoplasts, embryos, and callus). Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, embryos, meristematic zones, callus, leaves, roots, branches, gametophytes, sporophytes, pollen, and microspores. Plant tissues may be located within the plant or in plant organs, tissues, or cell cultures.

[0436] As used herein, the term "plant tissue" refers to any part of a plant. Examples of plant organs include, but are not limited to, leaves, stems, roots, tubers, seeds, branches, hairs, tubercles, leaf axils, flowers, pollen, stamens, pistils, petals, pedicels, stalks, stigmas, styles, bracts, fruits, trunks, carpels, sepals, anthers, ovules, pedicels, needles, cones, rhizomes, stolons, branches, pericarps, endosperm, placentation, berries, stamens, and leaf sheaths.

[0437] The terms “polynucleotide,” “nucleic acid,” and “nucleotide sequence,” used interchangeably herein, refer to nucleotides (ribonucleotides or deoxyribonucleotides or similar compounds) of any length in polymeric form. The term refers to the primary structure of a molecule and therefore includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. The term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. It also includes modified nucleic acids, such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone-modified nucleic acids, etc. “Oligonucleotide” generally refers to a polynucleotide of about 5 to about 100 nucleotides in single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also called “oligomers” or “oligomers” and can be isolated from genes or chemically synthesized by methods known in the art. As applicable to the described implementation scheme, the terms “polynucleotide,” “nucleic acid,” and “nucleotide sequence” should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0438] As used in this article, the term "population" refers to a collection of genetically homogeneous or heterogeneous plants that share a common genetic origin.

[0439] As used herein, “potato” generally refers to the potato species. Furthermore, it will be apparent to those skilled in the art that some potato varieties include genetic introgression from related Solanum species, but unless otherwise stated, these varieties are still considered potatoes. The terms “potato” and “potato plant” include the whole potato plant or any part or derivative thereof, such as plant organs (e.g., harvested or unharvested flowers, leaves, etc.), plant cells, plant protoplasts, plant cells or tissue cultures that can regenerate into whole plants, regenerative or non-regenerative plant cells, plant callus, plant cell masses, and whole plant cells in or of a plant, such as embryos, pollen, ovules, ovaries (e.g., harvested tissues or organs), flowers, leaves, seeds, tubers, cloned plants, roots, stems, cotyledons, hypocotyls, root tips, meristems, nodes, stolons, etc. Potato plant parts or their derivatives may also include any of the aforementioned plant parts in an embedded form, such as branch meristems, nodes, stolons, etc., embedded in alginate, for example, embedded in artificial seeds. This also includes any developmental stage, such as seedling, immature, and mature stages.

[0440] As described herein, “potato food products” can be foods containing potato tuber tissue and / or food ingredients and / or components. Tuber tissue can be prepared for consumption and / or inclusion in food products by any of a variety of different artificial and / or mechanical processing methods, including “slicing,” which is defined herein as cutting or slicing potato tubers into various shapes. Other methods for preparing potato food products include washing, peeling, cutting, blanching, frying, freezing, and packaging.

[0441] Potato tubers are typically cut into cross-sections to create round, flat slices, which can then be processed into potato chips (commonly called potato chips in the US) or crisps (commonly called crisps in the UK). Potato tubers can also be cut into rod-shaped strips and processed into French fry (commonly called French fries in the US) or chips (commonly called chips in the UK). The invention described herein is not limited to any particular slice shape.

[0442] Examples of potato food products include tuber flesh that has been mashed, baked, boiled, fried, dehydrated, etc., and include potato starch, which can be used to bind meat mixtures, thicken sauces, stews, gravy and soups, and as a binder in cake mixes, dough, cookies and ice cream. Potato food products also include beverages made from fermented tuber tissue, including vodka and aquavit.

[0443] Examples of potato food products include tuber flesh that has been mashed, baked, boiled, fried, dehydrated, etc., and include potato starch, which can be used to bind meat mixtures, thicken sauces, stews, gravy and soups, and as a binder in cake mixes, dough, cookies and ice cream. Potato food products also include beverages made from fermented tuber tissue, including vodka and aquavit.

[0444] As used herein, "processed potato products" generally refers to biomass, oil, meal, edible starch, syrup, sugar, animal feed, flour, flakes, potato chips, French fries, potato wedges, potato cakes, potato balls, baked potatoes, mashed potatoes, dehydrated potatoes, granules, peels, cooked skins, potato pulp, mashed potatoes, filter cake, sieve residue, potato residue, potato protein isolate or concentrate, discarded French fries, discarded potato chips, scraps, batter, crumbs, defective pieces, or alcoholic beverages. In some embodiments, the processed potato product is potato chips. In some embodiments, the processed potato product is non-renewable.

[0445] As used herein, the term "primer" refers to an oligonucleotide that can anneal to an amplification target to allow DNA polymerase to attach, thereby acting as the starting point for DNA synthesis when under conditions that induce primer extension product synthesis (i.e., in the presence of nucleotides and reagents for polymerization (such as DNA polymerase) and at suitable temperature and pH). Primers are preferably single-stranded for maximum amplification efficiency. Preferably, primers are oligodeoxyribonucleotides. Primers must be long enough to initiate the synthesis of extension products in the presence of reagents for polymerization. The exact length of the primer will depend on many factors, including temperature and primer composition (A / T and G / C content). A bidirectional primer pair consists of a forward primer and a reverse primer, as commonly used in the field of DNA amplification (such as PCR amplification).

[0446] The term "protospacer" refers to the DNA sequence targeted by the guide sequence of crRNA or gRNA. In some implementations, the protospacer sequence hybridizes with the crRNA or gRNA guide (spacer) sequence of the CRISPR complex.

[0447] As used in this article, a "quadruple" mutation refers to a genetic modification that occurs in the four alleles of a given gene.

[0448] As used herein, the phrases “recombinant construct,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA construct” are used interchangeably. A recombinant construct comprises an artificial combination of nucleic acid fragments, such as regulatory and coding sequences that are not commonly found in nature. For example, a chimeric construct may contain regulatory and coding sequences derived from different sources, or from the same source but arranged in a manner different from that found in nature. Such constructs may be used alone or in combination with a vector. If a vector is used, the choice of vector depends on methods for transforming host cells as known to those skilled in the art. For example, a plasmid vector may be used. Those skilled in the art understand the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleic acid fragments of this disclosure. Those skilled in the art will also recognize that different independent transformation events will lead to different expression levels and patterns (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), therefore multiple events must be screened to obtain strains exhibiting the desired expression levels and patterns. This screening can be performed through Southern blotting analysis of DNA, Northern blotting analysis of mRNA expression, Western blotting analysis of protein expression, or phenotypic analysis. Vectors can be plasmids, viruses, bacteriophages, proviruses, phage particles, transposons, artificial chromosomes, etc., which can replicate autonomously or integrate into the chromosome of the host cell. Vectors can also be non-replicating naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of DNA and RNA within the same strand, polylysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, etc. As used in this article, the term “expression” refers to the production of functional end products, such as mRNA or proteins (precursors or mature forms).

[0449] The term "seed region" refers to the critical portion of the guide sequence of a crRNA or guide RNA most susceptible to mismatch with its target. In some embodiments, a single mismatch in the seed region of a crRNA / gRNA can render the CRISPR complex inactive at that binding site. In some embodiments, the seed region of the Cas9 endonuclease is located approximately 12 nt from the end of the 3' portion of the guide sequence, corresponding to (hybridization to) the protospacer target sequence portion adjacent to PAM. In some embodiments, the seed region of the Cpf1 endonuclease is located approximately 5 nt from the beginning of the 5' portion of the guide sequence, corresponding to (hybridization to) the protospacer target sequence portion adjacent to PAM.

[0450] As used in this article, the terms “self-pollinating,” “self-introducing,” or “self-pollinating” refer to the application of pollen from one flower on one plant (by artificial or natural means) to the ovules (stigmas) of the same or different flowers on the same plant.

[0451] The term "sequence identity" refers to the percentage of identical bases or amino acids at the same relative positions between two polynucleotide or polypeptide sequences. Therefore, one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another. For sequence comparisons, one sequence typically serves as a reference sequence to be compared with the test sequence. The term "reference sequence" refers to the molecule to which the test sequence is compared. When using the percentage of sequence identity to refer to proteins, it should be recognized that dissimilar residue positions are often due to conserved amino acid substitutions, where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), thus not altering the functional properties of the molecule. When differences in conserved substitutions exist in a sequence, the percentage of sequence identity can be adjusted upwards to correct for the conservatism of the substitution. Sequences that differ due to similar conserved substitutions are thus referred to as having "sequence similarity" or "similarity." Methods for making such adjustments are well known to those skilled in the art. Typically, this involves counting conserved substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Therefore, for example, when the score for the same amino acid is 1 and the score for non-conservative substitution is zero, the score for conservative substitution is between zero and 1. The score for conservative substitution is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).

[0452] As used in this article, a "single" mutation refers to a genetic modification that occurs in only one allele of a given gene.

[0453] As used in this article, the term “monoallelic transformed plant” refers to plants bred through a plant breeding technique called backcrossing, in which virtually all the desired morphological and physiological characteristics of the inbred species are restored, except for the monoallelic gene transferred to the inbred species via backcrossing.

[0454] As used in this article, "specific gravity" is a measure of density and a measure of potato quality. There is a strong correlation between tuber specific gravity and starch content and the percentage of dry matter or total solids. Higher specific gravity contributes to higher recovery rates and better processed product quality.

[0455] As used herein, the term "targeting" refers to the expectation that one article or molecule will interact with another article or molecule with a degree of specificity, thereby excluding untargeted articles or molecules. For example, a first polynucleotide targeting a second polynucleotide according to this disclosure has been designed to hybridize with the second polynucleotide in a sequence-specific manner (e.g., via Watson-Crick base pairing). In some embodiments, the selected hybridization region is designed such that the hybridization is unique for one or more target regions. If the target sequence (hybridization region) of the second polynucleotide is mutated or otherwise removed / separated from the second polynucleotide, the second polynucleotide may no longer be a target of the first targeting polynucleotide. Furthermore, "targeting" can be used interchangeably with "site specificity" or "site-specific," the latter referring to a molecular biological action that uses sequence information of the target genomic region to be modified and further relies on information about the mechanism of action of molecular tools, such as nucleases, including CRISPR nucleases and their variants, TALENs, ZFNs, a wide range of nucleases or recombinases, DNA-modifying enzymes (including base-modifying enzymes such as cytidine deaminases), histone-modifying enzymes, DNA-binding proteins, cr / tracr RNA, guide RNA, etc.

[0456] As used herein, the term "tissue culture" refers to a composition comprising isolated cells of the same or different types, or a collection of such cells organized into a part of a plant. Exemplary types of tissue cultures are protoplasts, callus, plant cell masses, and plant cells capable of generating complete tissue cultures in a plant or part of a plant, such as embryo, pollen, flower, seed, leaf, stem, root, root tip, anther, pistil, meristem, axillary bud, ovary, seed coat, endosperm, hypocotyl, cotyledon, etc. The term "plant organ" refers to a plant tissue or a group of tissues that constitute morphologically and functionally distinct parts of a plant. "Progeny" includes any subsequent generations of the plant.

[0457] The term "tracrRNA" refers to a small trans-coding RNA. TracrRNA is complementary to crRNA and pairs with it to form a crRNA / tracrRNA hybrid, which can recruit CRISPR endonucleases and / or CRISPR-related effectors to target sequences.

[0458] As used herein, the term “transgenic” or “genetically modified” refers to at least one nucleic acid sequence derived from the genome of an organism or synthesized from it, which is then introduced into a target host cell or organism or tissue and subsequently integrated into the host genome via a “stable” transformation or transfection method. In contrast, the term “transient” transformation, transfection, or introduction refers to a method of introducing a molecular tool comprising at least one nucleic acid (DNA, RNA, single-stranded or double-stranded, or a mixture thereof) sequence and / or at least one amino acid sequence, optionally containing a suitable chemical or biological agent, to achieve transfer to at least one target compartment of a cell (including, but not limited to, cytoplasm, organelles (including the nucleus), mitochondria, vacuoles, chloroplasts) or membrane, resulting in transcription and / or translation and / or association and / or activity of at least one introduced molecule, without achieving stable integration or incorporation of the corresponding at least one molecule introduced into the cell genome and thus heritable. The term “transgenic-free” means that a transgene is absent or not detected in the genome of the target host cell or tissue or organism.

[0459] As used in this article, a "triple" mutation refers to a genetic modification that occurs in the three alleles of a given gene.

[0460] "Variant" peptides are peptides derived from natural proteins by: deleting (so-called truncation) or adding one or more amino acids at the N-terminus and / or C-terminus of the natural protein; deleting or adding one or more amino acids at one or more sites in the natural protein; or replacing one or more amino acids at one or more sites in the natural protein.

[0461] The variant proteins covered by this disclosure are biologically active, meaning they retain the desired biological activities of the native protein, namely the regulatory or controllable activities as described herein. Such variants can be generated, for example, by genetic polymorphism or by artificial manipulation. The biologically active variants of the native R protein of this disclosure will have at least 40%, 50%, 60%, 70%, typically at least 75%, 80%, 85%, preferably about 90% to 95% or higher, and more preferably about 98% or higher sequence identity with the amino acid sequence of the native protein, as determined by the sequence alignment procedure described elsewhere herein using default parameters. The biologically active variants of the protein of this disclosure may differ from the protein by as few as 1-15 amino acid residues, as few as 1-10, for example 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0462] The nuclear proteins disclosed herein can be altered in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulation are generally known in the art. For example, amino acid sequence variants of the R protein can be prepared by mutation in the DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and the references cited therein. Guidelines on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), which is incorporated herein by reference. Conserved substitutions, such as exchanging one amino acid with another amino acid having similar properties, may be preferred.

[0463] A "variation of conserved modification" is a change, addition, or deletion of a single amino acid or a small percentage (usually less than 5%, more often less than 1%) in the coding sequence, where the change results in the substitution of the amino acid with a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conserved substitutions for each other: aliphatic amino acids: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I); aromatic amino acids: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing amino acids: methionine (M), cysteine ​​(C); basic amino acids: arginine I, lysine (K), histidine (H); and acidic amino acids: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q). See also Creighton, 1984. Additionally, an individual substitution, deletion, or addition of a single amino acid or a small percentage of amino acids in the coding sequence is also a "variation of conserved modification."

[0464] Methods for preparing and using nucleic acid probes and primers are described, for example, in Sambrook et al. (eds.), *Molecular Cloning: A Laboratory Manual*, 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., *Short Protocols in Molecular Biology*, 4th ed., John Wiley & Sons, Inc., 1999; and Innis et al., *PCR Protocols*, *A Guide to Methods and Applications*, Academic Press, Inc., San Diego, CA, 1990. Amplification primer pairs can be derived from known sequences, for example, by using computer programs designed for this purpose, such as PRIMER (version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). Those skilled in the art will understand that the specificity of a particular probe or primer increases with its length. Therefore, to obtain higher specificity, probes and primers containing at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of the target nucleotide sequence can be selected. For the PCR amplification of polynucleotides disclosed herein, oligonucleotide primers can be designed for PCR reactions to amplify the corresponding DNA sequences from cDNA or genomic DNA extracted from any target organism. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand (1999) PCRMethods Manual (Academic Press, New York).Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, and partially mismatched primers.

[0465] As used herein, the term “varietal” or “cultivated variety” means a group of similar plants that can be distinguished from other varieties within the same species by structural characteristics and phenotypes. As used herein, the term “varietal” has the same meaning as the corresponding definition in the International Convention for the Protection of New Varieties of Plants (UPOV Treaty) of 2 December 1961, which was amended in Geneva on 10 November 1972, 23 October 1978, and 19 March 1991. Therefore, “varietal” means a group of plants within a single known lowest level of plant taxonomy that, whether or not it fully meets the conditions for granting rights to breeders, is i) defined by the expression of characteristics derived from a given genotype or combination of genotypes, ii) distinguished from any other group of plants by the expression of at least one of said characteristics, and iii) considered a unit in relation to its unchanging reproductive suitability.

[0466] As used herein, the terms “vector,” “plasmid,” or “construct” refer broadly to any plasmid or virus encoding exogenous nucleic acid. The term should also be interpreted to include non-plasmid and non-viral compounds, such as polylysine compounds, that facilitate the transfer of nucleic acid into virions or cells. A vector may be a viral vector suitable as a delivery medium for delivering nucleic acid or its mutants into cells, or a non-viral vector suitable for the aforementioned purposes. Examples of viral and non-viral vectors for delivering DNA into cells and tissues are well known in the art and described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. USA 94:12744-12746). Examples of viral vectors include, but are not limited to, recombinant plant viruses. Non-limiting examples of plant viruses include: TMV-mediated (transient) transfection into tobacco (Tuipe, TH et al. (1993), J. Virology Meth, 42: 227-239), ssDNA genomic viruses (e.g., Geminiviridae), retroviruses (e.g., Caulimoviridae, Pseudoviridae, and Metaviridae), dsNRA viruses (e.g., Reoviridae and Partitiviridae), (-) ssRNA viruses (e.g., Rhabdoviridae and Bunyaviridae), (+) ssRNA viruses (e.g., Bromoviridae, Closteroviridae, Comoviridae, Luteoviridae, Potato Y Viridae, Sequiviridae, and Tobusviridae) and viroids (e.g., Pospiviroldae and Avsunviroidae)Detailed classification information on plant viruses can be found in Fauquet et al. (2008, "Geminivirus strain demarcation and nomenclature". Archives of Virology 153:783–821, the full text of which is incorporated herein by reference) and Khan et al. (Plantviruses as molecular pathogens; Publisher Routledge, 2002, ISBN 1560228954, 9781560228950). Examples of non-viral vectors include, but are not limited to, liposomes and polyamine derivatives of DNA.

[0467] Furthermore, "vector" is defined in particular as any plasmid, granule, phage, or Agrobacterium binary vector in double-stranded or single-stranded linear or circular form, which may or may not be self-propagating or mobile, and which can transform prokaryotic or eukaryotic hosts by integrating into the cell genome or existing outside the chromosome (e.g., autonomously replicating plasmids with a replication origin).

[0468] In particular, this includes shuttle vectors, which are DNA mediators that can replicate naturally or by design in two different host organisms, which may be actinomycetes and related species, bacteria and eukaryotes (e.g., higher plants, mammals, yeast or fungal cells).

[0469] The nucleic acid in the vector is controlled by and operably linked to an appropriate promoter or other regulatory element for transcription in a host cell (such as a microorganism, e.g., bacteria or plant cells). The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory element, while in the case of cDNA, it may be controlled by an appropriate promoter or other regulatory element for expression in a host cell.

[0470] As used herein, “wildtype” refers to the most common, typical form of a plant or gene found in nature. A “wildtype Vinv allele” is a naturally occurring Vinv allele that encodes a functional Vinv protein (e.g., found in naturally occurring potato plants), while a “non-functional mutant Vinv allele” is a Vinv allele that does not encode a functional Vinv protein. Such a “non-functional mutant Vinv allele” may include one or more mutations in its nucleic acid sequence, resulting in an undetectable amount of functional Vinv protein in the plant or plant cells.

[0471] General methods in molecular and cellular biochemistry can be found in the following standard textbooks: Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., Academic Press 1999); Viral Vectors (Kaplift and Loewy, Academic Press 1995); Immunology Methods Manual (I. Lefkovits, Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons 1998). The contents of these publications are incorporated herein by reference.

[0472] Potatoes and potato trimmings

[0473] In some embodiments, potato plants, plant parts, or plant cells contain one or more genetic modifications. Genetic modifications can be produced by modifying any nucleic acid sequence or genetic element through the insertion, deletion, or substitution of one or more nucleotides in a nucleic acid molecule. This can be done by the substitution of at least one nucleotide, the mutation of at least one nucleotide, the insertion of at least one nucleotide, the chemical alteration of at least one nucleotide, or a combination thereof, as long as the result is a detectable change in the nucleotide sequence compared to the sequence of the nucleic acid molecule before modification (e.g., by PCR, DNA sequencing, chromatography, etc.). Such modifications can be achieved by any of several well-known methods known in the art, including but not limited to random mutagenesis, genome editing, insertion of recombinant nucleic acids, hybridization of unmodified plants with modified plants to introduce the modification from the modified plant into the unmodified plant, etc. Genetic modifications can be naturally occurring or non-natural.

[0474] The genetic modifications described herein can be present in any known genetic element, including but not limited to protein-coding sequences, non-protein-coding sequences, promoter regions, 5' untranslated leader sequences, genes, exons, introns, poly-A signal sequences, 3' untranslated regions, regions encoding small RNAs (such as microRNAs and small interfering RNAs), and any other sequences that affect the transcription or translation of one or more nucleic acid sequences. In some embodiments, genetic modifications may include, but are not limited to, modifying or replacing target nucleotide sequences (such as regulatory elements), gene disruption, gene knockout, gene knockdown, gene knock-in, gene silencing (including, for example, expressing inverted repeat sequences in a target gene), RNA interference (including, for example, by inserting and / or expressing RNA interference constructs), modifying methylation states, modifying splice sites, introducing alternative splice sites, or any combination thereof. As used herein, gene disruption refers to altering a sequence or inserting a sequence into a gene or locus that results in reduced expression (including non-expression or altered activity) of a functional protein gene product. Gene disruption can be achieved by introducing genetic modifications into protein-coding sequences, including but not limited to as missense or nonsense mutations, or insertions, deletions, or substitutions. As used herein, knockout is a genetic modification in which a gene or gene product becomes completely invalid. Knockout of a gene product can be achieved by introducing a genetic modification into the protein-coding sequence of a gene or any non-protein-coding or regulatory sequence described herein. Knockdown, as used herein, is a genetic modification in which a gene or gene product is partially invalidated. Knockdown of a gene product can be achieved by introducing a genetic modification into the protein-coding sequence of a gene or a non-protein-coding or regulatory sequence; or by inserting a trans-acting element, such as a construct expressing an inverted repeat sequence of the gene product, or a construct expressing a DNA or RNA-binding protein (such as a transcriptional repressor), which may include, for example, an inactivated targeting nuclease, such as an inactivated Cas9 (dCas9). Knockin, as used herein, means the replacement or insertion of a DNA sequence at a specific DNA locus in a cell. Knockin can include, but is not limited to, the specific insertion of a heterologous amino acid coding sequence into the coding region of a gene, the insertion of a transcriptional regulatory element into a gene locus, or any of several methods of inserting a DNA sequence into a cell known to those skilled in the art.

[0475] In some embodiments, the potato plant, plant part, or plant cell contains one or more mutations that result in increased or decreased expression (including no expression or altered activity) of the gene product at a genomic locus. In some embodiments, genetic modifications that result in increased or decreased expression (including no expression or altered activity) of the gene product or locus may include, but are not limited to: modifications to enhancers, modifications to promoters, modifications to the 5' untranslated leader sequence, modifications to coding regions, modifications to non-coding regions, insertion and / or expression of RNA interference constructs targeting mRNA, modifications to regions encoding small RNA, modifications to the methylation state of genomic loci, expression of repressor proteins targeting DNA or mRNA sequences, and any other sequences that affect the transcription or translation of one or more nucleic acid sequences. In some implementations, genetic modifications that result in reduced expression (including non-expression or altered activity) of a gene product or locus may include, but are not limited to: modification or substitution of a target nucleotide sequence (such as a regulatory element), gene disruption, gene knockout, gene knockdown, gene knock-in, gene silencing (including, for example, by inserting and / or expressing an inverted repeat sequence in a target gene), RNA interference (including, for example, by inserting and / or expressing an RNA interference construct), expression of a repressor protein (e.g., dCas9), modification of the methylation state of a locus, modification of a splice site, introduction of an alternative splice site, or any combination thereof. As described herein, potato plants, plant parts, or plant cells generated using a guide endonuclease produce non-transgenic plants, plant parts, or plant cells. Given that the plants, plant parts, or plant cells do not carry any foreign DNA, they may be considered by regulatory agencies to be unmodified (unlike those defined herein) or non-transgenic.

[0476] In some embodiments, this document provides a potato plant, plant part, or plant cell containing a mutation in at least one, at least two, at least three, or four VINV alleles, wherein the mutation is generated by a guide endonuclease, such that the VINV alleles of the potato plant, plant part, or plant cell contain at least one, at least two, at least three, or four sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229. In this document, one or more, two or more, three or more, or four VINV alleles are generally referred to as “Hap1,” “Hap2,” “Hap3,” “Hap4,” and “Hap5,” referring to one, two, three, or four of the five VINV alleles on four different haplotypes. Furthermore, as used herein, potato plants, plant parts, or plant cells containing mutations in at least one, at least two, at least three, or four VINV alleles may be referred to, for example, as “Hap1,” “Hap1_Hap2,” “Hap1_Hap3,” “Hap1_Hap4,” “Hap2_Hap3,” “Hap2_Hap4,” “Hap1_Hap2_Hap3,” “Hap1_Hap2__Hap4,” “Hap1__Hap3_Hap4,” “Hap2_Hap3_Hap4,” “Hap1_Hap2_Hap3_Hap4,” or “Hap1_Hap2__Hap5 Hap5.” These sequences may contain scars generated by guide endonucleases. In some embodiments, the potato plant, plant part, or plant cell contains mutations in one, two, three, or four VINV alleles. In another embodiment, this document provides a potato plant, plant part, or plant cell containing a mutation in one, two, three, or four VINV alleles, wherein each mutation is generated by a guided endonuclease, and wherein each mutation contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO: 169.

[0477] In some implementations, the VINV allele comprises a set of one, two, three, or four sequences, including SEQ ID NO: 1-4, SEQ ID NO: 5-8, SEQ ID NO: 9-12, SEQ ID NO: 13-16, SEQ ID NO: 17-20, SEQ ID NO: 21-24, SEQ ID NO: 25-28, SEQ ID NO: 29-32, SEQ ID NO: 33-36, SEQ ID NO: 37-40, SEQ ID NO: 41-44, SEQ ID NO: 45-48, SEQ ID NO: 49-52, SEQ ID NO: 53-56, SEQ ID NO: 57-60, SEQ ID NO: 61-64, SEQ ID NO: 65-68, SEQ ID NO: 69-72, SEQ ID NO: 73-76, SEQ ID NO: 77-80, SEQ ID NO: 81-84, SEQ ID NO: 85-88, SEQ ID NO: 89-92, SEQ ID NO: 93-96, SEQ ID NO: 97-100, SEQ ID NO: 101-104, SEQ ID NO: 105-108, SEQ ID NO: 109-112, SEQ ID NO: 113-116, SEQ ID NO: 117-120, SEQ ID NO: 121-124, SEQ ID NO: 125-128, SEQ ID NO: 129-132, SEQ ID NO: 133-136, SEQ ID NO: 137-140, SEQ ID NO: 141-144, SEQ ID NO: 145-148, SEQ ID NO: 170-173, SEQ ID NO: 174-177, SEQ ID NO: 178-181, SEQ ID NO: 182-185, SEQ ID NO: 186-189, SEQ ID NO: 190-193, SEQ ID NO: 194-197, SEQ ID NO: 198-201, SEQ ID NO: 202-205, SEQ ID NO: 206-209, SEQ ID NO: 210-213, SEQ ID NO: 214-217, SEQ ID NO: 218-221, SEQ ID NO: 222-225 and SEQ ID NO: 226-229.

[0478] In some embodiments, the endonuclease uses a guide RNA to target the protospacer sequence. The selection of the protospacer sequence and the guide RNA is determined by the editing efficiency. Those skilled in the art will be able to select appropriate protospacer sequences and guide RNAs to achieve optimal editing efficiency of the target gene. In some embodiments, the endonuclease targets a protospacer containing a sequence selected from the group consisting of SEQ ID NO: 149-158. In some embodiments, the protospacer sequence contains SEQ ID NO: 149. In some embodiments, the endonuclease uses a guide RNA containing a sequence selected from the group consisting of SEQ ID NO: 159-168. In some embodiments, the guide RNA sequence contains SEQ ID NO: 159. In any of the above embodiments, the sequence may have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO.

[0479] In the genetic and phenotypic assays described herein, modified potato plants, plant parts, or plant cells may be compared with control plants, plant parts, or plant cells. Control plants should lack one or more, or all, of the deletions, inversions, or duplications in the VINV alleles. Control plants may also belong to the same breeding line as the modified potato plants, plants, parts, or plant cells. In some embodiments, control plants are unedited. In some embodiments, control plants are wild-type. In some embodiments, control plants are null isolates.

[0480] In some embodiments, the reduced expression is a decrease in the expression of the VINV gene product. In some embodiments, the reduced expression of the VINV gene product may be caused by deletion, duplication, or inversion. In some embodiments, the expression of the VINV gene product may be reduced by at least 50%, at least 85%, at least 95%, at least 99%, or 100% (e.g., no expression of the VINV protein or expression of only a non-functional truncated form). In some embodiments, the reduced expression of the VINV gene occurs throughout the plant. As used herein, the VINV gene product may include RNA and / or protein levels.

[0481] Plant and plant parts

[0482] Modified potato plants can be obtained from modified potato seeds. Modified potato plant parts can be obtained by cutting, breaking, grinding, or otherwise separating the part from the potato plant. Potato plant parts can be any plant part known in the art, including but not limited to: flowers, pistils, leaves, stems, petioles, cuttings, tissues, seed coats, ovules, microspores, pollen, tubers, stolons, meristems, roots, rootstocks, scions, fruits, cotyledons, hypocotyls, protoplasts, embryos, anthers, seeds, or any part thereof. In some embodiments, the modified potato plant parts provided herein are non-renewable portions of modified potato plant parts. As used in this context, a "non-renewable" portion of a modified potato plant part means a portion that cannot be induced to form a complete potato plant or cannot be induced to form a complete potato plant capable of sexual and / or asexual reproduction (e.g., through in vitro culture). The non-renewable parts of a modified potato plant may be flowers, pistils, leaves, stems, petioles, cuttings, tissues, seed coats, ovules, microspores, pollen, tubers, stolons, roots, rootstocks, scions, fruits, cotyledons, hypocotyls, protoplasts, embryos, anthers, or any part thereof.

[0483] In some embodiments, this document provides non-renewable or non-reproducible potato plant cells. As used herein, a "non-renewable plant cell" is a cell that cannot be regenerated to produce a complete potato plant capable of sexual and / or asexual reproduction through in vitro culture. Non-renewable potato cells may be located within the potato plant or plant part described herein. Non-renewable potato cells may be cells in a seed or in the seed coat of said seed. Mature potato plant organs, including mature leaves, mature stems, or mature roots, contain at least one non-renewable cell. In some embodiments, the non-renewable potato plant cell is a somatic cell.

[0484] This document also provides a potato cell culture or tissue culture of non-renewable or renewable potato cells or tissues from the potato plant or modified plant part described herein, wherein the non-renewable or renewable potato cells contain one or more genetic modifications that result in reduced expression of one or more VINV loci described herein. Preferably, the renewable potato cells are derived from the embryo, protoplast, meristem, callus, microspore, pollen, leaf, tuber, microtuber, stolon, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, and / or hypocotyl of the modified potato plant or modified plant part described herein.

[0485] In some embodiments, this document provides a processed potato product derived from the modified potato plant, plant part, or plant cell described herein, which contains one or more modifications or mutations that result in reduced expression of one or more VINV loci. In some embodiments, the processed potato product contains sufficient nucleic acid (e.g., DNA or RNA) and / or protein material from the modified potato plant, plant part, or plant cell to detect nucleic acid and / or protein sequences corresponding to one, two, three, or four haplotypes that result in reduced expression of one or more VINV loci. In some embodiments, the processed potato product is non-renewable, i.e., it cannot be induced to form a complete potato plant, or cannot be induced to form a complete potato plant capable of sexual and / or asexual reproduction.

[0486] Processed potato products may be seeds, tubers, microtubers, plant tissues, fruits, grains, roots, stolons, vegetables, or any potato plant part described herein, and may be blended as commodities or other products that circulate commercially and are derived from mutant or modified potato plants or plant parts. In some embodiments, commodities or other products can be traced in commercial circulation by detecting the nucleic acid and / or protein sequences from which they are derived. In some embodiments, processed potato products contain detectable amounts of nucleotide and / or protein sequences corresponding to one or more modifications or mutations that result in reduced expression of one or more VINV loci. In some embodiments, commodities or other potato products are produced or maintained in the modified potato plants or plant parts from which the commodities or other products are derived. Such goods or other commercial products include, but are not limited to: potato plant parts, biomass, oil, meal, edible starch, syrup, sugar, animal feed, flour, flakes, processed seeds, seeds, French fries, potato wedges, shredded potato products (e.g., potato cakes, potato balls), baked potatoes, fresh potatoes, mashed potatoes, dehydrated potatoes, granules, peels, cooked skins, potato pulp, mashed potatoes, filter cake, ash starch, sieve residue, potato pulp, potato concentrate, discarded fries, discarded potato chips, scraps, batter, crumbs, defective pieces, or fermented for use in the production of alcoholic beverages. Processed potato products can be food products processed by any means known in the art, such as canning, steaming, boiling, frying, blanching, and / or freezing. Potato products can be produced for any purpose or industry, including but not limited to human consumption, animal feed, dietary supplements, food ingredients, pharmaceuticals, textiles, wood, paper, adhesives, binders, texture agents, fillers, borehole washing, or biofuel production.

[0487] The potato products described herein may include potato food products such as potato chips and / or French fries. Industry-standard methods for producing potato chips and French fries are well known in the art. To make potato chips, the tuber is cut longitudinally from the sprout end to the stem end. In some embodiments, each half tuber is used to make 4-5 chips. In some embodiments, the chips are about 1 mm thick. In some embodiments, a mandolin slicer is used to slice the chips.

[0488] In some embodiments that can be combined with any of the foregoing embodiments, the potato plant part is a flower, pistil, leaf, stem, petiole, cutting, tissue, seed coat, ovule, microspore, pollen, tuber, stolon, meristem, root, rootstock, scion, fruit, cotyledon, hypocotyl, protoplast, embryo, anther, or part thereof.

[0489] In some embodiments, this document provides a processed potato product derived from potato plants, plant parts, or plant cells from any of the foregoing embodiments. In some embodiments, the product is selected from the group consisting of potato biomass, oil, meal, animal feed, flour, flakes, and processed seeds. In some embodiments, the processed potato product is non-renewable. In some embodiments, the processed potato product contains sufficient nucleic acid (e.g., DNA or RNA) and / or protein material from modified potato plants, plant parts, or plant cells to detect nucleic acid and / or protein sequences corresponding to one or both of the following: one, two, three, or four haplotypes, resulting in one or more genetic modifications leading to reduced expression of one or more VINV loci.

[0490] In some embodiments, the potato plants, plant parts, or plant cells are derived from a specific breeding line. This breeding line can be selected from the group consisting of Rustet Burbank and Atlantic varieties. In some embodiments, the modified potato plants, plant parts, or plant cells are derived from the Rustet Burbank variety. In some embodiments, the modified potato plants, plant parts, or plant cells are derived from the Atlantic variety.

[0491] Plant harvesting and storage

[0492] In some embodiments, modified potato tubers are harvested from modified potato plants. The tubers can be harvested between approximately 5 and 15 days after vine kill (e.g., approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days, e.g., approximately 10 days). In some embodiments, the size of the potato tubers is assessed using a potato size measuring board. The tubers can be size A (diameter > 4.8 cm), size B (3.8 cm < diameter < 4.8 cm), or size C (diameter < 3.8 cm). In some embodiments, the potato tubers selected for further processing are size A and have few or no external defects.

[0493] In some embodiments, harvested potato tubers are cold-stored. Cold storage conditions vary by variety / processor, ranging from 3°C to 13°C. Initial cold storage conditions may be approximately 55°F and approximately 95% relative humidity. This initial cold storage period may last approximately two weeks, after which the temperature is gradually reduced to approximately 4°C. In some embodiments, the temperature change rate is 0.5°F per 12 hours. Potato plants, plant parts, or plant cells removed after cold storage have been “cold-treated” for at least two weeks, at least four weeks, at least eight weeks, at least sixteen weeks, at least twenty-four weeks, or at least thirty-two weeks. In other embodiments, potato plants, plant parts, or plant cells removed after cold storage have been “cold-treated” for two weeks, four weeks, eight weeks, sixteen weeks, twenty-four weeks, or thirty-two weeks.

[0494] Tuber Sugar Spectrum

[0495] In some embodiments, tuber glycosylation profiles are obtained from modified potato plants, plant parts, or plant cells. In some embodiments, the tuber glycosylation profile obtained from said plants contains lower levels of glucose and / or fructose compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, the tuber glycosylation profile obtained from said plants contains higher levels of sucrose compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, the percentage of sucrose in the potato plants, plant parts, or plant cells increases by no more than 200%, no more than 100%, no more than 50%, or no more than 25% compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, for example, after sixteen weeks of cold treatment, the tuber glycosylation profile obtained from said plants contains lower levels of glucose compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, the percentage of fructose in potato plants, plant parts, or plant cells is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the tuber sugar profile obtained from control plants. In some embodiments, for example, after sixteen weeks of cold treatment, the tuber sugar profile obtained from said plants contains a lower level of fructose compared to the tuber sugar profile obtained from control plants. In some embodiments, the percentage of fructose in modified potato plants, plant parts, or plant cells is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the tuber sugar profile obtained from control plants.

[0496] The tuber sugar profile can be obtained by various methods known to those skilled in the art. In some embodiments, colorimetric determination is used to obtain the tuber sugar profile. In other embodiments, industrial-standard high-performance liquid chromatography (HPLC) is used to obtain the tuber sugar profile, for example, as described in (“HPLC determination of fructose, glucose, and sucrose in potatoes.” Journal of Food Science 46.1 (1981): 300-301). The tuber sugar profile can be obtained at any time after harvest. In some embodiments, the tuber sugar profile is obtained at harvest. In some embodiments, the tuber sugar profile is obtained after the tubers have undergone refrigeration.

[0497] Acrylamide content in tubers

[0498] In some embodiments, modified potato plants, plant parts, or plant cells contain lower levels of acrylamide compared to control potato plants, plant parts, or plant cells. Controlling acrylamide formation during heat treatment of potatoes is particularly important when the potatoes have been refrigerated for any period of time. In some embodiments, the acrylamide content after refrigeration (e.g., after sixteen weeks of refrigeration) is at least 50%, at least 75%, at least 85%, at least 95%, or at least 99% lower than that of control potato plants, plant parts, or plant cells. Acrylamide content can be measured at any time after harvest. In some embodiments, the acrylamide content is determined after refrigeration. In a further embodiment, the acrylamide content is obtained from the potato food product. Conventional techniques known in the art can be used to determine acrylamide content. For example, a combination of mass spectrometry and liquid chromatography can be used to detect acrylamide. [doi DOT org SLASH 10.3390SLASH foods10092038 for more details]

[0499] The determination of acrylamide content in potato products may also include comparing the acrylamide content of potato products derived from modified potato plants, plant parts, or plant cells that have been refrigerated for at least sixteen weeks with the acrylamide content of control potato products derived from control potato plants. During the determination, when compared with potato products derived from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells will show an acrylamide content that is at least 1 / 5, at least 1 / 6, at least 1 / 7, at least 1 / 8, at least 1 / 9, at least 1 / 10, at least 1 / 11, at least 1 / 12, at least 1 / 13, at least 1 / 14, at least 1 / 15, at least 1 / 20, at least 1 / 25, at least 1 / 30, at least 1 / 35, or at least... Reduced to 1 / 40, at least 1 / 45, at least 1 / 50, at least 1 / 55, at least 1 / 60, at least 1 / 65, at least 1 / 70, at least 1 / 75, at least 1 / 80, at least 1 / 85, at least 1 / 90, at least 1 / 95, at least 1 / 100, at least 1 / 150, at least 1 / 200, at least 1 / 250, at least 1 / 300, at least 1 / 350, at least 1 / 400, at least 1 / 450, or at least 1 / 500. More specifically, refrigeration can last for at least two weeks, at least four weeks, at least eight weeks, at least sixteen weeks, at least twenty-four weeks, or at least thirty-two weeks.In other embodiments, alternatively, when compared with potato products from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells that have been refrigerated for at least two hours will show a reduction in acrylamide content of 1 / 5 to 1 / 500, 1 / 5 to 1 / 450, 1 / 5 to 1 / 400, 1 / 5 to 1 / 400, 1 / 5 to 1 / 350, 1 / 5 to 1 / 300, 1 / 5 to 1 / 250, 1 / 5 to 1 / 200, etc. To 1 / 5 to 1 / 150, down to 1 / 5 to 1 / 100, down to 1 / 5 to 1 / 95, down to 1 / 5 to 1 / 90, down to 1 / 5 to 1 / 85, down to 1 / 5 to 1 / 80, down to 1 / 5 to 1 / 75, down to 1 / 5 to 1 / 70, down to 1 / 5 to 1 / 65, down to 1 / 5 to 1 / 60, down to 1 / 5 to 1 / 55, down to 1 / 5 to 1 / 50, down to 1 / 5 to 1 / 45, down to 1 / 5 to 1 / 40, down to 1 / 5 to 1 / 35, down to 1 / 5 to 1 / 30, down to 1 / 5 to 1 / 25. Decrease to 1 / 5 to 1 / 20, decrease to 1 / 5 to 1 / 15, decrease to 1 / 5 to 1 / 10, decrease to 1 / 10 to 1 / 500, decrease to 1 / 10 to 1 / 450, decrease to 1 / 10 to 1 / 400, decrease to 1 / 10 to 1 / 400, decrease to 1 / 10 to 1 / 350, decrease to 1 / 10 to 1 / 300, decrease to 1 / 10 to 1 / 250, decrease to 1 / 10 to 1 / 200, decrease to 1 / 10 to 1 / 150, decrease to 1 / 10 to 1 / 100, decrease to 1 / 10 to 1 / 95, decrease to 1 / 10 to 1 / 90, reduced to 1 / 10 to 1 / 85, reduced to 1 / 10 to 1 / 80, reduced to 1 / 10 to 1 / 75, reduced to 1 / 10 to 1 / 70, reduced to 1 / 10 to 1 / 65, reduced to 1 / 10 to 1 / 60, reduced to 1 / 10 to 1 / 55, reduced to 1 / 10 to 1 / 50, reduced to 1 / 10 to 1 / 45, reduced to 1 / 10 to 1 / 40, reduced to 1 / 10 to 1 / 35, reduced to 1 / 10 to 1 / 30, reduced to 1 / 10 to 1 / 25, reduced to 1 / 10 to 1 / 20, or reduced to 1 / 10 to 1 / 15. More specifically, refrigeration can be sustained for at least three hours, at least four hours, at least five hours, at least six hours, at least eight hours, at least ten hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, or longer.Alternatively, when compared to potato products from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells that have been refrigerated for at least two hours will show acrylamide content that is 25% to 75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 50%, 25% to 45%, 25% to 40%, 25% to 35%, 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 55%, 30% to 55%, 30% to 50%, 30% to 45%, 25% to 40%, and 30% to 35% lower. 35% to 75%, 35% to 70%, 35% to 65%, 35% to 60%, 35% to 55%, 35% to 55%, 35% to 50%, 35% to 45%, 35% to 40%, 40% to 75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 55%, 40% to 50%, 40% to 45%, 45% to 75%, 45% to 70%, 45% to 65%, 45% to 60%, 45% to 55%, 45% to 55%, 45% to 50%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55%. More specifically, refrigeration can last for a period of at least three hours, at least four hours, at least five hours, at least six hours, at least eight hours, at least ten hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours or longer.

[0500] In addition, it is believed that when measured as described above, potato products derived from modified or mutated potato plants, plant parts or plant cells and that have been refrigerated for at least two hours will show acrylamide content of less than 500 ppb (mg / Kg), less than 400 ppb (mg / Kg), less than 300 ppb (mg / Kg), less than 200 ppb (mg / Kg) or less than 100 ppb (mg / Kg). Alternatively, during the assay, potato products derived from potato plants produced by the above method will exhibit values ​​of approximately 90 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 100 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 200 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 250 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 100 ppb (mg / kg) to approximately 300 ppb (mg / kg), approximately 100 ppb (mg / kg) to approximately 250 ppb (mg / kg), approximately 200 ppb (mg / kg) to approximately 300 ppb (mg / kg), approximately 250 ppb (mg / kg) to approximately 300 ppb (mg / kg), and approximately 300 ppb (mg / kg) to approximately 500 ppb (mg / kg). Acrylamide content is between approximately 400 ppb (mg / kg) and approximately 500 ppb (mg / kg). More specifically, refrigeration can be sustained for at least three hours, at least four hours, at least five hours, at least six hours, at least eight hours, at least ten hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, or longer. Furthermore, it is believed that potato products derived from modified potato plants, plant parts, or plant cells that have been exposed to or stored at room temperature under these conditions may exhibit acrylamide content below 1100 ppb (mg / kg), below 1000 ppb (mg / kg), below 900 ppb (mg / kg), below 800 ppb (mg / kg), below 700 ppb (mg / kg), below 600 ppb (mg / kg), or below 500 ppb (mg / kg) when measured as described above.Alternatively, during the assay, potato products derived from potato plants produced by the above method will exhibit values ​​of approximately 400 ppb (mg / kg) to approximately 1100 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 1000 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 900 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 800 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 700 ppb (mg / kg), approximately 500 ppb (mg / kg) to approximately 1100 ppb (mg / kg), approximately 500 ppb (mg / kg) to approximately 1000 ppb (mg / kg), approximately 500 ppb (mg / kg) to approximately 900 ppb (mg / kg), and approximately 500 ppb (mg / kg) to approximately 800 ppb (mg / kg). The acrylamide content is between approximately 500 ppb (mg / Kg) and approximately 750 ppb (mg / Kg).

[0501] The determination of acrylamide content in potato products may also include comparing the acrylamide content of potato products derived from modified potato plants, plant parts, or plant cells that have been stored at or subjected to room temperature conditions with the acrylamide content of control potato products derived from control potato plants. During the determination, when compared with potato products derived from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells will show an acrylamide content that is at least reduced to 1, at least reduced to 1 / 2, at least reduced to 1 / 3, at least reduced to 1 / 4, at least reduced to 1 / 5, at least reduced to 1 / 6, at least reduced to 1 / 7, at least reduced to 1 / 8, at least reduced to 1 / 9, at least reduced to 1 / 10, at least reduced to 1 / 11, at least reduced to 1 / 12, at least reduced to 1 / 13, at least reduced to 1 / 14, or at least reduced to 1 / 15.

[0502] Alternatively, when compared with potato products from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells will show a reduction in acrylamide content, at least to 1 to 1 / 15, to 1 / 2 to 1 / 15, to 1 / 3 to 1 / 15, to 1 / 4 to 1 / 15, to 1 / 5 to 1 / 15, to 1 to 1 / 14, to 1 / 2 to 1 / 14, to 1 / 3 to 1 / 14, to 1 / 4 to 1 / 14, to 1 / 5 to 1 / 14, to 1 to 1 / 13, to 1 / 2 to 1 / 13. , down to 1 / 3 to 1 / 13, down to 1 / 4 to 1 / 13, down to 1 / 5 to 1 / 15, down to 1 to 1 / 12, down to 1 / 2 to 1 / 12, down to 1 / 3 to 1 / 12, down to 1 / 4 to 1 / 12, down to 1 / 5 to 1 / 12, down to 1 to 1 / 11, down to 1 / 2 to 1 / 11, down to 1 / 3 to 1 / 11, down to 1 / 4 to 1 / 11, down to 1 / 5 to 1 / 11, down to 1 to 1 / 10, down to 1 / 2 to 1 / 10, down to 1 / 3 to 1 / 10, down to 1 / 4 to 1 / 10 or down to 1 / 5 to 1 / 10.

[0503] Alternatively, when compared to potato products from control potato plants or sweet potato products from control sweet potato plants, potato products derived from modified potato plants, plant parts, or plant cells that have been stored or subjected to room temperature conditions have acrylamide content that is 25% to 75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 55%, 25% to 50%, 25% to 45%, 25% to 40%, 25% to 35%, 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 55%, 30% to 55%, 30% to 50%, 30% to 45%, 25% to 40%, or 30% lower. Up to 35%, low 35% to 75%, low 35% to 70%, low 35% to 65%, low 35% to 60%, low 35% to 55%, low 35% to 55%, low 35% to 50%, low 35% to 45%, low 35% to 40%, low 40% to 75%, low 40% to 70%, low 40% to 65%, low 40% to 60%, low 40% to 55%, low 40% to 55%, low 40% to 45%, low 45% to 75%, low 45% to 70%, low 45% to 65%, low 45% to 60%, low 45% to 55%, low 45% to 55%, 45% to 50%, low 50% to 75%, low 50% to 70%, low 50% to 65%, low 50% to 60%, or low 50% to 55%.

[0504] The above methods (both refrigerated and room temperature) may also include heat processing potatoes into chips, potato chips, French fries, potato sticks or shoelace fries or other edible potato products.

[0505] potato chip color

[0506] In some implementations, potato products produced from modified potato plants, plant parts, or plant cells are lighter in color than those produced from control plants. To determine the chip color, the tuber can be cut longitudinally from the bud tip to the stem tip. Using a mandolin slicer, 4-5 chips (1 mm thick) can be made from each half tuber, for a total of 8-10 chips. The chip slices are then fried in peanut oil at approximately 360°F for about 2 minutes and 10 seconds in a custom basket. The chips are then crushed to a specific particle size and placed in a measuring instrument. Immediately after frying and cooling, the chip color is quantified by reflectance using a Konica Minolta CR410 colorimeter (Konica Minolta, NJ, USA). Hunter Lab color space readings L, a, and b are obtained. L is relative lightness, a is the chromaticity range between red and green, and b is the chromaticity range between yellow and blue. The color of the potato product can then be reported as a chip lightness score. The color of potato chips can be determined at harvest, after cold storage, or at any intermediate point in time.

[0507] In some embodiments, the potato chip brightness score of potato products derived from modified potato plants, plant parts, or plant cells is 25% to 100% higher than that of products derived from control plants. In some embodiments, the potato chip brightness score of potato products derived from modified potato plants, plant parts, or plant cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 5000%, or at least 10000% higher than that of products derived from control plants. In some embodiments, the potato chip brightness score of potato products derived from modified potato plants, plant parts, or plant cells is higher than 63.

[0508] In some implementations, potato chips produced from modified potato plants, plant parts, or plant cells containing mutations in one, two, three, or four VINV alleles have a lighter chip brightness score than potato chips produced from modified potato plants, plant parts, or plant cells containing mutations in all four VINV alleles.

[0509] Methods for producing modified potatoes

[0510] In another aspect, this document provides methods for producing modified potato plants, plant parts, or plant cells. In some embodiments, this document provides a method for producing modified potato plants, plant parts, or plant cells, comprising providing a guiding endonuclease to the plant, plant part, or plant cell, and generating a mutation in at least one VINV allele, at least two VINV alleles, at least three VINV alleles, or four VINV alleles. In some embodiments, this document provides a method for generating modified potato plants, plant parts, or plant cells, comprising generating mutations, such as but not limited to deletions, edits, phase shifts, inversions, or duplications, in all at least one, at least two, at least three, or four VINV alleles, and further comprising using a guide endonuclease to generate the mutations, wherein the endonuclease targets a protospacer sequence comprising, or comprising, selected from, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or a sequence containing, or with, a protospacer sequence selected from, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, and SEQ ID NO: 158. Sequences 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO: 158 having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity. In another aspect, this document provides a method for producing modified potato plants, plant parts, or plant cells, comprising providing a guiding endonuclease to the plant, plant part, or plant cell, and generating mutations in at least one, at least two, at least three, or four VINV alleles, wherein each mutation is generated by the guiding endonuclease, and wherein each mutation contains a mutation of one or more nucleotides corresponding to the editing window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO: 169. In some embodiments, the guiding endonuclease is a Cas protein.

[0511] Potato strain selection

[0512] In some embodiments, methods for breeding polyploid hybrid potato lines containing one or more edits to the VINV allele include breeding lines using conventional plant breeding methods to generate a set of candidate potato lines. This method may include breeding the line using any conventional plant breeding method known in the art or described herein. In some embodiments, line breeding includes recurrent selection. In other embodiments, line breeding includes inbreeding one or more lines to achieve homozygosity. In some variations, line breeding includes hybridizing, self-pollinating (self-pollinating), and backcrossing lines to generate candidate lines. In other variations, line breeding includes hybridizing several pairs of lines to produce the F1 (first progeny) generation, followed by several generations of self-pollination (producing F2, F3, etc.). In other variations, line breeding includes a backcross (BC) step, whereby the progeny are backcrossed with one of the parent lines (referred to as the recurrent parent).

[0513] In the process of breeding potato lines using traditional plant breeding methods, many steps can be taken to generate a set of candidate potato lines. The choice of breeding method depends on the plant's reproductive pattern and the heritability of the trait to be improved. Backcrossing can be used to transfer one or more favorable genes for a highly heritable trait into the desired line. This method has been widely used to breed disease-resistant lines. Multiple recurrent selection techniques can be used to improve quantitatively heritable traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids obtained with each pollination, and the number of hybrid offspring produced with each successful hybridization. Breeders can initially select two or more parental lines and cross them, followed by repeated self-pollination and selection, thereby generating many new genetic combinations. In addition, breeders can generate a variety of different genetic combinations through hybridization, self-pollination, generating mutations, or any combination thereof. Plant breeders can then select which lines are chosen as candidate lines. A review of cyclic selection techniques is presented in Vasal et al. (2004. Population Improvement Strategies for Crop Improvement. Plant Breeding. Springer, pp. 391-406).

[0514] The development of candidate lines for the methods described herein may include obtaining parental lines, hybridizing these lines, and evaluating the hybridization. Lineage breeding and recurrent selection breeding methods can be used to develop candidate lines from breeding populations. Breeding programs can combine desired traits from two or more varieties or a wide range of sources into a breeding bank, from which lines are developed through self-pollination and selection for desired phenotypes. New lines can be further hybridized with other lines, and the potential of the hybrids from these hybridizations to be selected as candidate lines can be evaluated.

[0515] The choice of breeding or selection method depends on the plant's reproductive pattern and the heritability of the trait to be improved. For traits with high heritability, selection of superior individuals evaluated at a single location will be effective, while for traits with low heritability, selection should be based on the average value obtained through repeated evaluations of relevant plant families. Mainstream selection methods typically include pedigree selection, improved pedigree selection, population selection, and recurrent selection.

[0516] In some implementations, potato line breeding involves inbreeding one or more members of the line to achieve homozygosity. In some variations, achieving homozygosity through inbreeding of potato lines may include self-pollinating plants of the line for two or more generations, such as five to seven generations, to produce inbred or homozygous potato lines. Homozygous potato lines can also be bred by producing double haploids. Double haploids are produced by generating haploid plants from heterozygous plants and doubling the genome of the haploid plants to produce completely homozygous individuals. The process of producing haploid plants is also known as haploid induction. Haploid induction can be achieved in a variety of plants using methods well known in the art and described herein. After the production of haploid plants, genome doubling can occur spontaneously or artificially using, for example, colchicine, methyl parathion (APM), ammoniacalin, chlorpyrifos, trifluralin, or nitrous oxide. Methods for producing double haploids are well known in the literature, and examples are described in Wan et al. (1989. Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus. Theor. Appl. Genet., 77:889-892.) and Ren et al. (2017. Novel technologies in doubled haploid line development. PlantBiotechnol J 15, 1361-1370.) and the references cited therein.

[0517] Line breeding is commonly used to improve inbred lines of self-pollinating or cross-pollinating crops. Two parents with favorable complementary traits are crossed to produce the F1 population. The F2 population is produced by self-pollinating one or more F1 individuals or by intercrossing two F1 individuals (sib crossing). Selection for the best individuals can begin with the F2 population; then, starting with F3, the best individuals from the best families can be selected. Repeated testing of families or hybrid combinations involving individuals from these families can be performed in the F4 generation to improve the effectiveness of selection for traits with low heritability. In the advanced stages of inbreeding (i.e., F6 and F7), the potential of the best lines or mixtures of phenotypic similar lines to be selected as candidate lines can be tested.

[0518] Population selection and recurrent selection can be used to improve lines of self-pollinating or cross-pollinating crops. Populations of genetically variable heterozygous individuals can be identified or generated by intercrossing several different parents. Optimal plants are selected based on individual superiority, outstanding offspring, or heterosis. Selected plants are then intercrossed to create new populations, within which further selection cycles continue.

[0519] Backcross breeding can be used to transfer genes for easily inherited, highly heritable traits into a desired homozygous line (i.e., a recurrent parent). The source of the trait to be transferred is called the donor parent. The resulting plants are expected to possess the attributes of the recurrent parent and the desired trait transferred from the donor parent. After the initial hybridization, individuals exhibiting the donor parent phenotype are selected and repeatedly crossed with the recurrent parent (backcross). The resulting plants are expected to possess the attributes of the recurrent parent (e.g., a cultivated variety) and the desired trait transferred from the donor parent.

[0520] Strictly speaking, single-seed propagation refers to planting a segregating population, harvesting a sample of one seed from each plant, and using this single seed sample to propagate the next generation. Once the population has progressed from F2 to the desired inbreeding level, the plants from which the strain originated will be traced back to different F2 individuals. Because some seeds fail to germinate, or some plants fail to produce at least one seed, the number of plants in the population decreases with each generation. Therefore, when the generations are complete, not all the F2 plants initially sampled from the population will have offspring representatives.

[0521] In addition to phenotypic observation, plant genotypes can be examined during the breeding process to generate candidate lines. Many laboratory-based techniques are available for analyzing, comparing, and characterizing plant genotypes; these include isoenzyme electrophoresis, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), arbitrary primer polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence characterization of amplified regions (SCAR), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR—also known as microsatellites), and single nucleotide polymorphisms (SNPs).

[0522] Molecular markers can also be used in breeding to select for qualitative traits. For example, in backcross breeding programs, markers closely linked to alleles or containing sequences within the actual target allele can be used to select plants containing the target allele. Markers can also be used to select for the genome of the recurrent parent and for markers targeting the donor parent. This procedure aims to minimize the amount of the donor parent's genome retained in the selected plants. It can also be used to reduce the number of backcrosses required to the recurrent parent in a backcross program. The use of molecular markers in selection is often referred to as marker-assisted selection or marker-enhanced selection. Molecular markers can also be used to identify and exclude certain line origins as ancestors of parent varieties or plants by providing a means of tracing the genetic map during hybridization.

[0523] Mutation breeding can also be used to select potato lines to generate candidate lines. Spontaneous or artificially induced mutations can be a useful source of variability for plant breeders. The purpose of artificial mutagenesis is to increase the mutation rate of the desired trait. The mutation rate can be increased by a variety of different means, including: temperature, long-term seed storage, tissue culture conditions, radiation (such as X-rays, gamma rays, neutrons, beta radiation, or ultraviolet radiation), chemical mutagens (such as base analogs, such as 5-bromouracil), antibiotics, alkylating agents (such as sulfur mustard, nitrogen mustard, epoxides, ethyleneamine, sulfates, sulfonates, sulfones, or lactones), azides, hydroxylamine, nitrite, or acridine. Once the desired trait is observed through mutagenesis, it can be incorporated into existing lines using conventional breeding techniques. Details of mutation breeding can be found in Fehr's Principles of Cultivar Development, Macmillan Publishing Company, 1993.

[0524] Other non-limiting examples of breeding methods that may be used include, but are not limited to, those described in the following literature: Allard (1960. Principles of Plant Breeding, John Wiley and Son, pp. 115-161); Simmonds (1979. Principles of Crop Improvement, Longman Group Limited); Sneep (1979. Plant Breeding Perspectives, Unipub); and Fehr and Walt (1987. Principles of Cultivar Development, pp. 261-286).

[0525] In some implementations, breeding potato lines involves generating and maintaining one or more potato lines carrying the VINV mutation. The one or more potato lines carrying the VINV mutation can be maintained via vegetative propagation, self-pollination, apomixis, cell culture, or any combination thereof. The VINV allele of the one or more potato lines carrying the VINV mutation can be propagated during the breeding cycle to reduce the number of editing or transgenic events required to introduce the VINV mutation into potato candidate lines.

[0526] Methods of introducing genetic modification

[0527] In some embodiments, genetic modifications are introduced via gene editing. Any of several gene editing methods known in the art can be used to introduce genetic modifications into the VINV gene. In some variations, gene editing is performed using one or more natural or engineered nucleases, including but not limited to RNA-guided nucleases, broad-spectrum nucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector-based nucleases (TALENs). In further variations, gene editing is performed using RNA-guided nucleases, including but not limited to clustered regularly spaced short palindromic repeat (CRISPR)-associated nucleases. Gene editing methods are numerous, well-known, and conventional in the art and are described in US17 / 045747, US16 / 977020, and US16 / 961396, which are incorporated herein by reference in their entirety.

[0528] Engineered nucleases can be guide nucleases that function as ribonucleoprotein (RNP) complexes with guide RNA. According to some implementation schemes, the guide nuclease can be selected from the group consisting of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, and Cas9. (Also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, and their homologs or modified forms, Argonaute (non-limiting examples of Argonaute proteins include *Thermus thermophilus*). (TtAgo), *Pyrococcus furiosus* Argonaute (PfAgo), *Natronobacterium gregoryi* Argonaute (NgAgo), and their homologs or modified forms). According to some embodiments, the guide nuclease is a Cas9 or Cpf1 enzyme. The DNA construct or molecule encoding the guide nuclease, or the guide nuclease itself, can be delivered with or without a guide nucleic acid.

[0529] For guide nucleases, a guide nucleic acid molecule may be further provided to direct the guide nuclease to a target site in the plant genome via base pairing or hybridization, thereby creating a DSB or nick at or near the target site. The guide nucleic acid may be transformed or introduced into plant cells or tissues as a guide nucleic acid molecule or as a recombinant DNA molecule, construct, or vector containing a transcribed DNA sequence encoding a guide nucleic acid operably linked to a promoter or a plant-expressible promoter. The promoter may be a constitutive promoter, a tissue-specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter. The host cell may contain a recombinant DNA molecule, construct, or vector containing a transcribed DNA sequence encoding a guide nucleic acid operably linked to a promoter or a plant-expressible promoter. The host cell may be a bacterial cell or a plant cell. In some embodiments, the host cell is an Agrobacterium cell.

[0530] In some embodiments, the guide nucleic acid comprises a first segment and a second segment, the first segment comprising a nucleotide sequence complementary to a sequence in the target nucleic acid, and the second segment interacting with a guide nuclease protein. In some embodiments, the first segment of the guide containing a nucleotide sequence complementary to a sequence in the target nucleic acid corresponds to CRISPR RNA (crRNA or a crRNA repeat sequence). In some embodiments, the second segment of the guide containing a nucleic acid sequence interacting with a guide nuclease protein corresponds to trans-acting CRISPR RNA (tracrRNA). In some embodiments, the guide nucleic acid comprises two separate nucleic acid molecules that hybridize to each other (a polynucleotide complementary to a sequence in the target nucleic acid and a polynucleotide interacting with a guide nuclease protein). In other embodiments, the guide nucleic acid is a single polynucleotide. In some embodiments, the guide nucleic acid may comprise DNA, RNA, or a combination of DNA and RNA.

[0531] In some embodiments, the method utilizes a nuclease that targets the protospacer sequence with a guide RNA. The choice of protospacer sequence and guide RNA is determined by the editing efficiency. Those skilled in the art will be able to select appropriate protospacer sequences and guide RNA to achieve optimal editing efficiency of the target gene. In some embodiments, the endonuclease targets a protospacer comprising a sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 and SEQ ID NO: 158, or a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 and SEQ ID NO: 158.

[0532] In some embodiments, the endonuclease utilizes a guide RNA comprising a sequence selected from the group consisting of: a guide RNA comprising a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, or SEQ ID NO: 168.

[0533] As is known in the art, a protospacer adjacent motif (PAM) may be present in the genome, immediately upstream of the 5' end of the genomic target sequence complementary to the guide RNA's target sequence, and immediately downstream (3') of the sense (+) strand of the genomic target site (relative to the guide RNA's target sequence). See, for example, Wu, X. et al. 2014. “Targetspecificity of the CRISPR-Cas9 system,” Quant Biol. 2(2): 59-70. The genomic PAM sequence on the sense (+) strand adjacent to the target site (relative to the guide RNA's target sequence) may contain 5'-NGG-3'. However, the corresponding sequence of the guide nucleic acid (immediately downstream (3') of the guide RNA's target sequence) may not typically be complementary to the genomic PAM sequence.

[0534] Guide nucleic acids are typically non-coding RNA molecules that do not encode proteins. The target sequence of a guide nucleic acid can be at least 10 nucleotides long, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides long, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides long. The target sequence may be identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more consecutive nucleotides of the DNA sequence at the genomic target site, which is at least 95%, at least 96%, at least 97%, at least 99% or 100%.

[0535] In addition to the target sequence, the guide nucleic acid may also contain one or more other structural or scaffold sequences that can bind to or interact with RNA-guided endonucleases. These scaffold or structural sequences can further interact with other RNA molecules (e.g., tracrRNA). Methods and techniques for designing targeted constructs and guide nucleic acids for genome editing and site-specific integration at target sites within the plant genome using guided nucleases are known in the art.

[0536] Engineered nucleases can be site-specific nucleases. Several site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), broad-spectrum nucleases, and TALENs, are not nucleic acid-guided but rely on their protein structure to determine their target sites to create a DSB or nick, or they fuse, tether, or attach to DNA-binding protein domains or motifs. The protein structure of site-specific nucleases (or fused / attached / tethered DNA-binding domains) allows the site-specific nuclease to target its target site. According to many of these implementation schemes, non-nucleic acid-guided site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), broad-spectrum nucleases, and TALENs, can be designed, engineered, and constructed using known methods to target and bind to target sites at or near genomic loci of endogenous plant genes, thereby generating a DSB or nick at that genomic locus to knock out or down the expression of the gene through the repair of the DSB or nick. This repair can be achieved through cellular repair mechanisms and may be guided by donor template molecules to generate a mutated or inserted sequence at the DSB or nick site.

[0537] In some embodiments, the site-specific nuclease is a recombinase. The recombinase may be a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif, or other recombinases known in the art. The recombinase or transposase may be a DNA transposase or recombinase attached to or fused to a DNA-binding domain. Non-limiting examples of recombinases include tyrosine recombinases attached to the DNA recognition motif provided herein, selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one aspect, the Cre or Gin recombinase provided herein is tethered to a zinc finger DNA-binding domain or a transcription activator-like effector (TALE) DNA-binding domain or a Cas9 nuclease. In another aspect, the serine recombinase attached to the DNA recognition motif provided herein is selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In another respect, the DNA transposases attached to the DNA-binding domain provided herein are selected from the group consisting of TALE-piggyBac and TALE-Mutator.

[0538] Site-specific nucleases can be zinc finger nucleases (ZFNs). ZFNs are synthetic proteins composed of engineered zinc finger DNA-binding domains fused to a cleavage domain (or cleavage hemidomain), which may be derived from a restriction endonuclease (e.g., FokI). The DNA-binding domain can be classical (C2H2) or non-classical (e.g., C3H or C4). Depending on the target site, the DNA-binding domain may contain one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more). Multiple zinc fingers within the DNA-binding domain can be separated by an adapter sequence. ZFNs can be designed to cleave virtually any segment of double-stranded DNA by modifying the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of non-specific DNA cleavage domains (e.g., derived from FokI nucleases) fused to the DNA-binding domain, which contain an array of zinc fingers engineered to bind to the target site DNA sequence. The DNA-binding domain of a ZFN typically consists of 3-4 (or more) zinc fingers. Amino acids at positions -1, +2, +3, and +6 relative to the origin of the zinc finger α-helix facilitate site-specific binding to the target site and can be altered and tailored to specific target sequences. Other amino acids can form a common backbone to produce ZFNs with different sequence specificities.

[0539] Methods and rules for designing ZFNs that target and bind specific target sequences are known in the art. See, for example, U.S. Patent Applications Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. FokI nuclease domains may require dimerization to cleave DNA, thus requiring two ZFNs with their C-terminal regions to bind to the opposing DNA strands (5-7 bp apart) at the cleavage site. If the two ZF binding sites are palindromic, the ZFN monomer can cleave the target site. As used herein, ZFNs are broad and include monomeric ZFNs capable of cleaving double-stranded DNA without the assistance of another ZFN. The term ZFN can also be used to refer to one or both members of a pair of ZFNs engineered to work together to cleave DNA at the same site. Without being limited by any theory, because the DNA-binding specificity of zinc finger domains can be reengineered using one of a variety of methods, it is theoretically possible to construct custom ZFNs to target virtually any target sequence (e.g., at or near a gene in a plant genome). Publicly available methods for engineering zinc finger domains include context-dependent assembly (CoDA), oligomerization library engineering (OPEN), and modular assembly. In one aspect, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more ZFNs. In another aspect, the ZFNs provided herein are capable of generating targeted DSBs or notches.

[0540] Site-specific nucleases can be TALENs. TALENs are artificial restriction enzymes created by fusing a TALE DNA-binding domain with a nuclease domain (e.g., FokI). When each member of the TALEN pair binds to a DNA site flanking the target site, the FokI monomer dimers, causing a double-strand DNA break at the target site. In addition to the wild-type FokI cleavage domain, variants with mutated FokI cleavage domains have been engineered to improve cleavage specificity and activity. For the FokI domain to function as a dimer, two constructs with unique DNA-binding domains are required for sites in the target genome with appropriate orientation and spacing. The number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, as well as the number of bases between the two individual TALEN binding sites, are parameters for achieving high levels of activity.

[0541] TALENs are artificial restriction enzymes created by fusing a TALE DNA-binding domain with a nuclease domain. In some respects, nucleases are selected from the group consisting of PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to a DNA site flanking the target site, the FokI monomer dimerizes and causes a double-stranded DNA break at the target site. As used herein, the term TALEN is broad and includes monomeric TALENs capable of cleaving double-stranded DNA without the assistance of another TALEN. The term TALEN also refers to one or both members of a TALEN pair that work together to cleave DNA at the same site.

[0542] TALEs can be engineered to bind virtually any DNA sequence, such as at or near genomic loci of genes in plants. A TALE has a central DNA-binding domain consisting of 13–28 repeating monomers of 33–34 amino acids. Except for the hypervariable amino acid residues at positions 12 and 13, the amino acids in each monomer are highly conserved. These two variable amino acids are called repeating variable diresidues (RVDs). The amino acids of the RVD preferentially recognize adenine, thymine, cytosine, and guanine / adenine for NI, NG, HD, and NN, respectively, and regulation of the RVD allows for the recognition of consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allows for the engineering of specific DNA-binding domains by selecting combinations of repeating segments containing appropriate RVDs.

[0543] In addition to the wild-type FokI cleavage domain, variants with mutated FokI cleavage domains have been engineered to improve cleavage specificity and activity. For the FokI domain to function as a dimer, two constructs with unique DNA-binding domains are required for sites in the target genome with appropriate orientation and spacing. The number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, as well as the number of bases between the two individual TALEN binding sites, are parameters for achieving high levels of activity. PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants used in conjunction with TALE. When coupled with TALE, PvuII functions as a highly specific cleavage domain (see Yank et al. 2013. PloS One. 8: e82539). MutH can introduce a chain-specific cleavage into DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41: e83). TevI introduces double-strand breaks in DNA at the target site (see Beurdeley et al., 2013. Nature Communications. 4: 1762).

[0544] The relationship between amino acid sequences and DNA recognition of TALE-binding domains allows for the designability of proteins. Software programs such as DNAWorks can be used to design TALE constructs. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al., Nucleic Acids Research (2012) 40: W117-122.; Cermak et al., Nucleic Acids Research (2011) 39:e82; and tale-nt.cac.cornelledu / about. On the other hand, the TALEN presented herein is capable of generating targeted DSBs.

[0545] Site-specific nucleases can be broad-spectrum nucleases. Broad-spectrum nucleases are commonly identified in microorganisms; for example, the LAGLIDADG family of homing endonucleases are unique enzymes with highly active and long recognition sequences (>14 bp) that lead to site-specific digestion of target DNA. Engineered forms of naturally occurring broad-spectrum nucleases typically have extended DNA recognition sequences (e.g., 14 to 40 bp). According to some embodiments, broad-spectrum nucleases may include scaffold or basal enzymes selected from the group consisting of I-CreI, I-CeuI, I-MsoI, I-SceI, I-AniI, and I-DmoI. The engineering of broad-spectrum nucleases can be more challenging than that of ZFNs and TALENs because the DNA recognition and cleavage functions of broad-spectrum nucleases are intertwined within a single domain. Specialized methods using mutagenesis and high-throughput screening have been used to generate novel broad-spectrum nuclease variants that recognize unique sequences and possess enhanced nuclease activity. Therefore, a wide range of nucleases can be selected or engineered to bind to genomic target sequences in plants, such as at or near genomic loci of genes. In another aspect, the wide range of nucleases presented herein are capable of generating targeted DSBs.

[0546] In some embodiments, gene editing includes: (a) inducing a DSB at a cleavage site in the cellular genome by expressing a natural or engineered nuclease in a cell that recognizes a recognition site and inducing a DSB at the cleavage site, the cleavage site being located at or near the recognition site of the natural or engineered nuclease; (b) introducing a repair nucleic acid molecule into the cell comprising an upstream flanking region homologous to a DNA region upstream of a preselected site and / or a downstream flanking DNA region homologous to a DNA region downstream of a preselected site, to allow homologous recombination between the one or more flanking regions and the one or more DNA regions flanking the preselected site; and (c) selecting cells in which the repair nucleic acid molecule has been used as a template for modifying the genome at the preselected site. In other embodiments, gene editing includes: (a) inducing a DSB at a cleavage site in the cellular genome by introducing a natural or engineered nuclease that recognizes a recognition site into a cell and inducing a DSB at the cleavage site, the cleavage site being located at or near the recognition site of the natural or engineered nuclease; (b) introducing a repair nucleic acid molecule into the cell comprising an upstream flanking region homologous to a DNA region upstream of a preselected site and / or a downstream flanking DNA region homologous to a DNA region downstream of a preselected site, to allow homologous recombination between the one or more flanking regions and the one or more DNA regions flanking the preselected site; and (c) selecting cells in which the repair nucleic acid molecule has been used as a template for modifying the genome at the preselected site.

[0547] As used herein, the repair nucleic acid molecule is a single-stranded or double-stranded DNA molecule or RNA molecule that serves as a template for modifying genomic DNA at a preselected site near or at the cleavage site. As used herein, "template for modifying genomic DNA" means that the repair nucleic acid molecule is copied or integrated into the preselected site via homologous recombination between a flanking region and a corresponding homologous region in the target genome located flanking the preselected site, optionally also binding a non-homologous end join (NHEJ) at one of the two ends of the repair nucleic acid molecule (e.g., in the case of only one flanking region). Integration via homologous recombination allows the repair nucleic acid molecule to be precisely linked to the target genome at the nucleotide level, while NHEJ may result in small insertions / deletions at the junction between the repair nucleic acid molecule and the genomic DNA.

[0548] In some embodiments, genetic modifications introduced through gene editing result in reduced expression (including non-expression or altered activity) of one or more VINV loci. In gene editing, the introduction of a DSB or notch can be used to introduce targeted genetic modifications into the plant genome. According to this method, genetic modifications, such as deletions, insertions, inversions, and / or substitutions, can be introduced at the target site via imperfect repair of the DSB or notch, resulting in gene knockout or knockdown, or the production of VINV components with altered activity. Such genetic modifications can be generated via imperfect repair of the targeted locus even without the use of a donor template molecule, and can lead to reduced expression (including non-expression or altered activity) of endogenous gene products. For example, genetic modifications can be generated by insertions or deletions (insertion or deletion of nucleotide bases in the target DNA sequence via NHEJ), or by specific removal of sequences that reduce or completely disrupt the function of sequences or motifs at or near the target site, or result in altered activity of VINV components. Such embodiments may include deletions or insertions that alter one or more post-translational modifications on one or more VINV components. Post-translational modifications may include phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, lipidation, etc. Altered activity in the VINV component can be achieved, for example, by deleting or otherwise disrupting one or more phosphorylation sites (e.g., tyrosine phosphorylation sites or serine / threonine phosphorylation sites). In a further embodiment, the disrupted motif is a proteolytic cleavage site. Gene knockout can be achieved by inducing a DSB or cleavage at or near an endogenous locus of the gene, resulting in the non-expression of the gene product; similarly, gene knockdown can be achieved by inducing a DSB or cleavage at or near an endogenous locus of the gene, where the DSB or cleavage is imperfectly repaired at a site in a manner that does not eliminate the function of the gene product and does not affect the coding sequence of the gene. For example, the DSB or cleavage site within the endogenous locus may be located upstream of the gene or in the 5' region (e.g., promoter and / or enhancer sequences) to affect or reduce its expression level. Similarly, such targeted knockout or knockdown mutations in genes can be generated using donor template molecules to guide specific or desired mutations at or near the target site via DSB or nick repair. The donor template molecule may contain homologous sequences with or without the insertion sequence and, relative to the target genomic sequence at or near the DSB or nick site, contains one or more mutations, such as one or more deletions, insertions, inversions, and / or substitutions. For example, targeted knockout mutations in genes can be achieved by substituting, inserting, deleting, or inverting at least a portion of the gene, including but not limited to introducing a frameshift or early stop codon into the protein-coding sequence of the gene. The deletion of a portion of a gene can also be introduced by generating a DSB or nick at two target sites, resulting in the deletion of the intermediate target region between the two target sites.

[0549] In some embodiments, genetic modification includes introducing proteins, nucleic acids, or combinations thereof into plant cells, such as CRISPR / Cas RNP. Introducing proteins, nucleic acids, or combinations thereof into plant cells can be achieved by any of several methods known and conventionally used in the art. In some embodiments, introducing proteins, nucleic acids, or combinations thereof into plant cells includes isolating protoplasts, transfecting protoplasts, embedding protoplasts, and regenerating plants from protoplasts. In other embodiments, introducing proteins, nucleic acids, or combinations thereof into plant cells includes gene gun transformation. In some embodiments, introducing proteins, nucleic acids, or combinations thereof into plant cells includes isolating immature plant embryos, bombarding the embryos with particles containing nucleic acids, and regenerating plants from immature embryos. Many other transformation methods can be used to introduce proteins, nucleic acids, or combinations thereof into suitable plants or plant cells. Transformation methods include using liposomes, electroporation, chemicals that increase the uptake of cell-free DNA, direct injection of DNA into plant (cells) (such as microinjection), particle gun bombardment, transformation using viruses or pollen, and microprojection. Methods may include: calcium / polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296:72-74; Negrutiu et al. (1987) Plant. Mol. Biol. 8: 363-373); electroporation of protoplasts (Shillito et al. (1985) Bio / Technol. 3: 1099-1102); microinjection into plant material (Crossway et al. (1986) Mol. Gen. Genet. 202: 179-185); particle bombardment with DNA or RNA (Klein et al. (1987) Nature 327: 70); infection with (non-integrative) viruses; etc.

[0550] In this application, references to "isolated DNA molecule" or "recombinant DNA construct" or equivalent terms or phrases are intended to indicate that a DNA molecule exists alone or in combination with other compositions but is not in its natural environment. For example, nucleic acid elements (such as coding sequences, intron sequences, nontranslation leader sequences, promoter sequences, transcription termination sequences, etc.) naturally occurring in the genomic DNA of an organism are not considered "isolated" as long as the element is located within the organism's genome and in its naturally occurring location within the genome. However, each of these elements and sub-parts of these elements will be "isolated" within the scope of this disclosure, provided that the element is not within the organism's genome and is not located in its naturally occurring location within the genome. Similarly, the nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein will be an isolated nucleotide sequence, provided that the nucleotide sequence is not located within the DNA of the bacteria whose sequence encoding the protein is naturally present. For the purposes of this disclosure, synthetic nucleotide sequences encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered isolated. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present in a plasmid or similar structure used to transform the cell, in the genome of a plant or bacteria, or in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacteria.

[0551] As used herein, the term "control plant" (or similar "control" plant seed, plant part, plant cell, and / or plant genome) refers to a plant (or plant seed, plant part, plant cell, and / or plant genome) used for comparison with a modified plant (or modified plant seed, plant part, plant cell, and / or plant genome), and has the same or similar genetic background (e.g., the same parental line, hybrid cross, inbred line, testcross, etc.) as the modified plant (or plant seed, plant part, plant cell, and / or plant genome), except for transgenesis, expression cassette, mutation, and / or genome editing affecting one or more genes. For the purpose of comparison with a modified plant, plant seed, plant part, plant cell, and / or plant genome, "wild-type plant" (or similar "wild-type" plant seed, plant part, plant cell, and / or plant genome) refers to a non-transgenic, non-mutated, and non-genome-edited control plant, plant seed, plant part, plant cell, and / or plant genome. Alternatively, as may be specified herein, such a “control plant” (or similar “control” plant seed, plant part, plant cell and / or plant genome) may refer to a plant (or plant seed, plant part, plant cell and / or plant genome) that: (i) is used for comparison with a modified plant (or modified plant seed, plant part, plant cell and / or plant genome) having a stack of two or more transgenes, expression cassettes, mutations and / or genome edits; (ii) has the same or similar genetic background as the modified plant (or plant seed, plant part, plant cell and / or plant genome) (e.g., the same parental line, hybrid cross, inbred line, testcross, etc.); but (iii) lacks at least one of the two or more transgenes, expression cassettes, mutations and / or genome edits of the modified plant (e.g., a stack compared to a single member of the stack). As used herein, if the characteristics or traits to be analyzed are considered sufficiently similar, such “control” plants, plant seeds, plant parts, plant cells and / or plant genomes may also be plants, plant seeds, plant parts, plant cells and / or plant genomes that have a similar (but not identical or identical) genetic background to the modified plants, plant seeds, plant parts, plant cells and / or plant genomes.

[0552] In some embodiments, the methods described herein result in reduced expression of the VINV gene product. In some embodiments, the reduced expression of the VINV gene product may be caused by deletion, duplication, or inversion. In some embodiments, the expression of the VINV gene product may be reduced by at least 50%, at least 85%, at least 95%, at least 99%, or 100% (e.g., no expression of the VINV protein or expression of only a non-functional truncated form). In some embodiments, the reduced expression of the VINV gene occurs throughout the plant. As used herein, the VINV gene product may include RNA and / or protein levels.

[0553] In some embodiments, the methods described herein result in increased expression of the VINV gene product. In some embodiments, the increased expression of the VINV gene product may be caused by deletion, duplication, or inversion. In some embodiments, the expression of the VINV gene product may be increased by at least 50%, at least 85%, at least 95%, at least 99%, or 100% (e.g., no expression of the VINV protein or expression of only a non-functional truncated form). In some embodiments, the increased expression of the VINV gene occurs throughout the plant. As used herein, the VINV gene product may include RNA and / or protein levels.

[0554] In some implementations, the method described herein produces genetic modifications comprising one, two, three, or four sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.In some implementations, the VINV allele comprises a set of four sequences, including SEQ ID NO: 1-4, SEQ ID NO: 5-8, SEQ ID NO: 9-12, SEQ ID NO: 13-16, SEQ ID NO: 17-20, SEQ ID NO: 21-24, SEQ ID NO: 25-28, SEQ ID NO: 29-32, SEQ ID NO: 33-36, SEQ ID NO: 37-40, SEQ ID NO: 41-44, SEQ ID NO: 45-48, SEQ ID NO: 49-52, SEQ ID NO: 53-56, SEQ ID NO: 57-60, SEQ ID NO: 61-64, SEQ ID NO: 65-68, SEQ ID NO: 69-72, SEQ ID NO: 73-76, SEQ ID NO: 77-80, SEQ ID NO: 81-84, SEQ ID NO: 85-88, SEQ ID NO: 89-92, SEQ ID NO: 93-96, SEQ ID NO: 97-100, SEQ ID NO: 101-104, SEQ ID NO: 105-108, SEQ ID NO: 109-112, SEQ ID NO: 113-116, SEQ ID NO: 117-120, SEQ ID NO: 121-124, SEQ ID NO: 125-128, SEQ ID NO: 129-132, SEQ ID NO: 133-136, SEQ ID NO: 137-140, SEQ ID NO: 141-144, SEQ ID NO: 145-148, SEQ ID NO: 170-173, SEQ ID NO: 174-177, SEQ ID NO: 178-181, SEQ ID NO: 182-185, SEQ ID NO: 186-189, SEQ ID NO: 190-193, SEQ ID NO: 194-197, SEQ ID NO: 198-201, SEQ ID NO: 202-205, SEQ ID NO: 206-209, SEQ ID NO: 210-213, SEQ ID NO: 214-217, SEQ ID NO: 218-221, SEQ ID NO: 222-225 or SEQ ID NO: 226-229.

[0555] Product line inventory and maintenance

[0556] In some embodiments, methods for breeding polyploid potato lines include maintaining potato lines. In some variations, potato lines are maintained through vegetative propagation, self-pollination, cell culture, apomixis, or any combination thereof. Other methods for maintaining potato lines are well known in the art. In some variations, a stock of lines comprises one or more potato lines having at least one, at least two, at least three, or four VINV alleles described herein, which are maintained through vegetative propagation, hybridization with haploid inducers, or combinations thereof.

[0557] Plant harvesting and storage

[0558] In some embodiments, the method includes harvesting modified potato tubers from modified potato plants. The potato tubers may be harvested between approximately 5 and 15 days after vine kill (e.g., approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days, e.g., approximately 10 days). In some embodiments, the size of the potato tubers is assessed using a potato size measuring square. The potato tubers may be size A (diameter > 4.8 cm), size B (3.8 cm < diameter < 4.8 cm), or size C (diameter < 3.8 cm). In some embodiments, the potato tubers selected for further processing are size A and have few or no external defects.

[0559] In some embodiments, the method includes placing harvested potato tubers in cold storage. Cold storage conditions vary depending on the variety / processor, ranging from 3°C to 13°C. Initial cold storage conditions may be approximately 55°F and approximately 95% relative humidity. This initial cold storage period may last approximately two weeks, after which the temperature is gradually reduced to approximately 4°C. In some embodiments, the temperature change rate is 0.5°F per 12 hours. Potato plants, plant parts, or plant cells removed after cold storage have been “cold-treated” for at least two weeks, at least four weeks, at least eight weeks, at least sixteen weeks, at least twenty-four weeks, or at least thirty-two weeks. In other embodiments, potato plants, plant parts, or plant cells removed after cold storage have been “cold-treated” for two weeks, four weeks, eight weeks, sixteen weeks, twenty-four weeks, or thirty-two weeks.

[0560] Tuber Sugar Spectrum

[0561] In some embodiments, the method includes obtaining a tuber glycosylation profile from modified potato plants, plant parts, or plant cells. In some embodiments, the tuber glycosylation profile obtained from said plant contains lower levels of glucose and / or fructose compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, the tuber glycosylation profile obtained from said plant contains higher levels of sucrose compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 200%, no more than 100%, no more than 50%, or no more than 25% compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, for example, after sixteen weeks of cold treatment, the tuber glycosylation profile obtained from said plant contains lower levels of glucose compared to a tuber glycosylation profile obtained from a control plant. In some embodiments, the percentage of fructose in the modified potato plants, plant parts, or plant cells is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the tuber sugar profile obtained from control plants. In some embodiments, for example, after sixteen weeks of cold treatment, the tuber sugar profile obtained from said plants contains a lower level of fructose compared to the tuber sugar profile obtained from control plants. In some embodiments, the percentage of fructose in the modified potato plants, plant parts, or plant cells is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the tuber sugar profile obtained from control plants.

[0562] Tuber sugar profiles can be obtained using various methods known to those skilled in the art. In some embodiments, colorimetric determination is used to obtain tuber sugar profiles. In other embodiments, industrial-standard high-performance liquid chromatography (HPLC) is used to obtain tuber sugar profiles. Tuber sugar profiles can be obtained at any time after harvest. In some embodiments, tuber sugar profiles are obtained at harvest. In some embodiments, tuber sugar profiles are obtained after the tubers have undergone refrigeration.

[0563] Acrylamide content in tubers

[0564] In some embodiments, the method includes obtaining acrylamide content from edited potato plants, plant parts, or plant cells. In some embodiments, the modified potato plants, plant parts, or plant cells contain lower levels of acrylamide compared to control potato plants, plant parts, or plant cells. Controlling acrylamide formation during the heat treatment of potatoes is particularly important when the potatoes have been refrigerated for any period of time. In some embodiments, the acrylamide content after refrigeration (e.g., after sixteen weeks of refrigeration) is at least 50%, at least 75%, at least 85%, at least 95%, or at least 99% lower than that of control potato plants, plant parts, or plant cells. Acrylamide content can be measured at any time after harvest. In some embodiments, the acrylamide content is determined after refrigeration. In a further embodiment, the acrylamide content is obtained from potato food products. Conventional techniques known in the art can be used to determine acrylamide content. For example, a combination of mass spectrometry and liquid chromatography can be used to detect acrylamide. [doi DOT org SLASH10.3390 SLASH foods10092038 for more details]

[0565] The determination of acrylamide content in potato products may also include comparing the acrylamide content of potato products derived from modified potato plants, plant parts, or plant cells that have been refrigerated for at least sixteen weeks with the acrylamide content of control potato products derived from control potato plants. During the determination, when compared with potato products derived from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells will show an acrylamide content that is at least 1 / 5, at least 1 / 6, at least 1 / 7, at least 1 / 8, at least 1 / 9, at least 1 / 10, at least 1 / 11, at least 1 / 12, at least 1 / 13, at least 1 / 14, at least 1 / 15, at least 1 / 20, at least 1 / 25, at least 1 / 30, at least 1 / 35, or even lower. Reduced to at least 1 / 40, at least 1 / 45, at least 1 / 50, at least 1 / 55, at least 1 / 60, at least 1 / 65, at least 1 / 70, at least 1 / 75, at least 1 / 80, at least 1 / 85, at least 1 / 90, at least 1 / 95, at least 1 / 100, at least 1 / 150, at least 1 / 200, at least 1 / 250, at least 1 / 300, at least 1 / 350, at least 1 / 400, at least 1 / 450, or at least 1 / 500. More specifically, refrigeration can last for at least two weeks, at least four weeks, at least eight weeks, at least sixteen weeks, at least twenty-four weeks, or at least thirty-two weeks.In other embodiments, alternatively, when compared with potato products from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells that have been refrigerated for at least two hours will show a reduction in acrylamide content of 1 / 5 to 1 / 500, 1 / 5 to 1 / 450, 1 / 5 to 1 / 400, 1 / 5 to 1 / 400, 1 / 5 to 1 / 350, 1 / 5 to 1 / 300, 1 / 5 to 1 / 250, 1 / 5 to 1 / 200, etc. To 1 / 5 to 1 / 150, down to 1 / 5 to 1 / 100, down to 1 / 5 to 1 / 95, down to 1 / 5 to 1 / 90, down to 1 / 5 to 1 / 85, down to 1 / 5 to 1 / 80, down to 1 / 5 to 1 / 75, down to 1 / 5 to 1 / 70, down to 1 / 5 to 1 / 65, down to 1 / 5 to 1 / 60, down to 1 / 5 to 1 / 55, down to 1 / 5 to 1 / 50, down to 1 / 5 to 1 / 45, down to 1 / 5 to 1 / 40, down to 1 / 5 to 1 / 35, down to 1 / 5 to 1 / 30, down to 1 / 5 to 1 / 25. Decrease to 1 / 5 to 1 / 20, decrease to 1 / 5 to 1 / 15, decrease to 1 / 5 to 1 / 10, decrease to 1 / 10 to 1 / 500, decrease to 1 / 10 to 1 / 450, decrease to 1 / 10 to 1 / 400, decrease to 1 / 10 to 1 / 400, decrease to 1 / 10 to 1 / 350, decrease to 1 / 10 to 1 / 300, decrease to 1 / 10 to 1 / 250, decrease to 1 / 10 to 1 / 200, decrease to 1 / 10 to 1 / 150, decrease to 1 / 10 to 1 / 100, decrease to 1 / 10 to 1 / 95, decrease to 1 / 10 to 1 / 90, reduced to 1 / 10 to 1 / 85, reduced to 1 / 10 to 1 / 80, reduced to 1 / 10 to 1 / 75, reduced to 1 / 10 to 1 / 70, reduced to 1 / 10 to 1 / 65, reduced to 1 / 10 to 1 / 60, reduced to 1 / 10 to 1 / 55, reduced to 1 / 10 to 1 / 50, reduced to 1 / 10 to 1 / 45, reduced to 1 / 10 to 1 / 40, reduced to 1 / 10 to 1 / 35, reduced to 1 / 10 to 1 / 30, reduced to 1 / 10 to 1 / 25, reduced to 1 / 10 to 1 / 20, or reduced to 1 / 10 to 1 / 15. More specifically, refrigeration can be sustained for periods of at least three hours, at least four hours, at least five hours, at least six hours, at least eight hours, at least ten hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, or longer.Alternatively, when compared to potato products from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells that have been refrigerated for at least two hours will show acrylamide content that is 25% to 75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 50%, 25% to 45%, 25% to 40%, 25% to 35%, 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 55%, 30% to 55%, 30% to 50%, 30% to 45%, 25% to 40%, and 30% to 35% lower. 35% to 75%, 35% to 70%, 35% to 65%, 35% to 60%, 35% to 55%, 35% to 55%, 35% to 50%, 35% to 45%, 35% to 40%, 40% to 75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 55%, 40% to 50%, 40% to 45%, 45% to 75%, 45% to 70%, 45% to 65%, 45% to 60%, 45% to 55%, 45% to 55%, 45% to 50%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55%. More specifically, refrigeration can last for a period of at least three hours, at least four hours, at least five hours, at least six hours, at least eight hours, at least ten hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours or longer.

[0566] In addition, it is believed that when measured as described above, potato products derived from modified potato plants, plant parts or plant cells and that have been refrigerated for at least two hours will show acrylamide content of less than 500 ppb (mg / Kg), less than 400 ppb (mg / Kg), less than 300 ppb (mg / Kg), less than 200 ppb (mg / Kg) or less than 100 ppb (mg / Kg). Alternatively, during the assay, potato products derived from potato plants produced by the above method will exhibit values ​​of approximately 90 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 100 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 200 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 250 ppb (mg / kg) to approximately 500 ppb (mg / kg), approximately 100 ppb (mg / kg) to approximately 300 ppb (mg / kg), approximately 100 ppb (mg / kg) to approximately 250 ppb (mg / kg), approximately 200 ppb (mg / kg) to approximately 300 ppb (mg / kg), approximately 250 ppb (mg / kg) to approximately 300 ppb (mg / kg), and approximately 300 ppb (mg / kg) to approximately 500 ppb (mg / kg). Acrylamide content is between approximately 400 ppb (mg / kg) and approximately 500 ppb (mg / kg). More specifically, refrigeration can be sustained for periods of at least three hours, at least four hours, at least five hours, at least six hours, at least eight hours, at least ten hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, or longer. Furthermore, it is believed that potato products derived from modified potato plants, plant parts, or plant cells that have been exposed to or stored at room temperature under these conditions may exhibit acrylamide content below 1100 ppb (mg / kg), 1000 ppb (mg / kg), below 900 ppb (mg / kg), below 800 ppb (mg / kg), below 700 ppb (mg / kg), below 600 ppb (mg / kg), or below 500 ppb (mg / kg) when measured as described above.Alternatively, during the assay, potato products derived from potato plants produced by the above method will exhibit values ​​of approximately 400 ppb (mg / kg) to approximately 1100 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 1000 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 900 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 800 ppb (mg / kg), approximately 400 ppb (mg / kg) to approximately 700 ppb (mg / kg), approximately 500 ppb (mg / kg) to approximately 1100 ppb (mg / kg), approximately 500 ppb (mg / kg) to approximately 1000 ppb (mg / kg), approximately 500 ppb (mg / kg) to approximately 900 ppb (mg / kg), and approximately 500 ppb (mg / kg) to approximately 800 ppb (mg / kg). The acrylamide content is between approximately 500 ppb (mg / Kg) and approximately 750 ppb (mg / Kg).

[0567] The determination of acrylamide content in potato products may also include comparing the acrylamide content of potato products derived from modified potato plants, plant parts, or plant cells that have been stored at or subjected to room temperature conditions with the acrylamide content of control potato products derived from control potato plants. During the determination, when compared with potato products derived from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells will show an acrylamide content that is at least reduced to 1, at least reduced to 1 / 2, at least reduced to 1 / 3, at least reduced to 1 / 4, at least reduced to 1 / 5, at least reduced to 1 / 6, at least reduced to 1 / 7, at least reduced to 1 / 8, at least reduced to 1 / 9, at least reduced to 1 / 10, at least reduced to 1 / 11, at least reduced to 1 / 12, at least reduced to 1 / 13, at least reduced to 1 / 14, or at least reduced to 1 / 15.

[0568] Alternatively, when compared with potato products from control potato plants, potato products derived from modified potato plants, plant parts, or plant cells will show a reduction in acrylamide content, at least to 1 to 1 / 15, to 1 / 2 to 1 / 15, to 1 / 3 to 1 / 15, to 1 / 4 to 1 / 15, to 1 / 5 to 1 / 15, to 1 to 1 / 14, to 1 / 2 to 1 / 14, to 1 / 3 to 1 / 14, to 1 / 4 to 1 / 14, to 1 / 5 to 1 / 14, to 1 to 1 / 13, to 1 / 2 to 1 / 13. , down to 1 / 3 to 1 / 13, down to 1 / 4 to 1 / 13, down to 1 / 5 to 1 / 15, down to 1 to 1 / 12, down to 1 / 2 to 1 / 12, down to 1 / 3 to 1 / 12, down to 1 / 4 to 1 / 12, down to 1 / 5 to 1 / 12, down to 1 to 1 / 11, down to 1 / 2 to 1 / 11, down to 1 / 3 to 1 / 11, down to 1 / 4 to 1 / 11, down to 1 / 5 to 1 / 11, down to 1 to 1 / 10, down to 1 / 2 to 1 / 10, down to 1 / 3 to 1 / 10, down to 1 / 4 to 1 / 10 or down to 1 / 5 to 1 / 10.

[0569] Alternatively, when compared to potato products from control potato plants or sweet potato products from control sweet potato plants, potato products derived from modified potato plants, plant parts, or plant cells that have been stored or subjected to room temperature conditions have acrylamide content that is 25% to 75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 55%, 25% to 50%, 25% to 45%, 25% to 40%, 25% to 35%, 30% to 75%, 30% to 70%, 30% to 65%, 30% to 60%, 30% to 55%, 30% to 55%, 30% to 50%, 30% to 45%, 25% to 40%, or 30% lower. Up to 35%, low 35% to 75%, low 35% to 70%, low 35% to 65%, low 35% to 60%, low 35% to 55%, low 35% to 55%, low 35% to 50%, low 35% to 45%, low 35% to 40%, low 40% to 75%, low 40% to 70%, low 40% to 65%, low 40% to 60%, low 40% to 55%, low 40% to 55%, low 40% to 45%, low 45% to 75%, low 45% to 70%, low 45% to 65%, low 45% to 60%, low 45% to 55%, low 45% to 55%, 45% to 50%, low 50% to 75%, low 50% to 70%, low 50% to 65%, low 50% to 60%, or low 50% to 55%.

[0570] The above methods (both refrigerated and room temperature) may also include heat processing potatoes into chips, potato chips, French fries, potato sticks or shoelace fries or other edible potato products.

[0571] Potato chip color and brightness rating (CLS)

[0572] In some implementations, the method involves producing a potato product with a lighter color than that produced by a control plant, from modified potato plants, plant parts, or plant cells. When determining chip color, a tuber about the size of a baseball can be cut longitudinally in half from the bud to the stem tip. Four to five 1mm thick chips are cut, yielding a total of eight to ten chips. The chip slices are then fried in peanut oil at approximately 360°F for about 2 minutes and 10 seconds in a custom basket. The chips can then be crushed to a specific particle size and placed in a measuring instrument. Immediately after frying and cooling, the chip color is quantified by reflectance using a Konica Minolta CR410 colorimeter (Konica Minolta, NJ, USA). Hunter Lab color space readings L, a, and b are obtained. L is relative lightness, a is the chromaticity range between red and green, and b is the chromaticity range between yellow and blue. The results can then be reported as a chip lightness score. Chip color can be determined at harvest, after cold storage, or at any intermediate point in time.

[0573] In some embodiments, the method includes generating a potato product from modified potato plants, plant parts, or plant cells, the potato product having a chip brightness score that is 25% to 100% higher than that of a product from a control plant. In some embodiments, the potato product from the modified potato plants, plant parts, or plant cells has a chip brightness score that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 5000%, or at least 10000% higher than that of a product from a control plant. In some embodiments, the potato product from the modified potato plants, plant parts, or plant cells has a chip brightness score higher than 63.

[0574] In some implementations, potato chips produced from modified potato plants, plant parts, or plant cells containing mutations in fewer than four VINV alleles have a lighter chip brightness score than potato chips produced from modified potato plants, plant parts, or plant cells containing mutations in all four VINV alleles.

[0575] Potato genome

[0576] On the other hand, this document provides a potato genome. In some embodiments, these genomes are characterized by containing mutations in at least one, at least two, at least three, or four VINV alleles, the VINV alleles containing sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229. In some embodiments, the genome is characterized by containing mutations in at least one, at least two, at least three, or four VINV alleles, each mutation containing a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are aligned with SEQ ID NO: 169. In some embodiments, the potato genome is modified. In some embodiments, the potato genome is not present in viable nonmicrobial cells.

[0577] Kits for producing modified potatoes

[0578] On the other hand, this document provides kits for producing modified potato plants, plant parts, or plant cells. The kits may contain the guide RNA, recombinant DNA construct, vector, host cell, and / or guide RNA-endonuclease complex described herein. In some embodiments, the kits may also contain instructions for introducing one or more guide endonucleases into potato cells using the guide RNA, recombinant DNA construct, vector, host cell, composition, or combination thereof, which together bind to the protospacer sequence of each of at least one VINV allele, at least two VINV alleles, at least three VINV alleles, or four VINV alleles.

[0579] Example

[0580] The subject matter of this invention will be better understood by referring to the following embodiments, which are provided as examples of the invention and not as limitations.

[0581] Example 1: General Method

[0582] GM1: Identification of VINV editing target sites

[0583] GM1.1: Identification of target DNA sequences for crRNA design

[0584] Protein BLAST searches were performed using the "tblastn" BLAST function, specifically Protein Query - Post-translational Target Sequence BLAST 2.11.0+, with default parameters (vacancy open penalty = 11, vacancy extension penalty = 1, E value = 10, word length = 3, maximum score = 25, query filter = SEG, query genetic code = general, substitution matrix = BLOSUM 62). A custom shell script extracted the corresponding nucleotide sequences from this search for identified orthologs and any putative paralogs, including the upstream 5kb of each gene. These sequences were then aligned to each other using Clustal Omega-1.2.4 with default parameters (substitution matrix = GONNET). The most probable candidate sequences with the highest identity to the canonical sequence and the most conserved exon structure were used to design CRISPR RNA (crRNA) for appropriate CRISPR-associated (Cas) nucleases.

[0585] GM1.2: Design of crRNA for DNA editing using Cas nuclease

[0586] The most likely candidate VINV sequences identified from protein BLAST, tblastn, and Clustal Omega workflows were used as targets for crRNA design in Geneious Prime 2020.0.3, where protospacer neighboring motif (PAM) sites targeting the appropriate Cas nuclease were identified near each candidate sequence. Generally, crRNAs with high specificity targeting the first or second exon or promoter sequence are preferred, but high-scoring crRNAs targeting subsequent exons are also selected. The resulting sequences were exported and scaffolded for the appropriate Cas nuclease. Functional crRNAs were synthesized by IDT (IntegratedDNA Technologies, Newark, NJ, USA) using standard RNA synthesis methods. The editing efficiency of the crRNAs was screened in protoplasts.

[0587] Figure 2 This is a diagram showing the target site at the StVINV location on chromosome 3, where thick boxes indicate coding sequence exons and thin lines indicate introns.

[0588] Figure 3Visual representations of the allele sequences of six fully edited samples are shown (E-PED165-7186 Hap1, SEQ ID NO:5, Hap2, SEQ ID NO:6, Hap3, SEQ ID NO:7 and Hap8 SEQ ID NO:8; E-PED165-7398 Hap1, SEQ ID NO:53, Hap2, SEQ ID NO:54, Hap3, SEQ ID NO:55 and Hap8 SEQ ID NO:56; E-PED165-7475 Hap1, SEQ ID NO:97, Hap2, SEQ ID NO:98, Hap3, SEQ ID NO:99 and Hap8 SEQ ID NO:100; E-PED165-7459 Hap1, SEQ ID NO:89, Hap2, SEQ ID NO:90, Hap3, SEQ ID NO:91 and Hap8 SEQ ID NO:80). NO:92; E-PED165-7632 Hap1, SEQ ID NO:105, Hap2, SEQ ID NO:106, Hap3, SEQ ID NO:107 and Hap8 SEQ ID NO:108; E-PED165-7621 Hap1, SEQ ID NO:101, Hap2, SEQ ID NO:102, Hap3, SEQ ID NO:103 and Hap8 SEQ ID NO:104). Figure 3 The location of VINV on chromosome 3 of the potato genome is highlighted, with thick boxes indicating coding exons and thin lines indicating introns.

[0589] Figure 4The amino acid sequences of all 24 edited alleles in six fully edited samples are shown (E-PED165-7186 Hap1, SEQ ID NO: 5, Hap2, SEQ ID NO: 6, Hap3, SEQ ID NO: 7 and Hap4 SEQ ID NO: 8; E-PED165-7398 Hap1, SEQ ID NO: 53, Hap2, SEQ ID NO: 54, Hap3, SEQ ID NO: 55 and Hap4 SEQ ID NO: 56; E-PED165-7475 Hap1, SEQ ID NO: 97, Hap2, SEQ ID NO: 98, Hap3, SEQ ID NO: 99 and Hap4 SEQ ID NO: 100; E-PED165-7459 Hap1, SEQ ID NO: 89, Hap2, SEQ ID NO: 90, Hap3, SEQ ID NO: 99, Hap4 SEQ ID NO: 99, Hap2, SEQ ID NO: 9 ...3, SEQ ID NO: 99, Hap3, SEQ ID NO: 99, Hap3, SEQ ID NO: 99, Hap3, SEQ ID NO: 99, Hap3, SEQ ID NO: 99, Hap3, SEQ ID NO: 99, Hap3, SEQ ID NO: 99, Hap3, SEQ ID NO Visual representation of the alleles of Hap1 and Hap4 (SEQ ID NO: 92); E-PED165-7632 Hap1, SEQ ID NO: 105, Hap2, SEQ ID NO: 106, Hap3, SEQ ID NO: 107 and Hap4 (SEQ ID NO: 108); E-PED165-7621 Hap1, SEQ ID NO: 101, Hap2, SEQ ID NO: 102, Hap3, SEQ ID NO: 103 and Hap4 (SEQ ID NO: 104) and wild-type (WT) alleles. (e.g.) Figure 4 As shown, edit-induced deleted amino acid sequences are displayed as dashed lines in the allele sequences, while naturally occurring SNPs used for haplotype allocation are shown as darker regions outside the dashed lines. The functional results of the edit mutations are summarized on the left side of each allele.

[0590] GM2: Preparation of Ribonucleoprotein

[0591] To prepare ribonucleoprotein (RNP), 2 μL of New England Biolabs Buffer (NEBuffer™) 2.1 (10x stock solution) was placed in a 1.5 mL microcentrifuge tube containing 10–600 pmol crRNA and an equal volume of the selected Cas nuclease protein. The final volume was adjusted to 20 μL using nuclease-free water. The solution was freshly prepared and used after incubation at room temperature for 15 minutes.

[0592] GM3: Sequence-based Edit Confirmation

[0593] Primers were designed to amplify VINV and generate protoplasts. Further details regarding plant selection, plant growth, protoplast generation, and protoplast transfection are provided in the species-specific protocols described in the following examples.

[0594] Transfected protoplasts were incubated at room temperature for 24 to 48 hours, then lysed, and the crude lysates were subjected to one or more long-distance direct polymerase chain reactions (PCR). Other transfected protoplasts were regenerated, and DNA was extracted from the callus, leaves, or other plant tissues from these regenerated protoplasts. PCR products were pooled according to the transfected samples, and library preparation was performed using seqWell™ (Beverly, MA, USA) to generate Illumina (Illumina, San Diego, CA, USA) libraries. Samples were loaded onto Illumina iSeq (Illumina, San Diego, CA, USA) and sequenced using a paired-end 150nt sequencing kit. Sequences were analyzed by aligning FASTQ files to reference sequences, and mutations near the target sites for each gene were statistically analyzed relative to controls. Editing efficiency was calculated based on the mutation frequencies observed in the reads obtained from a given sample, and this information was used to calculate how many plants should be screened to identify the multi-gene knockouts required for induced clonal gamete production.

[0595] Illumina sequencing libraries were prepared using PCR amplicon sequencing with the plexWell 96 kit (seqWell™, Beverly, MA, USA) and sequenced on Illumina iSeq (Illumina, San Diego, CA, USA). FASTQ datasets were aligned to corresponding reference genomes using the BWA-MEM algorithm (Li, H. (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiv:1303.3997), and variants were visualized and quantified using a custom script. Editing efficiency was calculated as the fraction of reads with the targeted mutation in the edited sample relative to unedited control reads. The editing efficiency of each wizard was determined and used to estimate the minimum number of plants required to restore the desired multi-gene knockout.

[0596] GM4: Flow Cytometry

[0597] Flow cytometry was performed on parental plants and progeny using the method described by Galbraith et al. (Rapid flowcytometric analysis of the cell cycle in intact plant tissues. Science. 220(4601): 1049-1051). Briefly, intact cell nuclei were extracted, filtered, and stained with propidium iodide according to the instructions of the CyStain® PI Absolute P kit (Sysmex America, Lincolnshire, IL, USA). The DNA content of the cell nuclei was determined by applying the samples to a BD Accuri C6 flow cytometer. Gating was performed, and the genomic DNA content was calculated by comparing the peak area of ​​the sample to a known position in a control with a known ploidy. The ploidy of unknown samples was determined based on relative comparisons with each control.

[0598] GM5: Protoplast

[0599] GM5.1: Protoplast Separation Technology

[0600] Plants obtained from selected and tested potato germplasm resources included Russet Burbank and Atlantic types. Plant material was propagated in vitro via internode cuttings on a sucrose-containing Murashige & Skoog modified BC potato medium and grown under 16-hour light illumination with cool white fluorescent lamps. Approximately one gram of leaves was harvested from 2–3-week-old explants under sterile conditions and placed in sterile water.

[0601] Cut the leaves into thin slices about 1 mm wide and incubate them overnight in a digestive solution. (See, e.g., Clasen et al. (2016). Improving cold storage and processing traits in potato throughtargeted gene knockout. Plant biotechnology journal, 14(1), 169-176; Fossi et al. (2019). Regeneration of Solanum tuberosum plants from protoplasts induceswidespread genome instability. Plant physiology, 180(1), 78-86; Masson et al. (1987). Plant regeneration from protoplasts of diploid potato derived fromcrosses of Solanum tuberosum with wild Solanum species. Plant Science, 53(2), 167-176; Nicolia et al. (2015) Targeted gene mutation in tetraploidpotato through transient TALEN expression in protoplasts. Journal of biotechnology. 204: 17-24; Veillet et al. (2019). The Solanum tuberosum GBSSIgene: a target for assessing Gene and base editing in tetraploid potato. Plant Cell Reports, 38(9), 1065-1080). The leaf sections in the digestion solution were then incubated overnight at 24°C. The next day, protoplasts were released from the leaf tissue after shaking at 40 RPM for 15 minutes at room temperature.

[0602] Protoplasts were collected through a 100 µm sterile cell filter into sterile 50 mL conical tubes and centrifuged at 100 x g for 5 min. The supernatant was removed and replaced with washing solution (see, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019). The cells were then gently resuspended and slowly plated onto 0.43 M sucrose solution. The tubes were centrifuged at 100 x g for 15 min. After 15 min, a dense, dark band of protoplasts appeared at the interface between the two solutions. This band was collected with a sterile serum pipette in a continuous, smooth motion and combined with transformation buffer (see, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019). The collected cells were quantified using a Bürker hemocytometer and stored at 4°C protected from light before transfection. One sample was retained and cell viability was tested using FDA staining, as described by Larkin (1976. Purification and viability determinations of plant protoplasts. Planta. 128(3): 213-216).

[0603] GM5.2: Protoplast Transfection

[0604] Transfection was performed as described in the art (see, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019). Protoplasts were centrifuged at 50 xg for 10 min and resuspended in a volume of transformation buffer to achieve a cell density of 1 × 10⁶ protoplasts / mL. 20 μL of freshly prepared RNP as described in the GM2 section was added to the bottom of a 15 mL round-bottom tube, and 100 μL of protoplasts suspended in transformation buffer was mixed with the RNP solution. Next, 120 μL of PEG solution was added and gently mixed by rotating the tube. After incubation at room temperature for 15 min, the protoplasts were washed twice with a wash solution consisting of 0.4 M D-mannitol, 15 mM CaCl₂, and 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) at 50 xg for 10 min. Perform a final centrifugation and resuspend the transfected cells in culture medium. (See, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019).

[0605] GM6: Plant Encapsulation and Regeneration

[0606] Equal volumes of transfected cells were combined with a 3.2% sodium alginate solution and gently mixed. The resulting solution was then added dropwise to the top of a solidified agar to allow the alginate matrix to solidify, forming an alginate lens. The lens was incubated at room temperature for 30 minutes and then transferred to a new culture dish containing 20 mL of culture medium.

[0607] Plant regeneration protocols were performed as described in the art (see, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019). Alginate lenses in the culture medium were placed in the dark at 24°C for approximately 3–4 weeks, during which time signs of cell division and callus induction were observed using an inverted microscope. When the callus reached 1 mm in size, the culture medium was replaced with a first callus induction medium as described in the art (see, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019), and light exposure was gradually increased under cool white fluorescent light by first covering and then removing the gauze layer. After 3–6 weeks, or when the small callus reached approximately 2 mm in diameter, the small callus was released from the alginate using a citric acid solution consisting of 20 mM sodium citrate and 0.5 M sorbitol. The small callus was then washed in callus induction medium and incubated in a second callus induction medium, which was replaced weekly with a freshly prepared solution. (See, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019). Finally, the 3–5 mm green callus was removed and placed on a petri dish containing bud induction medium. (See, for example, Clasen et al. 2016; Fossi et al. 2019; Masson et al. 1987; Nicolia et al. 2015; and Veillet et al. 2019). Then, the callus tissue was transferred to fresh shoot induction medium every 2 weeks until shoots emerged after about 2-3 months. When the shoots grew to about 1 cm in size on the shoot induction medium, they were cut off from the callus tissue and transferred to Murashige & Skoog modified BC potato medium containing sucrose, and then irradiated under a cool white fluorescent lamp for 16 hours.

[0608] The plants were then propagated by segmentation and genotyped using the same sequencing method described in the GM3 section of Example 1.

[0609] GM7: Plant Growth and Hybridization Conditions

[0610] Once the plant has fully regenerated in vitro from the protoplast, the rooted plantlets are transferred from the tissue culture to 1-gallon pots filled with peat moss and allowed to continue growing under greenhouse conditions with a 16-hour light-per-day duration. Freshly transplanted plantlets should be covered with a moisture-retaining cover for up to 3 days.

[0611] Regenerated plants with editable VINV were identified and transplanted from tissue cultures into peat substrate as described in the previous paragraph. When the plants flowered, pollen was collected from male plants, and cross-pollination was performed.

[0612] GM8: Potato Harvesting and Storage

[0613] Potato tubers were harvested 10 days after vine kill. Potato size measuring boards were used to evaluate the tubers, categorizing them into size A (diameter > 4.8 cm), size B (3.8 cm < diameter < 4.8 cm), and size C (diameter < 3.8 cm). Size A tubers with few or no external defects were selected for frying tests. Tubers were stored at 55°F and 95% relative humidity for two weeks to allow wounds to heal. Refrigerated storage was set to gradually decrease the temperature to 39.2°F (4°C) at a rate of 0.5°F every 12 hours.

[0614] GM9: Frying Test and General Phenotype

[0615] Initial frying experiments were conducted two weeks after healing. Specific gravity was measured based on genotype prior to the first frying test. Tubers from the same genotype were collected and their weights were measured in air and water. Specific gravity was then calculated by dividing the weight in air by the difference between the weight in air and the weight in water (Wang et al., 2017. https: / / doi.org / 10.2135 / cropsci2016.12.0976).

[0616] GM10: Slicing Technology

[0617] Cut the tuber lengthwise from the bud to the stem. Using a mandoline slicer, cut 4-5 slices of 1mm thickness from each half of the tuber, for a total of 8-10 slices. Fry the sliced ​​potato chips in peanut oil at 360°F for 2 minutes and 10 seconds in a custom basket.

[0618] GM11: Determination of Sugar Content (HPLC)

[0619] Sugar content in potato tubers and products can be measured, for example, by HPLC, using the methods described by Wilson, AM et al. ("HPLC determination of fructose, glucose, and sucrose in potatoes." Journal of Food Science 46.1 (1981): 300-301.) or similar methods.

[0620] Example 2: Mitigating Low-Temperature-Induced Glycation via CRIPSR-Cas Editing

[0621] Primary spacer selection

[0622] As described in GM 1.2, the editing efficiency of ten protospacers at the VINV allele was tested. Protospacer PRS155 exhibited the best editing efficiency and was selected as the lead protospacer candidate based on this efficiency. (See [link to GM 1.2]) Figure 5 )

[0623] Plant selection, growth, harvesting and storage

[0624] Thirty-six unique genotypes of Atlantic potato varieties were selected and cultivated, including edited, unedited, and wild-type haplotypes.

[0625] Illumina iSeq amplicon sequencing was used to phase the edits. For effective phasing, sufficient variation among one, two, three, or four haplotypes near the protospacer cleavage site is typically required to be captured by identical reads of 200-300 bp. Phased edits were manually verified, and true / false results were determined using confidence levels. In addition to other sequencing analyses and manual verification, ddPCR drop phases were used to determine the haplotypes of selected plants.

[0626] Table 1. Phases of selected plants with edited VINV alleles.

[0627] Plant Material ID Atl_Hap1 Atl_Hap2 Atl_Hap3 Atl_Hap4 category E-PED165-7318 7.00 9.00 12.00 6.00 1_Hap1 E-PED165-7393 7.00 0.00 0.00 0.00 1_Hap1 E-PED165-7436 7.00 9.00 6.00 6.00 1_Hap1 E-PED165-7405 0.00 0.00 4.00 0.00 1_Hap3 E-PED165-7487 0.00 0.00 8.00 0.00 1_Hap3 E-PED165-7186 9.00 9.00 9.00 8.00 1_Hap4 E-PED165-7242 9.00 33.00 9.00 8.00 1_Hap4 E-PED165-7324 9.00 15.00 15.00 10.00 1_Hap4 E-PED165-7400 6.00 6.00 6.00 13.00 1_Hap4 E-PED165-7477 8.00 0.00 8.00 15.00 2_Hap1_Hap3 E-PED165-7302 7.00 9.00 6.00 5.00 2_Hap1_Hap4 E-PED165-7340 13.00 6.00 6.00 7.00 2_Hap1_Hap4 E-PED165-7471 8.00 6.00 6.00 13.00 2_Hap1_Hap4 E-PED165-7651 13.00 0.00 13.00 0.00 2_Hap1_Hap3 E-PED165-7240 0.00 8.00 10.00 12.00 2_Hap2_Hap3 E-PED165-7287 7.00 7.00 10.00 6.00 3_Hap1_Hap2_Hap3 E-PED165-7426 11.00 5.00 5.00 9.00 3_Hap1_Hap2_Hap3 E-PED165-7621 7.00 7.00 8.00 9.00 3_Hap1_Hap2_Hap3 E-PED165-7300 11.00 8.00 0.00 7.00 3_Hap1_Hap2_Hap4 E-PED165-7419 7.00 0.00 11.00 11.00 3_Hap1_Hap3_Hap4 E-PED165-7590 19.00 0.00 8.00 7.00 3_Hap1_Hap3_Hap4 E-PED165-7385 15.00 4.00 8.00 13.00 3_Hap2_Hap3_Hap4 E-PED165-7421 6.00 11.00 10.00 13.00 3_Hap2_Hap3_Hap4 E-PED165-7182 13.00 8.00 13.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7188 16.00 16.00 14.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7326 7.00 7.00 11.00 11.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7347 11.00 8.00 11.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7373 13.00 49.00 13.00 10.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7398 13.00 13.00 8.00 10.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7413 11.00 17.00 13.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7432 14.00 7.00 16.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7437 8.00 8.00 7.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7458 8.00 13.00 10.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7459 11.00 11.00 11.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7475 10.00 10.00 8.00 8.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7632 7.00 10.00 10.00 7.00 4_Hap1_Hap2_Hap3_Hap4 E-PED165-7286 0.00 0.00 0.00 0.00 Unedited E-PED165-7384 0.00 0.00 0.00 0.00 Unedited E-PED165-7594 0.00 0.00 0.00 0.00 Unedited

[0628] A total of 292 plants were harvested, and the tuber yield per plant was measured. Details of genotypes, plots, and plant numbers by haplotype are shown in Table 2.

[0629] Table 2. Genotype, plot, and number of plants.

[0630] Edited haplotypes Number of genotypes Number of residential communities Number of plants 1 3 5 15 3 2 6 18 4 4 4 12 1|3 2 4 12 1|4 3 3 9 2|3 1 1 3 1|2|3 3 7 21 1|2|4 1 1 3 1|3|4 2 2 6 2|3|4 2 9 27 1|2|3|4 13 35 105 Unedited 3 9 27 wild type 1 12 36

[0631] The tubers were evaluated for internal and external defects, healed at 55℉ for 2 weeks, and then stored at 4℃. Before refrigerated storage, some tubers were also sliced ​​and their sugar content, color, etc., were measured.

[0632] Sugar content measurement (colorimetric method)

[0633] Combine 50 mg of tuber sample with 100 μL of 100 mM Na phosphate (pH 7.0). Vortex the sample vigorously for 3 minutes to homogenize, then centrifuge at 10 k x G for 5 minutes at 4 °C. Collect the supernatant and transfer it to a new tube. Then boil the sample at 95 °C for 5 minutes to inactivate endogenous invertase, and then centrifuge to remove the precipitate.

[0634] The remaining assay steps follow the Sigma Cat # MAK013 protocol. In short, for glucose, add 10 μL of supernatant to edited samples or 0.5 μL to unedited samples, then bring the supernatant volume to 50 μL using glucose assay buffer. For sucrose, add 0.5 μL of supernatant to both edited and unedited samples, then bring the supernatant volume to 50 μL using glucose assay buffer.

[0635] Add 2 μL of invertase to each sucrose sample and sucrose standard. Add 2 μL of glucose assay buffer to each glucose sample. Incubate the plate at 37°C for 30 minutes. Add 50 μL of master reaction mixture to each well. Mix thoroughly using a horizontal shaker or by pipetting, and incubate the reaction at 37°C for 30 minutes. Measure the absorbance at 595 nm using a microplate reader. See Lindsay, H. A colorimetric estimation of reducing sugars in potatoes with 3,5-dinitrosalicylic acid. Potato Res 16, 176–179 (1973).

[0636] Sugar content results

[0637] Glucose and sucrose content were measured in Russet Burbank potatoes with a complete frameshift mutation in VINV and in potatoes with a single frameshift mutation in VINV. Figure 6 Compared to the unedited control, potatoes with complete frameshift showed a 57% reduction in glucose and a 30% reduction in sucrose. Compared to the wild-type control, potatoes with complete frameshift showed a 25% reduction in glucose and no change in sucrose content. Compared to the unedited control, potatoes with single frameshift showed a 57% reduction in glucose and a 60% reduction in sucrose. Compared to the wild-type control, potatoes with single frameshift showed a 25% reduction in glucose and a 33% reduction in sucrose.

[0638] Glucose and sucrose contents were measured in Atlantic potatoes from two plants with a complete frameshift mutation in VINV. Figure 7 Compared to the unedited control, potatoes with complete frameshift showed a 90% reduction in glucose and a 122% increase in sucrose. Compared to the wild-type control, potatoes with complete frameshift showed an 83% reduction in glucose and a 300% increase in sucrose. Compared to the unedited control, potatoes from another plant with complete frameshift showed a 100% reduction in glucose and an 11% increase in sucrose. Compared to the wild-type control, potatoes from another plant with complete frameshift showed a 100% reduction in glucose and a 100% increase in sucrose.

[0639] Example 3: Reduction of Acrylamide via CRIPSR-Cas Editing

[0640] Various methods known in the art can be used to assess the acrylamide content in potato products. Liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and Fourier transform-near-infrared spectroscopy (FT-NIR) have all been shown to accurately measure the acrylamide content in potatoes (see Skinner et al., Instrumentation for Routine Analysis of Acrylamide in French Fries: Assessing Limitations for Adoption, Foods, 2021 10(9), 2038).

[0641] Example 4: Producing lighter-colored fried potato products using CRIPSR-Cas editing

[0642] Potato Chip Color Measurement

[0643] After harvesting, the tubers are optionally refrigerated for a period of time. To measure the color of the chips, the tubers are cut longitudinally from the bud tip to the stem tip. Using a mandolin slicer, 4-5 chips (1 mm thick) are made from each half of the tuber, for a total of 8-10 slices. The chips are collected after discarding the first slice from each half. The chip slices are then washed twice in cold water to remove free starch granules and patted dry with paper towels. The chip slices are then fried in peanut oil at 360°F for 2 minutes and 10 seconds in a custom basket. The chips are then crushed to a certain particle size and placed in a measuring instrument. Immediately after frying and cooling, the color of the chips is quantified by reflectance using a Konica Minolta CR410 colorimeter (Konica Minolta, NJ, USA). Readings for L, a, and b in the Hunter Lab color space are obtained. L is relative lightness, a is the chromaticity range between red and green, and b is the chromaticity range between yellow and blue.

[0644] Color results

[0645] Figure 8 Photos of potato chips made from unedited Russet Burbank potatoes after frying for 2 minutes are provided, showing a noticeable darkening effect.

[0646] Figure 9 Photos of French fries produced from unedited plants were provided, showing a similar darkening change after frying, while French fries produced from VINV-edited Russet Burbank potatoes were noticeably lighter in color.

[0647] Figure 10 The color difference in edited potatoes was only noticeable after frying, because edited potatoes showed a similar color at harvest compared to unedited potatoes.

[0648] Figure 11 Similar color differences to those seen in the Russet Burbank variety were also observed in fried Atlantic potato chips; however, Figure 12 The harvested Atlantic variety shows a color similar to that of Rustet Burbank potatoes.

[0649] Figure 13 Edited Atlantic potato chips exhibiting two or three haplotypes were approximately 11% lighter in color after frying than unedited chips. Specifically, two combinations demonstrated this phenotype: knockout of alleles 1, 2, and 3 instead of 4 (Hap1_Hap2_Hap3), and knockout of alleles 2 and 3 instead of 1 and 4.

[0650] Figure 22 The images show potato chips from the edited line E-PED165-7398, grown in a greenhouse at harvest, containing four mutations (E-PED165-7398Hap1, SEQ ID NO: 53; Hap2, SEQ ID NO: 54; Hap3, SEQ ID NO: 55 and Hap4 SEQ ID NO: 56), compared with wild-type and unedited potatoes. Figure 22 As shown, the potato chips from the edited strain E-PED165-7398 quadruple mutant exhibit a significantly lighter color than those from the WT and unedited controls.

[0651] Figure 23The first row shows potato chips from the edited strain E-PED165-7398 quadruple mutant, grown in a greenhouse one month after harvest, compared with wild-type and unedited potatoes; the second row then shows potato chips from the edited strain E-PED165-7398 quadruple mutant, grown in a greenhouse two to three months after harvest, compared with wild-type and unedited potatoes. Figure 23 As shown, the potato chips from the edited strain E-PED165-7398 quadruple mutant exhibit a significantly lighter color than those from the WT and unedited controls.

[0652] Figure 24 The first row displays potato chips of the edited strain E-PED165-7398 quadruple mutant, grown one month after harvest in the field at location BG (Black Gold Farm, Camden, North Carolina); and the second row displays potato chips of the edited strain E-PED165-7398 quadruple mutant, grown one month after harvest in the field at location MR (Mills River Farm, Mills, River, North Carolina). Figure 24 As shown, the potato chips from the edited strain E-PED165-7398 quadruple mutant exhibit a significantly lighter color than those from the WT and unedited controls.

[0653] Figure 25 The first row shows potato chips of the edited strain E-PED165-7398 quadruple mutant, grown in the field at location BG for two to three months after harvest; and the second row shows potato chips of the edited strain E-PED165-7398 quadruple mutant, grown in the field at location MR (Mills River Farm, Mills, River, North Carolina) for two to three months after harvest. Figure 25 As shown, the potato chips from the edited strain E-PED165-7398 quadruple mutant exhibit a significantly lighter color than those from the WT and unedited controls.

[0654] Measure the color after refrigeration

[0655] After refrigerating at approximately 4°C for two, four, eight, sixteen, twenty-four, or thirty-two weeks, measure the color as described above.

[0656] Example 5: Field Trial

[0657] Figure 14Field trial data were provided, using greenhouse-grown microtubers as seed potatoes to initiate field trials at two sites in North Carolina, USA. One trial was conducted at Black Gold Farm (BG) in Camden, North Carolina, and the second trial was conducted at Mills River Farm (MR) in Mills River, North Carolina.

[0658] Table 3 below shows the planting date, harvesting date, and grading date of the micro-tubers.

[0659] Table 3 .

[0660] Place Planting Rice cutting Harvest Classification BG February 29 June 9 (101 DAP) June 10 (102 DAP) June 10 (102 DAP) MR March 19 June 21 June 27 June 28

[0661] In BG, a randomized complete block design experiment was conducted with 4 blocks, 15 plants / plot, and a plant spacing of 1 ft, including Atlantic. Eighteen different genotypes were evaluated, including one WT (wild type), one unedited control, one mini-tuber planted in the fourth year of field cultivation, and 15 genotypes containing different combinations of VINV alleles.

[0662] Tubers were harvested, counted by size, and yield was assessed by size. Specific gravity, hollow heart, soft rot, brown heart, heat necrosis, deformities, growth cracks, sunburn, pointed tips, and nodular protrusions were also evaluated. For each plot, 10 tubers were also assessed for symptoms of Rhizoctonia solani, common scab, and other infections.

[0663] Two tubers were taken from each plot, for a total of eight tubers per genotype, for slicing and color determination. Tubers were either fried at harvest (FRY0) or stored at 4°C for one month (FRY1) or three months (FRY2) before frying. Slicing, frying, and color assessment were performed as described herein.

[0664] At MR, a partially replicated randomized design experiment was conducted using eight commercial varieties: Adirondack Blue, DarkRed Norland, Rustet Burbank, Gold Rush, Kennebec, Superior, Yukon Gold, and Norwis. Each plot contained 15 plants, spaced 1 ft apart. Twenty-four distinct genotypes were evaluated, including one unedited control, one unedited control, and 22 edited genotypes containing different combinations of VINV alleles.

[0665] Tubers were harvested and their total weight, specific gravity, internal and external defects were assessed. Selected genotypes were fried at harvest (FRY0), or after 1 month of refrigeration at 4°C (FRY1) or 3 months of refrigeration (FRY2), and the color of the fried chips was assessed.

[0666] There were a total of 10 genotypes in the BG and MR trials: 1 WT, 1 unedited control, and 8 edits containing different combinations of VINV alleles.

[0667] Field trial results - yield

[0668] Figure 14 The experimental yield results revealed that, in several cases, plants with edits in one or more VINV alleles performed better than plants with wild-type (WT) and / or unedited genotypes. In all cases, single-mutant / single, double-mutant / double, or triple-mutant / triple-edited genotypes yielded more than quadruple-edited genotypes, suggesting that yield may be improved by selectively editing certain VINV alleles rather than knocking out all four alleles.

[0669] Surprisingly, the yields of several edited genotypes appeared to be the same as or better than the unedited control, and the average yields of the double-edited genotypes (2_Hap1_Hap2) and triple-edited genotypes (3_Hap1_Hap2_Hap3) exceeded those of the WT and unedited control.

[0670] potato chip color

[0671] Figure 15 The results of CLS analysis on tubers harvested from field trials were revealed.

[0672] Although no Fry0 data was collected from the BG trial, the quadruple-edited genotype produced the optimal chip color whenever it was tested.

[0673] Furthermore, regardless of when tested (FRY1 and FRY2), tubers with double / double mutant edited genotypes Hap1_Hap4_ or triple / triple mutant edited genotypes Hap1_Hap2_Hap3 unexpectedly performed roughly the same as or better than WT and unedited controls in MR trials.

[0674] proportion

[0675] Figure 21The proportionate weight (SpGr) score of plants harvested from the BG trial was revealed. The following twelve genotypes, containing edits in two or more VINV alleles, exhibited an average SpGr higher than the industry standard of 1.08 expected by most potato processors: 7240, 7300, 7347, 7398, 7413, 7419, 7437, 7458, 7459, 7475, 7590, and 7632. Compared to the wild-type and unedited control, the following seven genotypes, with edits in all four VINV alleles, showed a surprisingly improved average SpGr: 7347, 7398, 7458, 7459, 7475, and 7632. Compared to the unedited control, the following two genotypes, with edits in two 3 VINV alleles, showed a surprisingly improved average SpGr: 7300 and 7590. Plants with double editing include E-PED165-7240=Hap2_Hap3, plants with triple editing include E-PED165-7300=hap1_hap2_hap4 and E-PED165-7590=hap1_hap3_hap4, and all fully edited plants shown in brown are quadruple edited plants, which also show a surprisingly high average SpGr compared to wild type and unedited control.

[0676] Example 6: Greenhouse Experiment

[0677] Atlantic varietal miniature tubers were sown in 2-gallon pots in a greenhouse on August 9, 2023, and grown under artificial light, fertilization, and irrigation. Fertilization was stopped and irrigation was restricted at 80 days post-plant...

Claims

1. A potato plant containing at least one mutation in the VINV allele, wherein the plant exhibits an improved cold storage phenotype and increased yield compared to potato plants lacking said at least one VINV mutation.

2. A potato plant containing at least two mutations in the VINV alleles, wherein the plant exhibits an improved cold storage phenotype and increased yield compared to potato plants lacking said at least two VINV mutations.

3. A potato plant containing at least three mutations in the VINV alleles, wherein the plant exhibits an improved cold storage phenotype and improved yield compared to potato plants lacking said at least three VINV mutations.

4. A modified potato plant, plant part or plant cell containing a modification in at least one VINV allele, wherein the potato chip brightness score of a potato food product derived from said potato plant is at least 10% higher than that of a potato food product derived from a control potato plant.

5. The modified potato plant, plant part or plant cell as described in claim 4, wherein the brightness score of fried potato chips from the potato food product derived from said potato plant is 10% to 25% higher than that of potato food products derived from the control potato plant.

6. A modified potato plant, plant part or plant cell containing a modification in at least one VINV allele, wherein potato food products derived from said potato plant have a fried potato chip brightness score higher than 63.

7. A modified potato plant, plant part or plant cell comprising a modification in at least one VINV allele, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than that of a control plant, or both.

8. A modified potato plant, plant part or plant cell containing the modification in at least two VINV alleles, wherein the potato chip brightness score of a potato food product derived from said potato plant is at least 10% higher than that of a potato food product derived from a control potato plant.

9. The modified potato plant, plant part or plant cell as described in claim 8, wherein the brightness score of fried potato chips from the potato food product derived from said potato plant is 10% to 25% higher than that of potato food products derived from the control potato plant.

10. A modified potato plant, plant part or plant cell containing the modification in at least two VINV alleles, wherein potato food products derived from said potato plant have a fried potato chip brightness score higher than 63.

11. A modified potato plant, plant part or plant cell containing modification in at least two VINV alleles, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than that of a control plant, or both.

12. A modified potato plant, plant part or plant cell containing the modification in at least three VINV alleles, wherein the brightness score of fried potato chips from said potato plant is at least 10% higher than that of potato food products from control potato plants.

13. The modified potato plant, plant part or plant cell as described in claim 12, wherein the brightness score of fried potato chips from the potato food product derived from said potato plant is 10% to 25% higher than that of potato food products derived from the control potato plant.

14. A modified potato plant, plant part or plant cell containing modifications in at least three VINV alleles, wherein potato food products derived from said potato plant have a fried potato chip brightness score higher than 63.

15. A modified potato plant, plant part or plant cell comprising a modification in at least three VINV alleles, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than that of a control plant, or both.

16. A modified potato plant, plant part or plant cell containing modifications in four VINV alleles, wherein the brightness score of fried potato chips from said potato plant is at least 10% higher than that of potato product from control potato plant.

17. The modified potato plant, plant part or plant cell as described in claim 16, wherein the potato chip brightness score of the potato food product derived from said potato plant is 10% to 25% higher than that of the potato food product derived from the control potato plant.

18. A modified potato plant, plant part or plant cell containing modifications in four VINV alleles, wherein potato food products derived from said potato plant have a fried potato chip brightness score higher than 63.

19. A modified potato plant, plant part or plant cell comprising modifications in four VINV alleles, wherein the tuber of the modified potato plant contains at least 10% less glucose and at least 10% less fructose than that of a control plant, or both.

20. The modified potato plant, plant part or plant cell as claimed in any one of claims 4-19, wherein the potato food product is potato chips.

21. The modified potato plant, plant part or plant cell as described in any one of claims 4-19, wherein the potato food product is French fries.

22. A modified potato plant, plant part or plant cell comprising a modification in at least one VINV allele, wherein the VINV allele is selected from the group consisting of Hap1 VINV allele, Hap2 VINV allele, Hap3 VINV allele, Hap4 VINV and Hap5 VINV allele.

23. The modified potato plant, plant part or plant cell as described in any one of claims 1-22, wherein the fried potato chip has a brightness score higher than 65.

24. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-22, wherein at least one of the Hap1 VINV allele, the Hap2 VINV allele, the Hap3 VINV allele, the Hap4 VINV allele or the Hap5 VINV allele contains a mutation.

25. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-22, wherein at least one of the Hap1 VINV allele, the Hap2 VINV allele, the Hap3 VINV allele, the Hap4 VINV allele or the Hap5 VINV allele contains a mutation.

26. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-22, wherein at least one of the Hap1 VINV allele, the Hap2 VINV allele, the Hap3 VINV allele, the Hap4 VINV allele or the Hap5 VINV allele is wild-type.

27. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-22, wherein at least two of the VINV alleles are edited.

28. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-22, wherein at least three of the VINV alleles are edited.

29. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-22, wherein four of the VINV alleles are edited.

30. A modified potato plant, plant part, or plant cell containing a mutation in at least one of the Hap1, Hap2, Hap3, Hap4, and Hap5 VINV alleles, said mutation being generated by a guide endonuclease, and said endonuclease binding to a protospacer sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or with a protospacer sequence selected from SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO:

158. Sequence 157 or SEQ ID NO: 158 having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity.

31. A modified potato plant, plant part or plant cell containing a mutation in at least one or more of the Hap1, Hap2, Hap3, Hap4 and Hap5 VINV alleles, wherein each mutation is generated by a guide endonuclease, and wherein each mutation contains one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO:

169.

32. The modified potato plant, plant part, or plant cell according to any one of claims 1-31, wherein the Hap1 VINV allele comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO:

101. SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 1121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO:

145. SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, SEQ ID NO: 202, SEQ ID NO: 206, SEQ ID NO: 210, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 222 and SEQ ID NO:

226.

33. The modified potato plant, plant part, or plant cell according to any one of claims 1-31, wherein the Hap2 VINV allele comprises a sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO:

102. SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223 and SEQ ID NO:

227.

34. The modified potato plant, plant part, or plant cell according to any one of claims 1-31, wherein the Hap3 VINV allele comprises a sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO:

103. SEQ ID NO: 107, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 216, SEQ ID NO: 220, SEQ ID NO: 224 and SEQ ID NO:

224. ID NO:

228.

35. The modified potato plant, plant part, or plant cell according to any one of claims 1-31, wherein the Hap4 VINV allele comprises a sequence selected from the group consisting of the following SEQ ID NOs: SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 10 ...78, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 99, SEQ ID NO: 9 NO: 104, SEQ ID NO: 108, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225 and SEQ ID NO:

229.

36. The modified potato plant, plant part, or plant cell according to any one of claims 1-31, wherein the Hap5 VINV allele comprises a sequence selected from the group consisting of the following SEQ ID NOs: SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 147, and SEQ ID NO:

148.

37. The modified potato plant, plant part or plant cell as described in any one of claims 1-36, wherein the brightness score of the potato chip is determined before refrigeration.

38. The modified potato plant, plant part or plant cell as described in any one of claims 1-36, wherein the brightness score of the potato chip is determined after refrigeration.

39. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-36, wherein the control potato plant contains genetic modification in four of the Hap1, Hap2, Hap3, Hap4 and Hap5 VINV alleles.

40. The modified potato plant, plant part or plant cell according to any one of claims 1-36, wherein the control potato plant lacks one or more or all of the genetic modifications in the VINV allele.

41. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-36, wherein the control plant is unedited.

42. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-36, wherein the control plant is wild type.

43. The modified potato plant, plant part or plant cell as described in any one of claims 1-36, wherein the control plant is an invalid isolate.

44. A modified potato plant, plant part, or plant cell comprising an edit in four VINV alleles, wherein the edit is generated by a guided endonuclease such that the VINV alleles of the potato plant, plant part, or plant cell comprise four sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

45. A modified potato plant, plant part, or plant cell containing a mutation in at least one VINV allele, wherein the mutation is generated by a guide endonuclease, and wherein the endonuclease binds to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158, or a sequence consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO:

158. 158 sequences having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity.

46. ​​The modified potato plant, plant part or plant cell of claim 45, wherein the potato plant, plant part or plant cell contains mutations in two, three or four VINV alleles.

47. The modified potato plant, plant part, or plant cell as described in claim 45 or 46, wherein the endonuclease is complexed with a guide RNA, the guide RNA comprising a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or a sequence selected from SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, or SEQ ID NO:

168. The group consisting of 168 sequences has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity.

48. A modified potato plant, plant part or plant cell containing mutations in four VINV alleles, wherein each edit is generated by a guided endonuclease, and wherein each edit contains an edit of one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are compared with SEQ ID NO:

169.

49. The modified potato plant, plant part or plant cell as described in any one of claims 30-48, wherein the guiding endonuclease is a Cas protein.

50. The modified potato plant, plant part or plant cell according to any one of claims 30-48, wherein the VINV allele is selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

51. The modified potato plant, plant part or plant cell as claimed in any one of claims 30-48, wherein the potato plant, plant part or plant cell is derived from a breeding line selected from the group consisting of Russet Burbank and Atlantic.

52. The modified potato plant, plant part or plant cell as described in claim 51, wherein the breeding line is Atlantic.

53. The modified potato plant, plant part or plant cell as described in claim 51, wherein the breeding line is Russet Burbank.

54. The modified potato plant, plant part or plant cell as described in any one of claims 30-53, wherein the VINV activity in the potato plant, plant part or plant cell is reduced by at least 50% compared with the control potato plant, plant part or plant cell.

55. The modified potato plant, plant part or plant cell as claimed in claim 54, wherein the VINV activity in the potato plant, plant part or plant cell is reduced by at least 85% compared with the control potato plant, plant part or plant cell.

56. The modified potato plant, plant part or plant cell as claimed in claim 54, wherein the VINV activity in the potato plant, plant part or plant cell is reduced by at least 95% compared with the control potato plant, plant part or plant cell.

57. The modified potato plant, plant part or plant cell as claimed in claim 54, wherein the VINV activity in the potato plant, plant part or plant cell is reduced by at least 99% compared with the control potato plant, plant part or plant cell.

58. The modified potato plant, plant part or plant cell as described in any one of claims 30-57, wherein the plant, plant part or plant cell is non-GMO.

59. The modified potato plant, plant part or plant cell as claimed in any one of claims 30-58, wherein the tuber sugar profile obtained from said plant contains lower levels of glucose, fructose or both compared with the tuber sugar profile obtained from the control plant.

60. The modified potato plant, plant part, or plant cell of claim 59, wherein the tuber glycosylation obtained from the plant contains a lower level of glucose compared to the tuber glycosylation obtained from the control plant.

61. The modified potato plant, plant part or plant cell of claim 59, wherein the tuber sugar profile obtained from the plant contains a lower level of fructose compared with the tuber sugar profile obtained from the control plant.

62. The modified potato plant, plant part or plant cell as claimed in claim 59, wherein the specific gravity spectrum obtained from the plant contains a higher specific gravity than that obtained from the control plant.

63. The modified potato plant, plant part or plant cell as claimed in any one of claims 30-62, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared with the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part or plant cell increases by no more than 200% compared with the tuber sugar profile obtained from the control plant.

64. The modified potato plant, plant part or plant cell of claim 63, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared with the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part or plant cell increases by no more than 100% compared with the tuber sugar profile obtained from the control plant.

65. The modified potato plant, plant part, or plant cell of claim 63, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared to the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 50% compared to the tuber sugar profile obtained from the control plant.

66. The modified potato plant, plant part, or plant cell of claim 63, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared to the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 25% compared to the tuber sugar profile obtained from the control plant.

67. The modified potato plant, plant part or plant cell as described in claims 59-66, wherein the tuber sugar spectrum is obtained by colorimetric determination.

68. The modified potato plant, plant part or plant cell as described in claims 59-66, wherein the tuber glycosylation profile is obtained using high-performance liquid chromatography.

69. The modified potato plant, plant part or plant cell as described in any one of claims 59-68, wherein the tuber glycosylation is obtained at harvest.

70. The modified potato plant, plant part or plant cell as described in any one of claims 59-68, wherein the tuber glycosylation is obtained after refrigeration.

71. The modified potato plant, plant part or plant cell as described in any one of claims 30-70, wherein the acrylamide content after refrigeration is lower than the acrylamide content of the control potato plant, plant part or plant cell.

72. The modified potato plant, plant part or plant cell as claimed in claim 71, wherein the acrylamide content after refrigeration is at least 50% lower than the acrylamide content of the control potato plant, plant part or plant cell.

73. The modified potato plant, plant part or plant cell as claimed in claim 71, wherein the acrylamide content after refrigeration is at least 75% lower than the acrylamide content of the control potato plant, plant part or plant cell.

74. The modified potato plant, plant part or plant cell as claimed in claim 71, wherein the acrylamide content after refrigeration is at least 85% lower than the acrylamide content of the control potato plant, plant part or plant cell.

75. The modified potato plant, plant part or plant cell as claimed in claim 71, wherein the acrylamide content after refrigeration is at least 95% lower than the acrylamide content of the control potato plant, plant part or plant cell.

76. The modified potato plant, plant part or plant cell as claimed in claim 71, wherein the acrylamide content after refrigeration is at least 99% lower than the acrylamide content of the control potato plant, plant part or plant cell.

77. The modified potato plant, plant part or plant cell as described in claims 72-76, wherein the acrylamide content after refrigeration is obtained from potato food products.

78. The modified potato plant, plant part or plant cell as described in any one of claims 30-76, wherein the potato chip brightness score of the potato food product produced from said plant is higher than that of the potato food product produced from the control plant.

79. The modified potato plant, plant part or plant cell as claimed in claim 78, wherein the potato chip brightness rating is determined by colorimetric readings.

80. The modified potato plant, plant part or plant cell as claimed in claim 79, wherein the potato chip brightness score is between 25% and 100% higher than the brightness score of potato chips produced from processed potato products from control plants.

81. The modified potato plant, plant part or plant cell as described in any one of claims 1-80, wherein the control potato plant, plant part or plant cell lacks one or more or all of the mutations, edits, deletions, inversions or duplications in the VINV allele.

82. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-80, wherein the control plant is unedited.

83. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-80, wherein the control plant is wild type.

84. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-80, wherein the control plant is an invalid isolate.

85. The modified potato plant, plant part or plant cell as described in any one of claims 1-84, wherein the control potato plant, plant part or plant cell belongs to the same breeding line as the modified potato plant, plant, plant part or plant cell.

86. The modified potato plant, plant part or plant cell as claimed in any one of claims 1-85, wherein the plant part or plant cell is non-renewable.

87. A processed potato product derived from a modified potato plant, plant part, or plant cell as described in any one of claims 1-86, wherein the processed potato product contains a detectable amount of at least one of the VINV alleles of the modified plant, plant part, or plant cell.

88. The processed potato product of claim 87, wherein the processed potato product is selected from the group consisting of: biomass, oil, meal, edible starch, syrup, sugar, animal feed, flour, flakes, potato chips, French fries, potato wedges, potato cakes, potato balls, baked potatoes, mashed potatoes, dehydrated potatoes, granules, peeled, cooked peels, potato pulp, mashed potatoes, filter cake, sieve residue, potato residue, potato protein isolate or concentrate, discarded French fries, discarded potato chips, scraps, batter, debris, defective pieces, or alcoholic beverages.

89. The processed potato product of claim 88, wherein the processed potato product is potato chips.

90. The processed potato product as claimed in any one of claims 87-89, wherein the processed potato product is non-renewable.

91. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, the guide endonuclease binding together with the prospacer sequence of at least one VINV allele; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains editing in at least one VINV allele such that the at least one VINV allele of the modified potato plant, plant part or plant cell contains at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

92. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, the guide endonuclease binding together with the prospacer sequences of at least two VINV alleles; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains editing in at least two VINV alleles such that the at least two VINV alleles of the modified potato plant, plant part or plant cell contain at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

93. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, the guide endonuclease binding together with the prospacer sequences of at least three VINV alleles; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains editing in at least three VINV alleles such that the at least three VINV alleles of the modified potato plant, plant part or plant cell contain at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

94. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, the guide endonuclease binding together with the protospacer sequences of four VINV alleles; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains edits in four VINV alleles such that the four VINV alleles of the modified potato plant, plant part or plant cell contain at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

95. The method of claims 91-94, wherein the method comprises: The at least one guiding endonuclease is introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts that have edited at least one VINV allele.

96. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: A guiding endonuclease is introduced into potato cells, said guiding endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 and SEQ ID NO: 158, or a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158; and Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains a mutation in at least one VINV allele.

97. The method of claim 96, wherein the method comprises: The guiding endonuclease was introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts that have a mutation in at least one VINV allele.

98. The method of claim 96 or 97, wherein the modified potato plant, plant, part or plant cell contains editing of at least one VINV allele.

99. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: A guiding endonuclease is introduced into potato cells, said guiding endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 and SEQ ID NO: 158, or a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158; and Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains mutations in at least two VINV alleles.

100. The method of claim 99, wherein the method comprises: The guiding endonuclease was introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts that have mutations in at least two VINV alleles.

101. The method of claim 99 or 100, wherein the modified potato plant, plant, part or plant cell contains editing of at least two VINV alleles.

102. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: A guiding endonuclease is introduced into potato cells, said guiding endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 and SEQ ID NO: 158, or a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158; and Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains mutations in at least three VINV alleles.

103. The method of claim 102, wherein the method comprises: The guiding endonuclease was introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts that have mutations in at least three VINV alleles.

104. The method of claim 102 or 103, wherein the modified potato plant, plant, part or plant cell contains editing of at least three VINV alleles.

105. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: A guiding endonuclease is introduced into potato cells, said guiding endonuclease binding to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 and SEQ ID NO: 158, or a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158; and Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contained mutations in four VINV alleles.

106. The method of claim 105, wherein the method comprises: The guiding endonuclease was introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts that have mutations in the four VINV alleles.

107. The method of claim 105 or 106, wherein the modified potato plant, plant, part or plant cell contains editing in four VINV alleles.

108. The method of any one of claims 91-106, wherein the endonuclease is complexed with guide RNA, the guide RNA comprising a sequence of the following SEQ ID NO: selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, or having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 or SEQ ID NO:

168.

109. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, the guide endonuclease binding to the prospacer sequence of at least one VINV allele; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains a mutation in at least one VINV allele such that when the modified VINV allele is compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

110. The method of claim 109, wherein the method comprises: The at least one guiding endonuclease is introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts the following: when the modified VINV allele is compared with SEQ ID NO: 169, each VINV allele has a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

111. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, said guide endonuclease binding to the prospacer sequences of at least two VINV alleles; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains mutations in at least two VINV alleles such that when the modified VINV alleles are compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

112. The method of claim 111, wherein the method comprises: The at least one guiding endonuclease is introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts the following: when the modified VINV allele is compared with SEQ ID NO: 169, each VINV allele has a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

113. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, said guide endonuclease binding to the protospacer sequences of at least three VINV alleles; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains mutations in at least three VINV alleles such that when the modified VINV alleles are compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

114. The method of claim 113, wherein the method comprises: The at least one guiding endonuclease is introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts the following: when the modified VINV allele is compared with SEQ ID NO: 169, each VINV allele has a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

115. A method for producing modified potato plants, plant parts, or plant cells, the method comprising: At least one guide endonuclease is introduced into potato cells, the guide endonuclease binding together with the protospacer sequences of four VINV alleles; as well as Modified potato plants, plants, or parts or cells of plants can be regenerated from the potato cells. The modified potato plant, plant, part or plant cell contains mutations in four VINV alleles such that when the modified VINV alleles are compared with SEQ ID NO: 169, each edit contains a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

116. The method of claim 115, wherein the method comprises: The at least one guiding endonuclease is introduced into multiple potato cells; Multiple potato plants or plant parts are regenerated from the multiple potato cells; as well as Select from the plurality of regenerated potato plants or plant parts the following: when the modified VINV allele is compared with SEQ ID NO: 169, each VINV allele has a mutation of one or more nucleotides corresponding to the edit window of SEQ ID NO:

169.

117. The method of any one of claims 91-116, wherein the guiding endonuclease is a Cas protein.

118. The method of any one of claims 91-116, wherein the at least one VINV allele is selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

119. The method of any one of claims 91-116, wherein the at least two VINV alleles are selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

120. The method of any one of claims 91-116, wherein the at least three VINV alleles are selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

121. The method of any one of claims 91-116, wherein the four VINV alleles are selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

122. The method of any one of claims 91-116, wherein the endonuclease binds to a protospacer sequence selected from the group consisting of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 and SEQ ID NO: 158, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO:

158.

123. The method of any one of claims 91-116, wherein the endonuclease is complexed with a guide RNA, the guide RNA comprising a sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, or SEQ ID NO:

168.

124. The method of any one of claims 91-116, wherein the potato plant, plant part or plant cell is derived from a breeding line selected from the group consisting of Russet Burbank and Atlantic.

125. The method of claim 124, wherein the breeding line is Atlantic.

126. The method of claim 124, wherein the breeding line is Russet Burbank.

127. The method of any one of claims 91-124, wherein the VINV activity in the modified potato plant, plant part or plant cell is reduced by at least 50% compared with that in the control potato plant, plant part or plant cell.

128. The method of claim 127, wherein the VINV activity in the modified potato plant, plant part or plant cell is reduced by at least 85% compared with that in a control potato plant, plant part or plant cell.

129. The method of claim 127, wherein the VINV activity in the modified potato plant, plant part or plant cell is reduced by at least 95% compared with that in the control potato plant, plant part or plant cell.

130. The method of claim 127, wherein the VINV activity in the modified potato plant, plant part, or plant cell is reduced by at least 99% compared to the control potato plant, plant part, or plant cell.

131. The method of any one of claims 91-130, wherein the plant, plant part or plant cell is non-GMO.

132. The method of any one of claims 91-130, wherein the tuber sugar profile obtained from said plant contains lower levels of glucose, fructose, or both compared to the tuber sugar profile obtained from the control plant.

133. The method of claim 132, wherein the tuber glycosylation obtained from the plant contains a lower level of glucose compared to the tuber glycosylation obtained from the control plant.

134. The method of claim 132, wherein the tuber sugar profile obtained from the plant contains a lower level of fructose compared to the tuber sugar profile obtained from the control plant.

135. The method of any one of claims 91-134, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared to the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part or plant cell increases by no more than 200% compared to the tuber sugar profile obtained from the control plant.

136. The method of claim 135, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared to the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 100% compared to the tuber sugar profile obtained from the control plant.

137. The method of claim 135, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared to the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 50% compared to the tuber sugar profile obtained from the control plant.

138. The method of claim 135, wherein the tuber sugar profile obtained from the plant contains a higher level of sucrose compared to the tuber sugar profile obtained from the control plant, wherein the percentage of sucrose in the modified potato plant, plant part, or plant cell increases by no more than 25% compared to the tuber sugar profile obtained from the control plant.

139. The method of claims 132-138, wherein the tuber sugar spectrum is obtained using colorimetric determination.

140. The method of claims 132-138, wherein the tuber glycosylation profile is obtained using high-performance liquid chromatography.

141. The method of any one of claims 132-140, wherein the tuber glycosylation is obtained at harvest.

142. The method of any one of claims 132-140, wherein the tuber glycosylation is obtained after refrigeration.

143. The method of any one of claims 91-142, wherein the specific gravity spectrum obtained from the plant contains a higher specific gravity than that obtained from the control plant.

144. The method according to any one of claims 91-140, wherein the acrylamide content after refrigeration is lower than the acrylamide content in the control potato plants, plant parts or plant cells.

145. The method of claim 144, wherein the acrylamide content after refrigeration is at least 50% lower than the acrylamide content of the control potato plant, plant part or plant cell.

146. The method of claim 144, wherein the acrylamide content after refrigeration is at least 75% lower than the acrylamide content of the control potato plant, plant part or plant cell.

147. The method of claim 144, wherein the acrylamide content after refrigeration is at least 85% lower than the acrylamide content of the control potato plant, plant part or plant cell.

148. The method of claim 144, wherein the acrylamide content after refrigeration is at least 99% lower than the acrylamide content of the control potato plant, plant part or plant cell.

149. The method of claims 144-149, wherein the refrigerated acrylamide content is obtained from potato food products.

150. The method of any one of claims 91-149, wherein the potato chip brightness score of the potato food product produced by said plant is higher than that of the potato food product produced by the control plant.

151. The method of claim 150, wherein the potato chip brightness rating is determined by colorimetric readings.

152. The method of claim 150 or 151, wherein the potato chip brightness score is between 25% and 100% higher than the potato chip brightness score of processed potato products produced from control plants.

153. The method of any one of claims 91-152, wherein the control potato plant, plant part or plant cell lacks one or more or all of the deletions, inversions or duplications in the VINV alleles.

154. The method of any one of claims 91-152, wherein the control plant is unedited.

155. The method of any one of claims 91-152, wherein the control plant is wild-type.

156. The method of any one of claims 91-152, wherein the control plant is an invalid isolate.

157. The method of any one of claims 91-152, wherein the control potato plant, plant part or plant cell belongs to the same breeding line as the modified potato plant, plant, part or plant cell.

158. The method of any one of claims 91-152, wherein the plant part or plant cell is non-renewable.

159. A potato genome, characterized in that... The mutation is contained in at least one VINV allele, which contains at least one sequence selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

160. A potato genome, characterized in that... The mutation is contained in at least two VINV alleles, which contain at least two sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

161. A potato genome, characterized in that... The mutation is contained in at least three VINV alleles, which contain at least three sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

162. A potato genome, characterized in that... The mutation is contained in four VINV alleles, which contain four sequences selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

163. A potato genome, characterized in that... The VINV allele contains a mutation, and each mutation contains one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV allele is compared with SEQ ID NO:

169.

164. A potato genome, characterized in that... Mutations are contained in at least two VINV alleles, and each mutation contains one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are compared with SEQ ID NO:

169.

165. A potato genome, characterized in that... The mutation is contained in at least three VINV alleles, and each mutation contains one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are compared with SEQ ID NO:

169.

166. A potato genome, characterized in that... Mutations are contained in the four VINV alleles, and each mutation contains one or more nucleotides corresponding to the edit window of SEQ ID NO: 169 when the modified VINV alleles are compared with SEQ ID NO:

169.

167. The potato genome as claimed in claims 159-166, wherein the potato genome is modified.

168. The potato genome as claimed in any one of claims 159-166, wherein the potato genome is not present in viable nonmicrobial cells.

169. The potato genome according to any one of claims 159-166, wherein the VINV allele is selected from the group consisting of SEQ ID NO: 1-148 and SEQ ID NO: 170-229.

170. A potato product comprising the potato genome as described in any one of claims 159-169.

171. A guide RNA comprising a targeting sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 or SEQ ID NO:

168.

172. A guide RNA comprising a target sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with 20 consecutive nucleotides of SEQ ID NO:

169.

173. A recombinant DNA construct comprising a first expression cassette, the first expression cassette comprising a first DNA sequence encoding a guide RNA as claimed in claim 168 or claim 169.

174. The recombinant DNA construct of claim 173, wherein the first DNA sequence is operatively linked to a first plant-expressible promoter.

175. The recombinant DNA construct of claim 174, further comprising an expression cassette containing a second DNA sequence encoding a guide endonuclease, wherein the second DNA sequence is operatively linked to a second plant-expressible promoter.

176. The recombinant DNA construct of claim 175, wherein the guiding endonuclease is a Cas protein.

177. A vector comprising a recombinant DNA construct as described in any one of claims 173-176.

178. A host cell comprising the vector as described in claim 177.

179. The host cell of claim 178, wherein the host cell is a bacterial cell.

180. The host cell of claim 179, wherein the bacterial cell is an Agrobacterium cell.

181. The host cell of claim 180, wherein the host cell is a plant cell.

182. A composition comprising the guide RNA as described in claim 171 or 172, conjugated with a guiding endonuclease.

183. The composition of claim 182, wherein the guiding endonuclease is a Cas protein.

184. A kit for producing modified potato plants, plant parts or plant cells, the kit comprising one or more of the following: a guide RNA as claimed in claim 171 or 172, a recombinant DNA construct as claimed in any one of claims 173-176, a vector as claimed in claim 177, a host cell as claimed in any one of claims 178-181, and a composition as claimed in claim 182 or 183.

185. The kit of claim 184, further comprising instructions for use of the guide RNA, the recombinant DNA construct, the vector, the host cell, the composition, or a combination thereof to introduce one or more guide endonucleases into potato cells, the one or more guide endonucleases together binding to the original spacer sequence of one or more VINV alleles, two or more VINV alleles, three or more VINV alleles, or each of four VINV alleles.

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