Plants with improved digestibility and marker haplotype

Abstract

The present invention relates to plants, in particular maize, having improved digestibility, in particular improved stalk digestibility. The present invention relates to QTL alleles associated with improved digestibility, as well as specific marker alleles associated with the QTL alleles. The present invention also relates to plants in which the F35H gene is mutated or in which F35H expression is reduced or absent. The present invention also relates to methods for identifying plants having improved digestibility as well as methods for obtaining such plants.

Description

Technical Field

[0001] This invention relates to quantitative trait loci (QTLs) and associated markers that relate to and / or are associated with increased digestibility of plants and plant parts (such as maize). The invention also relates to the use of such QTLs or markers for identification and / or selection purposes, and to transgenic or non-transgenic plants. Background Technology

[0002] Maize (Zea mays L.) is the world's most important annual forage crop. Primarily in Northern Europe, over 3 million hectares of maize are ensiled annually. Due to its high energy content and feed conversion efficiency, forage maize is a crucial food crop for dairy and beef cattle, significantly influencing milk and meat production. Extensive genetic variation exists in the forage characteristics of both the entire maize plant and its stalks (Geiger et al., 1992; Barrière et al., 2003).

[0003] Therefore, improving digestibility is a primary goal of forage maize breeding programs. The energy supplied from forage to the diets of ruminants or herbivores is related to forage intake and digestibility. The digestibility of any forage component (dry matter, organic matter, or cell walls) is measured as the percentage of silage absorbed by the animal's digestive tract (Barrière et al., 2003). The overall digestibility of forage maize is influenced by highly digestible cereal and straw fractions. Straw composition and digestibility limit the quality of forage maize. The main straw fractions are hemicellulose, cellulose, and lignin. Modern forage maize cultivars combine high dry matter yield with high straw digestibility.

[0004] Especially when conducting large-scale evaluations of germplasm in plant breeding programs, performing digestibility measurements on animals is too costly. Biological and chemical methods have been developed for determining the digestibility of maize and other forage crops (Van Soest et al., 1963). Neutral detergent fiber (NDF), as a residue after the removal of soluble cell contents, is an important indicator of plant cell wall and cellulose. In vitro NDF digestibility (IVNDFD) of forage crops is an estimate of cell wall digestibility—assuming that the non-NDF portion of the plant material is completely digestible (Méchin et al., 2000). Furthermore, digestibility traits of various forage crops, including maize, have been reported to be accurately measured using NIRS (Lübberstedt et al., 1997a, b; Zimmer et al., 1990).

[0005] Lübberstedt et al. (1997a,b) first published QTLs related to agronomic and quality traits in forage maize, as well as QTLs for whole-plant digestibility. Marker-assisted selection (MAS) utilizing genetic variation available for straw digestibility appears to be a promising approach to improving forage digestibility. Besides genetic variation, environmental variation may also be a cause of those heterogeneous traits. QTL analysis of forage traits in four different maize populations revealed that only a few QTLs showed epistatic interactions or interactions with the environment (Lübberstedt et al., 1998). Seven QTLs for DNDF were detected and two major QTLs were identified through self-value experiments using 242 RILs derived from the F838 × F286 cross under six environments (Barrière et al., 2010). Additional QTL analyses were performed using RIL progeny derived from crosses between the old dentate and modern Iodent strains, and novel QTLs in boxes 2.06 and 5.04 of ADL / NDF and DNDF were reported for the first time (Barrière et al., 2012).

[0006] WO 2019 / 206927 describes the QTL for digestibility in maize plants, the identification of the F35H gene linked to and responsible for the QTL for plant digestibility, and the description of a unique marker haplotype for enhanced digestibility. An insertion of approximately 187 nucleotides within F35H was found to cause reduced enzyme activity, resulting in enhanced digestibility. However, surprisingly, the inventors of this application found that the insertion behaves similarly to a transposon. Transposons are known to change their position within the genome, thus limiting the stability of such insertions. Therefore, the reliable use of this trait cannot be fully guaranteed.

[0007] Therefore, one object of the present invention is to overcome one or more shortcomings of the prior art. There has always been a need to improve the digestibility of forage crops, and to identify plants (including specific plant parts or derivatives) with increased digestibility. Specifically, one object of the present invention is to provide novel and stable major QTL alleles for digestibility, and to provide markers that allow for the economical use of these QTLs in maize development and breeding. Summary of the Invention

[0008] This invention is based on the identification of the major QTLs of plant digestibility, the identification of a novel F35H allele linked to and responsible for the QTLs of plant digestibility, and the description of unique marker haplotypes for improving digestibility.

[0009] Molecular markers associated with plant digestibility have been identified, and marker alleles associated with improved digestibility have been described.

[0010] The inventors have discovered that insertions in alleles previously described in WO 2019 / 206927 can behave like transposons and may be lost, and that excision may lead to the loss of knockout mutations that are the cause of the improved digestibility trait. In fact, in the inventors' analysis of the presence of SILO-09-02 (the QTL allele for improved digestibility described in WO2019 / 206927) in 1720 double haploid (DH) lines from 42 populations, excision occurred in approximately 2% of all lines carrying the QTL allele. This demonstrates an unacceptably high risk of losing this trait.

[0011] The inventors have developed another QTL with a more advantageous type of insertion (7-8 base pairs), which is much more stable than the first QTL explored in WO2019 / 206927 and simultaneously shows advantageously no yield loss. The novel insertion within the F35H gene has now been described, and markers for its detection have been developed. Furthermore, for the use of the new QTL in maize breeding in dent and durum maize libraries, the present invention provides a comprehensive set of claims, thereby allowing the transformation of any maize breeding library with the newly developed QTL. Therefore, the markers of the present invention are uniquely suitable for detecting high digestibility phenotypes in many different maize lines / libraries / haplotypes.

[0012] This invention relates in particular to methods for detecting identified QTL alleles associated with improved digestibility, and methods for detecting any described marker alleles. The invention also relates to the described marker alleles and polynucleotides, such as primers and probes, that can be used to detect marker alleles, and kits containing them. The invention further relates to methods for improving plant digestibility, particularly by naturally or artificially introducing the marker alleles described herein into plants and / or selecting plants containing the marker alleles described herein, such as, in particular, inducing F35H mutations, preferably mutations that alter F35H expression or F35H enzyme activity, for example, by reducing or eliminating F35H expression or F35H activity or otherwise reducing or increasing F35H expression or F35H activity. The invention also relates to plants with improved digestibility, and plant parts with improved digestibility, particularly straw, such as seeds deposited with NCIMB accession number NCIMB 43997.

[0013] The present invention is specifically embodied by one or more of the following numbered statements [1] to

[91] and any or any combination of any other statement and / or embodiment.

[0014] [1] A method for identifying maize plants or plant parts, comprising screening for the presence of polynucleotides containing the (molecular) marker (allele) ma61134d15 and / or ma61134d16 in the (genome) of maize plants or plant parts; wherein ma61134d15 is an 8-nucleotide insertion between positions 134254381 and 134254382 on chromosome 9 of reference strain PH207 or at position 76.19 cM on chromosome 9 of reference strain PH207, and / or detectable by the molecular marker SEQ ID NO:125; preferably the insertion as shown in SEQ ID NO:1, and ma61134d16 is a 7-nucleotide insertion between positions 134254381 and 134254382 on chromosome 9 of reference strain PH207 or at position 76.19 cM on chromosome 9 of reference strain PH207, and / or detectable by the molecular marker SEQ ID NO:125; preferably the insertion as shown in SEQ ID NO:1, and ma61134d16 is a 7-nucleotide insertion between positions 134254381 and 134254382 on chromosome 9 of reference strain PH207, and / or detectable by the molecular marker SEQ ID NO:125. The molecular marker NO:124 was detected; preferably, the insertion is as shown in SEQ ID NO:4.

[0015] [2] According to the method described in statement [1], the polynucleotide is contained in or is contained in a QTL (allele) that is particularly associated with improved digestibility on chromosome 9 and contains and / or is flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or is contained in a polynucleotide or QTL (allele) containing and / or flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01; wherein SYN38529 is a single nucleotide polymorphism (SNP) at position 56.38 cM on chromosome 9 of the reference strain PH207, and / or can be identified by SEQ ID. The molecular marker NO:13 was detected; PZE-109103504 is a single nucleotide polymorphism (SNP) at position 96.06 cM on chromosome 9 of the reference strain PH207, and / or can be detected by the molecular marker SEQ ID NO:195; PZE-109076467 is a single nucleotide polymorphism (SNP) at position 75.85 cM on chromosome 9 of the reference strain PH207, wherein the nucleotide is A or C, and / or can be detected by the molecular marker SEQ ID NO:109; and ma61161s01 is a single nucleotide polymorphism (SNP) at position 77.04 cM on chromosome 9 of the reference strain PH207, wherein the nucleotide is A or G, and / or can be detected by the molecular marker SEQ ID NO:154.

[0016] [3] A method for identifying maize plants or plant parts, comprising screening for the presence of a QTL (allele) specifically associated with improved digestibility on chromosome 9 and containing and / or flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or contained in a polynucleotide or QTL (allele) containing and / or flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01; wherein SYN38529 is a single nucleotide polymorphism (SNP) at position 56.38 cM on chromosome 9 of the reference strain PH207, and / or can be identified by SEQ ID NO. The molecular marker NO:13 was detected; PZE-109103504 is a single nucleotide polymorphism (SNP) at position 96.06 cM on chromosome 9 of the reference strain PH207, and / or can be detected by the molecular marker SEQ ID NO:195; PZE-109076467 is a single nucleotide polymorphism (SNP) at position 75.85 cM on chromosome 9 of the reference strain PH207, wherein the nucleotide is A or C, and / or can be detected by the molecular marker SEQ ID NO:109; and ma61161s01 is a single nucleotide polymorphism (SNP) at position 77.04 cM on chromosome 9 of the reference strain PH207, wherein the nucleotide is A or G, and / or can be detected by the molecular marker SEQ ID NO:154.

[0017] [4] According to the method described in statement [3], the QTL (allele) comprises (molecular) markers (alleles) ma61134d15 and / or ma61134d16; wherein ma61134d15 is an 8-nucleotide insertion between positions 134254381 and 134254382 on chromosome 9 of reference strain PH207 or at position 76.19 cM on chromosome 9 of reference strain PH207, and / or can be detected by the molecular marker of SEQ ID NO:125; preferably as shown in SEQ ID NO:1, and ma61134d16 is a 7-nucleotide insertion between positions 134254381 and 134254382 on chromosome 9 of reference strain PH207 or at position 76.19 cM on chromosome 9 of reference strain PH207, and / or can be detected by the molecular marker of SEQ ID NO:124; preferably as shown in SEQ ID NO:1. The insertion shown in NO:4.

[0018] [5] The method according to any one of the statements [1] to [4] includes screening for the presence of one or more (molecular) markers (alleles) selected from Table A.

[0019] [6] The method according to any one of the statements [1] to [4] includes screening for the presence of one or more (molecular) markers (alleles) selected from Table B.

[0020] [7] The method according to any one of the statements [1] to [4] includes screening for the presence of one or more (molecular) markers (alleles) selected from Table C.

[0021] [8] The method according to statement [1] or [2], wherein the polynucleotide comprises one or more (molecular) markers (allelices) as defined in any one of statements [5] to [7].

[0022] [9] The method described in claim [3] or [4], wherein the QTL (allele) comprises one or more (molecular) markers (alleles) as defined in any one of claims [5] to [7].

[0023]

[10] A method for identifying a maize plant or plant part comprising screening or detecting the presence of one or more (molecular) markers (alleles) as defined in statement [5] in the maize plant or plant part (the genome).

[0024]

[11] According to the method described in statement

[10] , the marker is contained in a QTL on chromosome 9, particularly associated with improved digestibility, the QTL being side-linked and / or containing the (molecular) markers (alleles) SYN38529 and PZE-109103504; wherein SYN38529 is a single nucleotide polymorphism (SNP) at position 56.38 cM on chromosome 9 of the reference strain PH207, and / or can be detected by the molecular marker of SEQ ID NO:13; and PZE-109103504 is a single nucleotide polymorphism (SNP) at position 96.06 cM on chromosome 9 of the reference strain PH207, and / or can be detected by the molecular marker of SEQ ID NO:195.

[0025]

[12] A method for identifying a maize plant or plant part, comprising screening for the presence of polynucleotides in the genome of the maize plant or plant part containing sequences selected from:

[0026] a) The nucleotide sequence of SEQ ID NO:7;

[0027] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0028] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0029] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0030] The nucleotide sequence wherein the nucleotide sequence has one or more nucleotides at the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or at the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides.

[0031]

[13] According to the method described in statement

[12] , the insertion of 8 nucleotides has a sequence of gcggttct, or the insertion of 7 nucleotides has a sequence of gcggtct.

[0032]

[14] The method according to statement

[12] or

[13] , wherein the nucleotide sequence is selected from:

[0033] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0034] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0035] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0036]

[15] A method for identifying maize plants or plant parts, comprising screening for the presence of polynucleotides in the genome of maize plants or plant parts containing sequences selected from:

[0037] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0038] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0039] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0040]

[16] The method according to any one of the statements [1] to

[15] is a method for identifying plants or plant parts with improved digestibility.

[0041]

[17] The method according to any one of the statements [1] to

[16] is a method for identifying plants or plant parts having improved straw digestibility.

[0042]

[18] The method according to statement

[16] or

[17] , wherein the digestibility is improved compared to that of maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, the QTL (allele), or the one or more (molecular) markers (alleles) mentioned above.

[0043]

[19] The method according to statement

[13] , wherein the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0044]

[20] The method according to any one of statements [1] to

[19] includes isolating genomic DNA from said plant or plant parts.

[0045]

[21] The method according to any one of statements [1] to

[20] includes selecting a plant or plant part containing one or more of the polynucleotide, the (molecular) marker (allele), or the QTL (allele).

[0046]

[22] The method according to any one of statements [1] to

[21] , wherein the plant part is not a propagation material.

[0047]

[23] The method according to any one of statements [1] to

[22] , wherein the plant part is straw.

[0048]

[24] The method according to any one of statements [1] to

[23] , wherein the plant or plant part comprises a polynucleotide having a sequence selected from:

[0049] a) The nucleotide sequence of SEQ ID NO:7;

[0050] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0051] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0052] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0053] Wherein the nucleotide sequences described in a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 in SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0054] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0055] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0056] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0057]

[25] The method according to any one of statements [1] to

[24] , wherein if one or more of the polynucleotide, the (molecular) marker (allele), or the QTL (allele) is present in the genome of the plant or plant part, the plant or plant part is identified as having improved digestibility.

[0058]

[26] The method according to any one of statements [1] to

[25] , wherein if one or more of the polynucleotide, the (molecular) marker alleles, or the QTL (allele) is present in the genome of the plant or plant part, the plant or plant part is identified as having improved straw digestibility.

[0059]

[27] A maize plant or plant part comprising one or more (molecular) markers (alleles) as defined in Table A.

[0060]

[28] A maize plant or plant part comprising one or more (molecular) markers (alleles) as defined in Table B.

[0061]

[29] A maize plant or plant part comprising one or more (molecular) markers (alleles) as defined in Table C.

[0062]

[30] A maize plant or plant part comprising the polynucleotide or the QTL (allele) as defined in any one of statements [1] to [4], [8], [9] or

[12] to

[15] .

[0063]

[31] A plant or plant part according to any one of the statements

[27] to

[30] , wherein the plant or plant part is derived from a plant containing the polynucleotide, the (molecular) marker (allele) or the QTL (allele) obtained by introduction or introgression.

[0064]

[32] A plant or plant part according to any one of the statements

[27] to

[30] , wherein the plant or plant part is obtained by mutagenesis mediated by transposons or transposable factors.

[0065]

[33] A plant or plant part according to any one of the statements

[27] to

[30] , wherein the plant or plant part is genetically modified or gene-edited.

[0066]

[34] A method for generating or producing maize plants or plant parts and / or for improving (straw) digestibility, comprising introducing into the genome of the maize plant or plant part the polynucleotide, one or more (molecular) markers (alleles), or the QTL (allele) as defined in any one of the statements [1] to [7].

[0067]

[35] A method for generating or producing maize plants or plant parts and / or for improving (straw) digestibility, comprising introducing a polynucleotide having a sequence selected from the genome of the maize plant or plant part:

[0068] a) The nucleotide sequence of SEQ ID NO:7;

[0069] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0070] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0071] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0072] Wherein the nucleotide sequences described in a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 in SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0073] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions corresponding to positions 103-109 of SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions corresponding to positions 103-110 of SEQ ID NO:1;

[0074] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0075] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0076]

[36] The method described in statement

[34] or

[35] , wherein the introduction into the genome comprises introgression.

[0077]

[37] The method according to any one of statements

[34] to

[36] comprises (a) providing a first maize plant having the polynucleotide, one or more (molecular) markers (alleles), or the QTL (allele) as defined in any one of statements 1 to 9, or a first maize plant (or its offspring) obtained from maize seeds such as those deposited with NCIMB accession number NCIMB 43997, (b) hybridizing the first maize plant with a second maize plant, and (c) selecting offspring plants having the polynucleotide, one or more (molecular) markers (alleles), or the QTL (allele) as defined in any one of statements [1] to [9].

[0078]

[38] The method described in statement

[37] further includes (d) harvesting plant portions from the offspring.

[0079]

[39] The method described in statement

[34] or

[35] , wherein the introduction into the genome comprises mutagenesis mediated by transposons or transposon factors.

[0080]

[40] The method described in statement

[34] or

[35] , wherein the introduction into the genome comprises transgene or gene editing.

[0081]

[41] The method according to any one of the statements

[34] to

[40] , wherein the plant part is a plant cell, tissue, organ or seed.

[0082]

[42] The method according to any one of the statements

[34] to

[41] , wherein the plant part is an immature or mature embryo, inflorescence, protoplast, leaf, straw material, root material, rachis, seed, grain or callus.

[0083]

[43] The method according to any one of the statements

[34] ,

[35] , or

[39] to

[42] includes transforming a plant or plant part, preferably plant cells, more preferably immature or mature embryos, inflorescences, protoplasts or callus, with the polynucleotide, one or more (molecular) markers (alleles), or the QTL (allele), and optionally regenerating a plant from the plant cells, preferably immature or mature embryos, inflorescences, protoplasts or callus.

[0084]

[44] The method according to any one of the statements

[34] to

[43] is a method for improving the digestibility of plants or plant parts.

[0085]

[45] The method according to any one of the statements

[34] to

[44] is a method for improving straw digestibility.

[0086]

[46] The method according to any one of the statements

[34] to

[45] , wherein the plant part is straw.

[0087]

[47] A maize plant or plant part that can be obtained by any one of the methods described in any one of statements

[34] to

[46] .

[0088]

[48] ​​The method, plant or plant part according to any one of the statements [1] to

[47] , wherein the polynucleotide, the one or more (molecular) markers (alleles), or the QTL (allele) is homozygous.

[0089]

[49] The method, plant or plant part according to any one of the statements [1] to

[47] , wherein the polynucleotide, the one or more (molecular) markers (alleles), or the QTL (allele) is heterozygous.

[0090]

[50] Use of one or more molecular markers as defined in statement [5] for the identification or selection of maize plants or plant parts.

[0091]

[51] Use of one or more molecular markers as defined in statement [6] for the identification or selection of maize plants or plant parts.

[0092]

[52] Use of one or more molecular markers as defined in statement [7] for the identification or selection of maize plants or plant parts.

[0093]

[53] The use according to any one of the statements

[50] to

[52] is for identifying or selecting maize plants or plant parts with improved digestibility.

[0094]

[54] The use according to any one of the statements

[50] to

[53] is for identifying or selecting maize plants or plant parts with improved straw digestibility.

[0095]

[55] The use according to any one of the statements

[50] to

[54] , wherein the digestibility is improved compared to that of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, the QTL (allele), or the one or more (molecular) markers (alleles) mentioned above.

[0096]

[56] For use according to any one of the statements

[50] to

[55] , wherein the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, the QTL (allele), or the one or more (molecular) markers (alleles) mentioned above.

[0097]

[57] For any of the uses described in any of the statements

[50] to

[56] , the plant part is straw.

[0098]

[58] The use of the polynucleotide or QTL (allele) as defined in any one of statements 1 to 9 for the generation or production of maize plants or plant parts.

[0099]

[59] As stated in

[58] , it is used to generate or produce maize plants or plant parts with improved digestibility.

[0100]

[60] As stated in

[58] or

[59] , it is used to generate or produce maize plants or plant parts with improved straw digestibility.

[0101]

[61] According to the use described in statement

[59] or

[60] , the digestibility is improved compared to that of maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, the QTL (allele), or the one or more (molecular) markers (alleles) mentioned above.

[0102]

[62] For use according to any one of the statements

[59] to

[61] , wherein the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, the QTL (allele), or the one or more (molecular) markers (alleles) mentioned above.

[0103]

[63] For any of the uses described in any of the statements

[58] to

[62] , the plant part is a seed or straw.

[0104]

[64] A (isolated) polynucleotide comprising a polynucleotide having a sequence selected from:

[0105] a) The nucleotide sequence of SEQ ID NO:7;

[0106] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0107] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0108] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0109] Wherein the nucleotide sequences described in a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 in SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0110] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions corresponding to positions 103-109 of SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions corresponding to positions 103-110 of SEQ ID NO:1;

[0111] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0112] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0113]

[65] The use of the (isolated) polynucleotides described in statement

[64] for the generation or production of maize plants or plant parts.

[0114]

[66] As stated in

[65] , it is used to generate or produce maize plants or plant parts with improved digestibility.

[0115]

[67] As stated in

[65] or

[66] , it is used to generate or produce maize plants or plant parts with improved straw digestibility.

[0116]

[68] As described in statement

[66] or

[67] , the digestibility is improved compared to that of maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, the QTL (allele), or the one or more (molecular) markers (alleles) mentioned above.

[0117]

[69] For use according to any one of the statements

[66] to

[68] , wherein the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, the QTL (allele), or the one or more (molecular) markers (alleles) mentioned above.

[0118]

[70] For any of the uses described in any of the statements

[65] to

[69] , the plant part is straw.

[0119]

[71] A (isolated) polynucleotide comprising a (molecular) marker (allele) as defined in statement [5], its complementary or reverse complementary sequence, or a fragment thereof.

[0120]

[72] A (isolated) polynucleotide comprising a (molecular) marker (allelic) as defined in statement [6], its complementary or reverse complementary sequence, or a fragment thereof.

[0121]

[73] A (isolated) polynucleotide comprising a (molecular) marker (allelic) as defined in statement [7], its complementary or reverse complementary sequence, or a fragment thereof.

[0122]

[74] The (isolated) polynucleotide according to any one of statements

[71] to

[73] comprises 10 to 500 nucleotides, preferably 15 to 250 nucleotides, more preferably 18 to 250 nucleotides, and most preferably 20 to 250 nucleotides.

[0123]

[75] The (isolated) polynucleotides according to any one of statements

[71] to

[74] are particularly suitable as molecular markers, comprising at least 15, preferably at least 18, more preferably at least 20 consecutive nucleotides of any one of SEQ ID NO: 13 to 195, or complementary to, or anticomplemental to, consecutive nucleotides of any one of SEQ ID NO: 13 to 195, and preferably comprising at least one nucleotide of the corresponding polymorphism (donor allele) as provided in Table A.

[0124]

[76] The (isolated) polynucleotides according to any one of statements

[71] to

[74] are particularly suitable as molecular markers, comprising at least 15, preferably at least 18, more preferably at least 20 consecutive nucleotides of any one of SEQ ID NO: 109 to 154, or complementary to, or anticomplemental to, consecutive nucleotides of any one of SEQ ID NO: 109 to 154, and preferably comprising at least one nucleotide of the corresponding polymorphism (donor allele) as provided in Table A.

[0125]

[77] The (isolated) polynucleotides according to any one of statements

[71] to

[74] are particularly suitable as molecular markers, comprising at least 15, preferably at least 18, more preferably at least 20 consecutive nucleotides of any one of SEQ ID NO: 124 to 125, or complementary to, or anticomplemental to, consecutive nucleotides of any one of SEQ ID NO: 124 to 125, and preferably comprising at least one nucleotide of the corresponding polymorphism (donor allele) as provided in Table A.

[0126]

[78] The (isolated) polynucleotide according to any one of statements

[71] to

[78] is specific for plants or plant parts having improved straw digestibility.

[0127]

[79] A (isolated) polynucleotide that specifically hybridizes with a molecular marker, its complementary or reverse complementary sequence as defined in statement [5].

[0128]

[80] A (isolated) polynucleotide that specifically hybridizes with a molecular marker, its complementary or reverse complementary sequence as defined in statement [6].

[0129]

[81] A (isolated) polynucleotide that specifically hybridizes with a molecular marker, its complementary or reverse complementary sequence as defined in statement [7].

[0130]

[82] The (isolated) polynucleotide according to any one of the statements

[71] to

[81] is a primer or probe.

[0131]

[83] The (isolated) polynucleotide according to any one of the statements

[71] to

[82] is an allele-specific primer.

[0132]

[84] The (isolated) polynucleotide according to any one of the statements

[71] to

[83] is a KASP primer.

[0133]

[85] A primer or probe that is capable of specifically detecting the polynucleotide, one or more (molecular) markers (alleles), or QTLs (alleles) as defined in any one of the statements [1] to [9].

[0134]

[86] A primer set that is capable of specifically detecting the polynucleotide, one or more (molecular) markers (alleles), or QTLs (alleles) as defined in any one of the statements [1] to [9].

[0135]

[87] A type of corn seed, such as that deposited with NCIMB accession number NCIMB 43997.

[0136]

[88] A (K0001) corn seed, a representative sample of which has been deposited in NCIMB No. NCIMB 43997.

[0137]

[89] A corn plant grown or obtained from seeds as described in statement 87 or 88, or its offspring.

[0138]

[90] A corn plant part obtained from seeds grown or obtained according to statement

[87] or

[88] or from a plant (or its offspring) according to statement

[89] .

[0139]

[91] The corn plant part as described in statement

[90] , wherein the plant part is straw. Attached Figure Description

[0140] Figure 1 DNDF (Digestible Neutral Detergent Fiber) at the level of maize (Corn) inbred lines. Analysis was performed using NIRS calibration as further described above. Left column: Wild type without a 7bp insert; Right column: Inbred lines with a 7bp insert according to an embodiment of the invention.

[0141] Figure 2 Total Dry Matter Yield (TDY), in dt / ha, for four maize hybrids: No-Ins1, No-Ins2, Ins1, and Ins2. No-Ins1 and No-Ins2 do not have insertions in the F35H gene. According to an embodiment of the invention, the 7-base-pair insertions in the F35H gene for Ins1 and Ins2 are heterozygous.

[0142] Figure 3The percentage of total dry matter yield (TDY) of four maize hybrids, No-Ins1, No-Ins2, Ins1, and Ins2, relative to the control mean (rcm). No-Ins1 and No-Ins2 do not have insertions in the F35H gene. According to an embodiment of the invention, the 7-base-pair insertions in the F35H gene of Ins1 and Ins2 are heterozygous. Detailed Implementation

[0143] Before describing the systems and methods of the present invention, it should be understood that the invention is not limited to the specific systems, methods, or combinations described, as such systems, methods, and combinations can certainly vary. It should also be understood that the terminology used herein is not intended to be limiting, as the scope of the invention will be defined only by the appended claims.

[0144] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural meanings, unless the context clearly specifies otherwise.

[0145] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” “containing,” and “contains,” and are inclusive or open-ended, not excluding additional unlisted members, elements, or method steps. It should be understood that, as used herein, the terms “comprising,” “comprises,” and “comprised of” include the terms “consisting of,” “consists,” and “consists of,” as well as the terms “consisting essentially of,” “consists essentially,” and “consistses essentially of.”

[0146] The range of values ​​listed by endpoints includes all numbers and fractions contained within the corresponding range, as well as the listed endpoints.

[0147] As used herein, the terms “about” or “approximately” when referring to measurable values ​​such as parameters, quantities, durations of time, etc., are intended to cover variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, and still more preferably + / -1% or less, within which such variations are suitable for implementation in the disclosed invention. It should be understood that the values ​​referred to by the modifiers “about” or “approximately” are themselves specifically and preferably disclosed.

[0148] Given that the terms “one or more” or “at least one” are self-evident as to one or more or at least one member of a group of members by means of further examples, the term specifically covers any one of the members, or any two or more of the members, such as any ≥3, ≥4, ≥5, ≥6 or ≥7 of the members, as well as references to up to all of the members.

[0149] All references cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings of all references specifically mentioned herein are incorporated by reference.

[0150] Unless otherwise defined, all terms used in this disclosed invention, including technical and scientific terms, have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Definitions of terms are included in this further guidance to better understand the teachings of this invention.

[0151] Standard reference works explaining the general principles of recombinant DNA technology include: Molecular Cloning: A Laboratory Manual, 2nd edition, Volumes 1-3, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Current Protocols in Molecular Biology, edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (updated regularly) (“Ausubel et al. 1992”); the series of publications Methods in Enzymology (Academic Press, Inc.); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; PCR 2: A Practical Approach, edited by MJ MacPherson, B.D. Hames and G.G. Taylor (1995); Harlow and Lane (1988); Antibodies, a Laboratory Manual; and Animal Cell Culture, edited by R.Freshney (1987). The general principles of microbiology are explained, for example, in Davis, BD et al., Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa. (1980).

[0152] The various aspects of the invention are defined in more detail in the following paragraphs. Each aspect thus defined may be combined with any other one or more aspects unless expressly indicated to the contrary. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0153] Throughout this specification, the phrase "in one embodiment" or "in an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in one or more embodiments in any suitable manner, as will be apparent to those skilled in the art from this disclosure. Moreover, while some embodiments described herein include features that are not included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the invention and form different embodiments, as will be understood by those skilled in the art. For example, in the appended claims, any claimed embodiment can be used in any combination.

[0154] In the following detailed description, reference is made to the accompanying drawings, which form a part thereof, and which are illustrated only by way of example of specific embodiments in which the invention may be practiced. It should be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the invention. Therefore, the following detailed description is not to be considered limiting, and the scope of the invention is defined by the appended claims.

[0155] Preferred claims (features) and embodiments of the invention are set forth below. Each claim and embodiment of the invention as thus defined may be combined with any other claim and / or embodiment unless expressly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with one or more other features or statements indicated as preferred or advantageous.

[0156] As used in this article, “maize” refers to the species maize, preferably the plant of Zea mays ssp mays.

[0157] The term "plant" includes the whole plant, including its offspring or progeny. The term "plant part" includes any part or derivative of a plant, including specific plant tissues or structures, plant cells, immature or mature plant embryos, plant inflorescences, plant callus, plant protoplasts, plant cell or tissue cultures from which plants can regenerate, plant callus, intact plant masses and plant cells in a plant or plant part, such as seeds, grains, rachis, flowers, cotyledons, leaves, stems, buds, roots, root tips, straw, etc. Plant parts may include processed plant parts or derivatives, including flowers, oils, extracts, etc. In some embodiments, the plant part or derivative mentioned herein is straw. In some embodiments, the plant part or derivative mentioned herein is a seed or part of a seed.

[0158] As used herein, the term plant community may be used interchangeably with the term plant population. A plant community preferably comprises a number of individual plants, such as preferably at least 10 plants, such as 20, 30, 40, 50, 60, 70, 80, or 90 plants, more preferably at least 100 plants, such as 200, 300, 400, 500, 600, 700, 800, or 900 plants, and even more preferably at least 1,000 plants, such as at least 10,000 or at least 100,000 plants.

[0159] As used herein, straw has its common meaning as known in the art. With further guidance, but not limited thereto, straw may comprise, consist of, or consist essentially of the leaves and stalks of field crops such as corn that are typically left in the field after grain harvest. Straw may also include the cob (e.g., the central core of a corn ear, without kernels). Straw may also exclude the cob. Straw may also include the husk or outer shell (e.g., the leaf-like outer layer of a corn ear). Straw may also exclude the husk or outer shell. Straw is similar to wheat straw, being the residue left after the seeds of any grain or grass are harvested at maturity. It can be used directly for cattle grazing or dried for use as forage. (Corn) straw can be used as feed, whether as grazing forage, chopped for silage for later use as forage, or collected for direct (non-silage) forage use. Corn fodder is typically ensiled in colder regions, but it can be harvested year-round in tropical regions and used as green forage for animals. In silage use cases, the whole plant (grain and stalk together) is typically chopped into small pieces at harvest and then crushed between rollers. In addition to the stems, leaves, outer skin, and cob left in the field, grains may also remain after harvest. These remaining grains, along with the corn stalks, serve as an additional feed source for grazing cattle.

[0160] In some embodiments, the plant part or derivative comprises, is composed of, or is substantially composed of one or more of stems, leaves, and rachis, preferably all of them. In some embodiments, the plant part or derivative is a leaf. In some embodiments, the plant part or derivative is a stem. In some embodiments, the plant part or derivative is a rachis. In some embodiments, the plant part or derivative comprises, is composed of, or is substantially composed of one or more of stems and leaves, preferably all of them. In some embodiments, the plant part or derivative comprises, is composed of, or is substantially composed of one or more of stems and rachis, preferably all of them. In some embodiments, the plant part or derivative comprises, is composed of, or is substantially composed of one or more of leaves and rachis, preferably all of them. In some embodiments, the plant part or derivative is not (functional) propagation material, such as germplasm, seeds, or plant embryos or other material from which the plant can regenerate. In some embodiments, the plant part or derivative does not contain (functional) male and female reproductive organs. In some embodiments, the plant part or derivative is or contains propagation material, except for propagation material that cannot or can no longer be used to produce or generate new plants, such as propagation material that has already been chemically, mechanically or otherwise (e.g., by heat treatment, acid treatment, compaction, crushing, chopping, ensiling, etc.) exhibiting non-functionality.

[0161] As used herein, “digestibility” refers to and is measured as the percentage of product (such as maize plant or plant parts or derivatives, including, for example, the dry matter, organic matter, or cell walls of the product) absorbed into the digestive tract of an animal (Barrière et al., 2003). Biological and chemical methods have been developed for determining the digestibility of maize and other forage crops (Van Soest et al., 1963). Neutral detergent fiber (NDF), as a residue after the removal of soluble cell contents, is an important indicator of plant cell walls and cellulose. In vitro NDF digestibility of forage (IVNDFD) is an estimate of cell wall digestibility—assuming that the non-NDF portion of the plant material is completely digestible (Méchin et al., 2000). Additionally, digestibility traits of various forage crops, including maize, have been reported to be accurately measured using NIRS (Lübberstedt et al., 1997a, b; Zimmer et al., 1990). In some embodiments, the animal is a mammal. In some embodiments, the animal is a ruminant. In some embodiments, the animal is a herbivore. In some embodiments, the animal is a herbivorous mammal.

[0162] As used herein, “improved digestibility” refers to an increase in the digestibility of a plant or plant part, such as straw, or its derivative, that has the characteristics according to the invention, such as polynucleotides, mutations, markers, SNPs, or QTLs as described elsewhere herein, compared to a plant or plant part or derivative that does not have such characteristics, such as a reference plant (or plant part). In some embodiments, improved or increased (straw) digestibility means an increase in average DNDF of at least 1%, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%, preferably at least 2%, more preferably at least 3%, such as at least 4%, most preferably at least 5%, such as at least 10%. In some embodiments, the reference plant (or plant part) is the maize inbred line PH207, as described in “Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize”, Hirsch et al., Plant Cell. Nov 2016; 28(11):2700-2714, published online November 1, 2016, doi:10.1105 / tpc.16.00353, or a gene line that does not contain (or is close to) the polynucleotides, mutations, markers, SNPs, or QTLs of the invention as described elsewhere herein. Those skilled in the art will understand that when reference is made to improved straw digestibility in the context of plant parts other than straw (e.g., seeds), it should be understood that this refers to the straw of the plant from which such plant parts are derived. For example, the seed mentioned as having "improved straw digestibility" refers to straw from the plant from which the seed originates, and / or straw from the plant from which the seed grows, preferably at least from the plant from which the seed grows.

[0163] The term “locus” (multiple loci) refers to one or more specific locations or sites on a chromosome where, for example, a QTL, gene, or genetic marker is found. As used herein, the terms “quantitative trait locus” or “QTL” have their common meaning as known in the art. With further guidance and not limitation, a QTL may refer to a region of DNA associated with differential expression of a quantitative phenotypic trait in at least one genetic context (e.g., in at least one breeding population). A QTL region encompasses one or more genes that affect or are closely linked to the trait under consideration. “Alleles of a QTL” may include multiple genes or other genetic factors, such as haplotypes, within a contiguous genomic region or linkage group. Alleles of a QTL may represent haplotypes within a specified window, wherein the window is a contiguous genomic region that can be defined and tracked using one or more monomorphic and / or polymorphic markers. A haplotype may be defined by a unique fingerprint of the allele at each marker within the specified window. A QTL may encode one or more alleles that affect the expression levels of a contiguously distributed (quantitative) phenotypic trait. In some embodiments, the QTLs, polynucleotides, markers, etc., of the present invention as described herein may be homozygous. In some implementations, the QTLs, polynucleotides, markers, etc. of the present invention as described herein may be heterogeneous.

[0164] As used in this article, the term "allele" or "alleles" refers to one or more alternative forms of a locus, i.e., different nucleotide sequences.

[0165] As used herein, the term "mutant allele" or "mutation" of an allele includes an allele having one or more mutations, such as insertions, deletions, stop codons, base changes (e.g., transitions or transversions), or alterations to splice sites / splicing signals, which may or may not produce an altered gene product. Allele modifications may occur in coding or non-coding regions (e.g., promoter regions, exons, introns, or splice sites).

[0166] As used herein, the terms “introgression,” “introgressed,” and “introgressing” refer to both natural and artificial processes in which a segment of chromosome or gene of one species, variety, or cultivar is transferred into those species through hybridization with another species, variety, or cultivar. This process may optionally be accomplished by backcrossing with a recurrent parent. For example, introgression of a desired allele at a designated locus can be transmitted to at least one offspring via sexual hybridization between two parents of the same species, where at least one parent has the desired allele in its genome. Alternatively, for example, allele transfer can occur via recombination between two donor genomes (e.g., in fused protoplasts), where at least one donor protoplast has the desired allele in its genome. The desired allele can be detected, for example, by markers associated with phenotypes at QTLs, transgenes, etc. In any case, offspring containing the desired allele can be repeatedly backcrossed into a line with the desired genetic background and selected for the desired allele, resulting in an allele that is fixed in the selected genetic background. An "introgression fragment" or "introgression region" refers to a segment (or part or region) of chromosome that has been artificially or naturally introduced into another plant of the same or related species (e.g., through hybridization or conventional breeding techniques such as backcrossing). That is, an introgression fragment is the result of the breeding method referred to by the verb "introgression" (e.g., backcrossing). It should be understood that the term "introgression fragment" never includes an entire chromosome, but only a portion of it. Introgression fragments can be large, for example, even three-quarters or half of a chromosome, but are preferably small, such as about 50 Mb or less, such as about 30 Mb or less, about 20 Mb or less, about 25 Mb or less, about 10 Mb or less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2.5 Mb or less. 2 Mb or less, about 1 Mb (equal to 1,000,000 base pairs) or less, or about 0.5 Mb (equal to 500,000 base pairs) or less, such as about 200,000 bp (equal to 200,000 base pairs) or less, about 100,000 bp (100 kb) or less, about 50,000 bp (50 kb) or less, about 25,000 bp (25 kb) or less.

[0167] If a genetic element, locus, introgression fragment, QTL, or gene or allele that confers a trait (such as improved digestibility) can be transferred from a plant in which it exists to another plant (such as a line or variety) where it does not exist using conventional breeding techniques without causing a phenotypic change in the recipient plant (other than the addition of the trait conferred by the genetic element, locus, introgression fragment, QTL, gene, or allele), then the genetic element, locus, introgression fragment, QTL, or gene or allele is referred to as “available from” or “possibly available from” or “derived from” or “as if present” or “as if found in” the plant or plant part described elsewhere herein. These terms are used interchangeably, and a genetic element, locus, introgression fragment, QTL, gene, or allele can thus be transferred to any other genetic background lacking that trait. Not only plants containing genetic elements, loci, introgression fragments, QTLs, genes, or alleles can be used, but also the offspring / progeny of such plants selected to retain genetic elements, loci, introgression fragments, QTLs, genes, or alleles, and they are covered herein. Whether a plant (or its genomic DNA, cells, or tissues) contains the same genetic elements, loci, introgression fragments, QTLs, genes, or alleles as available from such plants can be determined by a person skilled in the art using one or more techniques known in the art, such as phenotyping, whole-genome sequencing, molecular marker analysis, trait mapping, chromosome painting, allele testing, or a combination of these techniques. It should be understood that transgenic or gene-edited plants may also be covered.

[0168] As used herein, the terms “genetic engineering,” “transformation,” and “genetic or transgenic modification” are used as synonyms in this document for transferring isolated and cloned genes into the DNA (usually chromosomal DNA or genome) of another organism.

[0169] In the context of this invention, "introduction" includes: stable or transient integration by means of transformation, including Agrobacterium-mediated transformation, transfection, microinjection, gene gun bombardment; insertion using gene editing technologies such as CRISPR systems (e.g., CRISPR / Cas, particularly CRISPR / Cas9 or CRISPR / Cas12, CRISPR / CasX, or CRISPR / CasY), TALEN, zinc finger nucleases, or a wide range of nucleases; optionally by means of one of the gene editing technologies mentioned below, preferably including template repair for homologous recombination; modification of endogenous genes using random or targeted mutagenesis such as TILLING, or the gene editing technologies mentioned above.

[0170] As used herein, a “transgenic” or “genetically modified organism” (GMO) is an organism whose genetic material has been altered using a technique commonly referred to as “recombinant DNA technology.” Recombinant DNA technology encompasses the ability to combine DNA molecules from different sources in vitro (e.g., in a test tube) into a single molecule. This term typically does not cover organisms whose genetic makeup has been altered through conventional hybridization breeding or through “mutation” breeding, as these methods predate the discovery of recombinant DNA technology. As used herein, “non-transgenic” refers to plants and foods derived from plants that are not “transgenic” or “genetically modified” organisms as defined above.

[0171] "Transgenic" or "exogene" refers to a genetic locus containing a DNA sequence, such as a recombinant gene, that has been introduced into the plant genome through transformation (e.g., Agrobacterium-mediated transformation). Plants containing transgenes stably integrated into their genome are called "transgenic plants." "Endogenous" refers to nucleic acid molecules or genetic loci naturally present in the plant genome.

[0172] "Gene editing" or "genome editing" refers to genetic engineering that involves inserting, deleting, modifying, or replacing DNA or RNA in the genome of an organism. Gene editing can include directed or non-directed (random) mutagenesis. Directed mutagenesis can be achieved, for example, using designed nucleases, such as broad-spectrum nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and regularly spaced clustered short palindromic repeats (CRISPR / Cas) systems. These nucleases produce site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired by non-homologous end joining (NHEJ), homologous recombination (HR), or homologous directed repair (HDR), resulting in targeted mutations or nucleic acid modifications. The use of designed nucleases is particularly well-suited for generating gene knockouts or knock-outs. In some embodiments, designed nucleases have been developed to specifically induce mutations in the F35H gene, as described elsewhere herein, such as to generate mutated F35H or knockout of the F35H gene. In some embodiments, designed nucleases that specifically target F35H mRNA have been developed, particularly RNA-specific CRISPR / Cas systems, such as those for cleaving F35H mRNA and generating F35H gene / mRNA / protein knockdown. Delivery and expression systems for these designed nuclease systems are well known in the art.

[0173] In some embodiments, the nuclease or target / site-specific / homing nuclease is, comprises, substantially consists of, or is composed of (modified) a CRISPR / Cas system or complex, (modified) Cas proteins, (modified) zinc finger proteins, (modified) zinc finger nucleases (ZFNs), (modified) transcription factor-like effector (TALE), (modified) transcription factor-like effector nuclease (TALEN), or (modified) a wide range of nucleases. In some embodiments, the (modified) nuclease or target / site-specific / homing nuclease is, comprises, substantially consists of, or is composed of (modified) RNA-guided nucleases. It should be understood that in some embodiments, the nuclease may be codon-optimized for expression in plants. As used herein, the term "targeted" refers to a selected nucleic acid sequence, meaning that the nuclease or nuclease complex functions in a nucleotide sequence-specific manner. For example, in the context of a CRISPR / Cas system, the guide RNA is capable of hybridizing with a selected nucleic acid sequence. As used herein, “hybridization” or “hybridized” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific mechanism. The complex can contain two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a self-hybridized single strand, or any combination thereof. Hybridization reactions can constitute steps in broader processes such as the initiation of PGR or enzymatic cleavage of polynucleotides. A sequence capable of hybridizing with a given sequence is called the “complementary sequence” of the given sequence.

[0174] Gene editing can involve transient, inducible, or constitutive expression of gene-editing components or systems. Gene editing can involve genomic integration of gene-editing components or systems or the presence of free genes. Gene-editing components or systems can be provided on vectors such as plasmids, which can be delivered via suitable delivery media as known in the art. Preferred vectors are expression vectors.

[0175] Gene editing may include providing a recombination template to enable homology-mediated repair (HDR). For example, genetic elements may be replaced by gene editing (where a recombination template is provided). DNA may be cut upstream and / or downstream of the sequence to be replaced. Thus, the sequence to be replaced is excised from the DNA. With HDR, the excised sequence is then replaced with the template. In some embodiments, the QTL alleles of the present invention, as described herein, may be provided on / as a template. By designing the system such that double-strand breaks are introduced upstream and downstream of a corresponding region in the genome of a plant that does not contain a QTL allele, the region is excised and can be replaced with a template containing the QTL alleles of the present invention. Thus, the introduction of the QTL alleles of the present invention into plants does not require multiple backcrosses, particularly in plants with a specific genetic background. Similarly, the mutant F35H of the present invention may be provided on / as a template. However, more advantageously, the mutant F35H of the present invention may be generated without the use of a recombination template, but simply by the action of an endonuclease that causes a double-strand DNA break, which is repaired by NHEJ, resulting in the generation of an insertion / deletion.

[0176] In some implementations, nucleic acid modification or mutation is achieved through a (modified) transcription activator-like effector nuclease (TALEN) system. Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence. Exemplary methods for genome editing using the TALEN system can be found, for example, Cermak T. Doyle EL. Christian M. Wang L. Zhang Y. Schmidt C et al., Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011; 39:e82; Zhang F. Cong L. Lodato S. Kosuri S. Church GM. Arlotta P. Efficient construction of sequence-specific TALeffectors for modulating mammalian transcription. Nat Biotechnol. 2011; 29:149-153, and US Patents Nos. 8,450,471, 8,440,431, and 8,440,432, all of which are specifically incorporated herein by reference. With further guidance, but not limited thereto, naturally occurring TALEs, or “wild-type TALEs,” are nucleic acid-binding proteins secreted by various species of proteobacteria. TALE polypeptides contain a nucleic acid-binding domain consisting of a highly conserved tandem repeat sequence of monomeric polypeptides, primarily 33, 34, or 35 amino acids in length, and differing primarily from each other at amino acid positions 12 and 13. In an advantageous embodiment, the nucleic acid is DNA. As used herein, the term “polypeptide monomer” or “TALE monomer” will be used to refer to a highly conserved repeating polypeptide sequence within the TALE nucleic acid-binding domain, and the term “repeated variable diresidue” or “RVD” will be used to refer to the highly variable amino acid at positions 12 and 13 of the polypeptide monomer. As provided throughout this disclosure, the amino acid residues of RVDs are described using the IUPAC single-letter codes for amino acids. A general representation of a TALE monomer contained within a DNA-binding domain is X1-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates the amino acid position and X represents any amino acid. X12 and X13 indicate RVD. In some polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such polypeptide monomers, RVD consists of a single amino acid.In this case, RVD can alternatively be represented as X*, where X represents X12, and (*) indicates that X13 is absent. The DNA-binding domain contains several repeating sequences of the TALE monomer, and this can be represented as (X1-11-(X12X13)-X14-33 or 34 or 35)z, wherein in an advantageous embodiment, z is at least 5 to 40. In another advantageous embodiment, z is at least 10 to 26. The TALE monomer has a nucleotide binding affinity determined by the amino acid identity in its RVD. For example, a polypeptide monomer with RVD NI preferentially binds adenine (A), a polypeptide monomer with RVD NG preferentially binds thymine (T), a polypeptide monomer with RVD HD preferentially binds cytosine (C), and a polypeptide monomer with RVD NN preferentially binds both adenine (A) and guanine (G). In yet another embodiment of the invention, a polypeptide monomer with RVD IG preferentially binds T. Therefore, the number and order of repeating sequences of the polypeptide monomer in the nucleic acid binding domain of TALE determine its nucleic acid target specificity. In some embodiments of the present invention, RVD is a polypeptide monomer of NS that recognizes all four base pairs and can bind A, T, G, or C. The structure and function of TALE are further described, for example, in Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated herein by reference in its entirety.

[0177] In some implementations, nucleic acid modification or mutation is achieved through a (modified) zinc finger nuclease (ZFN) system. The ZFN system uses an artificial restriction enzyme generated by fusing a zinc finger DNA-binding domain with a DNA-cutting domain; this enzyme can be engineered to target the desired DNA sequence. Exemplary methods for genome editing using ZFNs can be found, for example, in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, all of which are specifically incorporated herein by reference. With the aid of further guidance, but not limited thereto, artificial zinc finger (ZF) technology involves arrays of ZF modules to target novel DNA binding sites in the genome. Each finger module in the ZF array targets three DNA bases. An array of custom-designed individual zinc finger domains is assembled into a ZF protein (ZFP). ZFPs may contain functional domains. The first synthetic zinc finger nuclease (ZFN) was developed by fusing a ZF protein with the catalytic domain of the IIS-type restriction enzyme FokI (Kim, YG et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. USA 91, 883-887; Kim, YG et al., 1996, Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156-1160). By using paired ZFN heterodimers, enhanced cleavage specificity can be achieved by reducing off-target activity, with each heterodimer targeting different nucleotide sequences separated by short spacers (Doyon, Y. et al., 2011, Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat. Methods 8, 74-79). ZFPs can also be engineered as transcriptional activators and repressors and have been used to target many genes in a variety of organisms.

[0178] In some embodiments, nucleic acid modification is achieved by a (modified) broad-spectrum nuclease, which is a deoxyribonuclease endonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs). Exemplary methods using broad-spectrum nucleases can be found in US Patent Nos. 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134, which are specifically incorporated herein by reference.

[0179] In some implementations, nucleic acid modification is achieved through a (modified) CRISPR / Cas complex or system. General information concerning the CRISPR / Cas system, its components, and the delivery of such components, including methods, materials, delivery media, carriers, particles, and their preparation and use (including with regard to quantity and formulation), and eukaryotic cells expressing Cas9CRISPR / Cas, eukaryotic organisms expressing Cas-9 CRISPR / Cas, such as mice, see: US Patent Nos. 8,999,641, 8,993,233, 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233, and 8,999,641;US Patent Publications: US2014-0310830 (US Application Serial No. 14 / 105,031), US2014-0287938 A1 (US Application Serial No. 14 / 213,991), US2014-0273234 A1 (US Application Serial No. 14 / 293,674), US2014-0273232A1 (US Application Serial No. 14 / 290,575), US2014-0273231 (US Application Serial No. 14 / 259,420), US2014-0256046A1 (US Application Serial No. 14 / 226,274), US2014-0248702 A1 (US Application Serial No. 14 / 258,458), US2014-0242700 A1 (US Application Serial No. 14 / 222,930), US2014-0242699 A1 (US Application Serial No. 14 / 183,512), US2014-0242664 A1 (US Application Serial No. 14 / 104,990), US2014-0234972A1 (US Application Serial No. 14 / 183,471), US2014-0227787 A1 (US Application Serial No. 14 / 256,912), US2014-0189896 A1 (US Application Serial No. 14 / 105,035), US2014-0186958 (US Application Serial No. 14 / 105,017), US2014-0186919 A1 (US Application Serial No. 14 / 104,977), US US2014-0186843A1 (US Application Serial No. 14 / 104,900), US2014-0179770 A1 (US Application Serial No. 14 / 104,837), US2014-0179006 A1 (US Application Serial No. 14 / 183,486), US2014-0170753 (US Application Serial No. 14 / 183,429); US2015-0184139 (US Application Serial No. 14 / 324,960); 14 / 054,414; European patent applications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6), and EP 2 784 162 (EP14170383.5);WO 2014 / 093661 (PCT / US2013 / 074743), WO 2014 / 093694 (PCT / US2013 / 074790), WO 2014 / 093595 (PCT / US2013 / 074611), WO 2014 / 093718 (PCT / US2013 / 074825), WO 2014 / 093709 (PCT / US2013 / 074812), WO 2014 / 093622 (PCT / US2013 / 074667), WO 2014 / 093635 (PCT / US2013 / 074691), WO 2014 / 093655(PCT / US2013 / 074736), WO 2014 / 093712(PCT / US2013 / 074819), WO 2014 / 093701(PCT / US2013 / 074800), WO 2014 / 018423(PCT / US2013 / 051418), WO 2014 / 204723(PCT / US2014 / 041790), WO 2014 / 204724(PCT / US2014 / 041800), WO 2014 / 204725(PCT / US2014 / 041803), WO 2014 / 204726(PCT / US2014 / 041804), WO 2014 / 204727(PCT / US2014 / 041806), WO 2014 / 204728(PCT / US2014 / 041808), WO 2014 / 204729(PCT / US2014 / 041809), WO 2015 / 089351(PCT / US2014 / 069897), WO 2015 / 089354(PCT / US2014 / 069902), WO 2015 / 089364(PCT / US2014 / 069925), WO 2015 / 089427(PCT / US2014 / 070068), WO 2015 / 089462(PCT / US2014 / 070127), WO 2015 / 089419(PCT / US2014 / 070057), WO 2015 / 089465(PCT / US2014 / 070135), WO 2015 / 089486(PCT / US2014 / 070175), PCT / US2015 / 051691, PCT / US2015 / 051830. For reference: January 30, 2013; March 15, 2013; March 28, 2013; April 20, 2013;US provisional patent applications filed May 6, 2013 and May 28, 2013, respectively, are referenced to: 61 / 758,468; 61 / 802,174; 61 / 806,375; 61 / 814,263; 61 / 819,803 and 61 / 828,130. Also referenced is US provisional patent application filed June 17, 2013, 61 / 836,123. Additionally, references are made to US provisional patent applications filed June 17, 2013, respectively, 61 / 835,931, 61 / 835,936, 61 / 835,973, 61 / 836,080, 61 / 836,101 and 61 / 836,127. Further reference is made to US provisional patent applications 61 / 862,468 and 61 / 862,355, filed August 5, 2013; 61 / 871,301, filed August 28, 2013; 61 / 960,777, filed September 25, 2013; and 61 / 961,980, filed October 28, 2013. Further references were made to: PCT / US2014 / 62558, filed October 28, 2014; and US provisional patent application series Nos.: 61 / 915,148, 61 / 915,150, 61 / 915,153, 61 / 915,203, 61 / 915,251, 61 / 915,301, 61 / 915,267, 61 / 915,260, and 61 / 915,397, each filed December 12, 2013; 61 / 757,972 and 61 / 768,959, filed January 29, 2013, and February 25, 2013, respectively; 62 / 010,888 and 62 / 010,879 were both submitted on June 11, 2014; 62 / 010,329, 62 / 010,439 and 62 / 010,441 were each submitted on June 10, 2014; 61 / 939,228 and 61 / 939,242 were each submitted on February 12, 2014; 61 / 980,012 was submitted on April 15, 2014; 62 / 038,358 was submitted on August 17, 2014; 62 / 055,484, 62 / 055,460 and 62 / 055,487 were each submitted on September 25, 2014.References to U.S. Provisional Patent Application No. 62 / 069,243, filed October 27, 2014. References also to PCT application No. 62 / 069,243, filed June 10, 2014, with particular designation of U.S. Application No. PCT / US14 / 41806. References also to U.S. Provisional Patent Application No. 61 / 930,214, filed January 22, 2014. References also to PCT application No. 62 / 069,243, filed January 22, 2014, with particular designation of U.S. Application No. PCT / US14 / 41806. It also mentions US Application 62 / 180,709 (June 17, 2015), PROTECTED GUIDE RNAS (PGRNAS); US Application 62 / 091,455 (filed December 12, 2014), PROTECTED GUIDE RNAS (PGRNAS); US Application 62 / 096,708 (December 24, 2014), PROTECTED GUIDE RNAS (PGRNAS); US Applications 62 / 091,462 (December 12, 2014), 62 / 096,324 (December 23, 2014), 62 / 180,681 (June 17, 2015), and 62 / 237,496 (October 5, 2015), DEAD GUIDES FOR CRISPR. TRANSCRIPTIONFACTORS; US Applications 62 / 091,456 (December 12, 2014) and 62 / 180,692 (June 17, 2015), ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; US Application 62 / 091,461 (December 12, 2014), DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); US Application 62 / 094,903 (December 19, 2014), UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING;US Application 62 / 096,761 (December 24, 2014), ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; US Applications 62 / 098,059 (December 30, 2014), 62 / 181,641 (June 18, 2015), and 62 / 181,667 (June 18, 2015), RNA-TARGETING SYSTEM; US Applications 62 / 096,656 (December 24, 2014) and 62 / 181,151 (June 17, 2015), CRISPR HAVING ORASSOCIATED WITH DESTABILIZATION DOMAINS; US Application 62 / 096,697 (December 24, 2014), CRISPR HAVING OR ASSOCIATED WITH AAV; US Application 62 / 098,158 (December 30, 2014), ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; US Application 62 / 151,052 (April 22, 2015), CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; US Application 62 / 054,490 (September 24, 2014), DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THECRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS; US application 61 / 939,154 (February 12, 2014), SYSTEMS, METHODSAND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;US Application No. 62 / 055,484 (September 25, 2014), SYSTEMS, METHODS AND COMPOSITIONSFOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; US Application No. 62 / 087,537 (December 4, 2014), SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; US Application No. 62 / 054,651 (September 24, 2014), DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCERMUTATIONS IN VIVO; US application 62 / 067,886 (October 23, 2014), Delivery, use and therapeutic applications of the CRISPR-CAS systems and compositions for modeling competition of multiple cancer mutations in VIVO; US application 62 / 054,675 (September 24, 2014) and 62 / 181,002 (June 17, 2015), Delivery, use and therapeutic applications of the CRISPR-CAS systems and compositions in Neuronal Cells / Tissues; US application 62 / 054,528 (September 24, 2014), Delivery, use and therapeutic applications of the CRISPR-CAS systems and compositions in IMMUNE DISEASES ORDISORDERS;US Application 62 / 055,454 (September 25, 2014), Delivery, Use, and Therapeutic Applications of the CRISPR-CAS Systems and Compositions for Targeting Disorders and Disseases Using Cell Penetration PEPTIDES (CPP); US Application 62 / 055,460 (September 25, 2014), Multificational-CRISPR Completions and / or Optimized Enzymelinked Functional-CRISPR Completions; US Applications 62 / 087,475 (December 4, 2014) and 62 / 181,690 (June 18, 2015), Functional Screening with Optimized Functional CRISPR-CAS SYSTEMS; US Application 62 / 055,487 (September 25, 2014), FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; US Applications 62 / 087,546 (December 4, 2014) and 62 / 181,687 (June 18, 2015), MULTIFUNCTIONAL CRISPR COMPLEXES AND / OR OPTIMIZED ENZYMELINKED FUNCTIONAL-CRISPR COMPLEXES;And US filing 62 / 098,285 (December 30, 2014), Criticized in VIVO MODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS. It also mentions US filings 62 / 181,659 (June 18, 2015) and 62 / 207,318 (August 19, 2015), ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS, ENZYME AND GUIDESCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FOR SEQUENCE MANIPULATION. The references include US Applications 62 / 181,663 (June 18, 2015) and 62 / 245,264 (October 22, 2015), NOVEL CRISPR ENZYMES AND SYSTEMS; US Application 62 / 181,675 (June 18, 2015); and Agent's File No. 46783.01.2128 (filed October 22, 2015), NOVEL CRISPR ENZYMES AND SYSTEMS.US Applications 62 / 232,067 (September 24, 2015), 62 / 205,733 (August 16, 2015), 62 / 201,542 (August 5, 2015), 62 / 193,507 (July 16, 2015), and 62 / 181,739 (June 18, 2015), each entitled "NOVELCRISPR ENZYMES AND SYSTEMS," and US Application 62 / 245,270 (October 22, 2015), entitled "NOVEL CRISPRENZYMES AND SYSTEMS." It also mentions US Application 61 / 939,256 (February 12, 2014) and WO 2015 / 089473 (PCT / US2014 / 070152) (December 12, 2014), each titled "ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FOR SEQUENCEMANIPULATION". It also mentions PCT / US2015 / 045504 (August 15, 2015), US Application 62 / 180,699 (June 17, 2015), and US Application 62 / 038,358 (August 17, 2014), each titled "GENOME EDITING USING CAS9NICKASES". European Patent Application EP3009511. Further referenceMultiplex genome engineering using CRISPR / Cas systems.Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA,&Zhang,F.Science Feb 15;339(6121):819-23(2013);RNA-guided editing of bacterial genomes using CRISPR-Cassystems.Jiang W.,Bikard D.,Cox D.,Zhang F,Marraffini LA.Nat Biotechnol Mar;31(3):233-9(2013);[ PMC free article ] [ PubMed ] Wang H, Yang H, ShivalilaCS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R.Cell May 9153(4):910-8(2013)Optical control of Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA, Church GM, Zhang F.Nature.2013August22:500(7463):472-6.doi:10.1038 / Nature12466.Epub 2013August23:Double Nicking by RNA-Guided CRISPRCas9 for Enhanced Genome Editing Specificity.Ran , FA , Hsu , PD , Lin , CY , Gootenberg , JS , Konermann , S , Trevino , AE , Scott , DA , Inoue , A , Matoba , S , Zhang , Y , & Zhang , F. Cell Aug 28.pii:S0092-8674(13)01015-5.(2013)DNA targetingspecificity of RNA-guided Cas9 nucleases.Hsu, P., Scott, D., Weinstein, J., Ran, F., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, TJ., Marraffini, LA., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038 / nbt.2647(2013)Genome engineering using the CRISPR-Cas9 system. Ran, FA., Hsu, PD., Wright, J., Agarwala, V., Scott, DA., Zhang, F. Nature Protocols Nov;8(11):2281-308. (2013);Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, NE., Hartenian, E., Shi, X., Scott, DA., Mikkelson, T., Heckl, D., Ebert, BL., Root, DE., Doench, JG., Zhang, F. Science Dec 12. (2013). [Epub ahead of print];Crystal structure of cas9 in complex with guide RNA and target DNA. Nishimasu, H., Ran, FA., Hsu, PD., Konermann, S., Shehata, SI., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell Feb 27. (2014). 156(5):935-49;Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Wu X., Scott DA., Kriz AJ., Chiu AC., Hsu PD., Dadon DB., Cheng AW., Trevino AE., Konermann S., Chen S., Jaenisch R., Zhang F., Sharp PA. Nat Biotechnol. (2014) Apr 20. doi:10.1038 / nbt.2889;CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling, Platt et al., Cell 159(2):440-455(2014) DOI:10.1016 / j.cell.2014.09.014;Development and Applications of CRISPR-Cas9 for Genome Engineering, Hsu et al., Cell 157, 1262-1278 (June 5, 2014) (Hsu 2014); Geneticscreens in human cells using the CRISPR / Cas9 system, Wang et al., Science. January 3, 2014; 343(6166):80–84. doi:10.1126 / science.1246981; Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench et al., Nature Biotechnology 32(12):1262-7 (2014), published online September 3, 2014; doi:10.1038 / nbt.3026; and In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9, Swiech et al., Nature Biotechnology 33,102–106 (2015), published online on October 19, 2014; doi:10.1038 / nbt.3055, Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Zetsche et al., Cell 163,1-13 (2015); Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems, Shmakov et al., Mol Cell 60(3):385-397 (2015);C2c2is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Abudayyeh et al., Science (2016), published online June 2, 2016, doi:10.1126 / science.aaf5573. Each of these publications, patents, patent publications and applications, and all documents cited therein or during the application period (“Application Reference Documents”), and all documents cited or referenced in the Application Reference Documents, and any descriptions, specifications and product tables of any products mentioned in or in any of these documents and incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of this invention. All documents (e.g., these patents, patent publications and applications and Application Reference Documents) are incorporated herein by reference to the extent that each individual document is specifically and individually indicated to be incorporated by reference.

[0180] In some embodiments, the CRISPR / Cas system or complex is a type II, type V, or type VI CRISPR / Cas system or complex. The CRISPR / Cas system does not require the generation of a custom protein targeting a specific sequence; instead, a single Cas protein can be programmed via an RNA guide (gRNA) to recognize a specific nucleic acid target. In other words, the short RNA guide can be used to recruit the Cas enzyme protein to a specific nucleic acid target locus of interest (which may contain or be composed of RNA and / or DNA).

[0181] Generally, as used in the preceding literature herein, CRISPR / Cas or the CRISPR system refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-related (“Cas”) genes, including sequences encoding Cas genes, and tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active tracrRNA), tracr pairing sequences (in the context of endogenous CRISPR systems, encompassing “positive repeat sequences” and partially positive repeat sequences processed by tracrRNA), guide sequences (also referred to as “spacer regions” in the context of endogenous CRISPR systems), or as used herein, “RNA” (e.g., RNA directing Cas such as Cas9, such as CRISPR RNA, and, where applicable, trans-activating (tracr) RNA or single-stranded guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from CRISPR loci. Generally, a CRISPR system is characterized by elements that promote the formation of the CRISPR complex at the target sequence site (also referred to as pre-spacer regions in the context of endogenous CRISPR systems). In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which the guide sequence is designed to be complementary, where hybridization between the target and guide sequences promotes CRISPR complex formation. The target sequence can contain any polynucleotide, such as DNA or RNA polynucleotides.

[0182] In some embodiments, the gRNA is a chimeric guide RNA or a single-stranded guide RNA (sgRNA). In some embodiments, the gRNA comprises a guide sequence and a tracr pairing sequence (or a forward repeat sequence). In some embodiments, the gRNA comprises a guide sequence, a tracr pairing sequence (or a forward repeat sequence), and a tracr sequence. In some embodiments, the CRISPR / Cas system or complex as described herein does not contain and / or is independent of the presence of the tracr sequence (e.g., if the Cas protein is Cpf1).

[0183] As used herein, the terms “crRNA” or “guide RNA” or “single-stranded guide RNA” or “sgRNA” or “one or more nucleic acid components” (if applicable) for CRISPR / Cas locus effector proteins encompass any polynucleotide sequence that is sufficiently complementary to the target nucleic acid sequence to hybridize with the target nucleic acid sequence and guide the nucleic acid targeting complex to bind sequence-specifically to the target nucleic acid sequence. In some embodiments, the degree of complementarity is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or greater when optimal alignment is performed using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for sequence alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows WheelerAligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of the guide sequence (within the nucleic acid targeting guide RNA) to direct the nucleic acid targeting complex to bind sequence-specifically to the target nucleic acid sequence can be assessed by any suitable assay.

[0184] A guide sequence, and therefore the nucleic acid targeting guide RNA, can be selected to target any target nucleic acid sequence. The target sequence can be DNA. The target sequence can be genomic DNA. The target sequence can be mitochondrial DNA. The target sequence can be any RNA sequence. In some embodiments, the target sequence can be a sequence within an RNA molecule selected from messenger RNA (mRNA), premRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), nucleolar small RNA (snoRNA), double-stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmic RNA (scRNA). In some preferred embodiments, the target sequence can be a sequence within an RNA molecule selected from mRNA, premRNA, and rRNA. In some preferred embodiments, the target sequence can be a sequence within an RNA molecule selected from ncRNA and lncRNA. In some more preferred embodiments, the target sequence can be a sequence within an mRNA molecule or premRNA molecule.

[0185] In some embodiments, the gRNA comprises a stem-loop, preferably a single stem-loop. In some embodiments, the forward repeat sequence forms a stem-loop, preferably a single stem-loop. In some embodiments, the spacer region of the guide RNA is 15 to 35 nt in length. In some embodiments, the spacer region of the guide RNA is at least 15 nucleotides in length. In some embodiments, the spacer region is 15 to 17 nt in length, such as 15, 16, or 17 nt; 17 to 20 nt in length, such as 17, 18, 19, or 20 nt; 20 to 24 nt in length, such as 20, 21, 22, 23, or 24 nt; 23 to 25 nt in length, such as 23, 24, or 25 nt; 24 to 27 nt in length, such as 24, 25, 26, or 27 nt; 27-30 nt in length, such as 27, 28, 29, or 30 nt; 30-35 nt in length, such as 30, 31, 32, 33, 34, or 35 nt; or 35 nt or longer. In certain embodiments, the CRISPR / Cas system requires tracrRNA. The term "tracrRNA" or similar terms includes any polynucleotide sequence that is sufficiently complementary to the crRNA sequence for hybridization. In some embodiments, when optimally aligned, the complementarity between the tracrRNA and crRNA sequences along the shorter of the two is about or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the length of the tracr sequence is about or greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides. In some embodiments, the tracr and gRNA sequences are contained in a single transcript, such that hybridization between the two produces a transcript with secondary structure, such as a hairpin. In one embodiment of the invention, the transcript or the transcribed polynucleotide sequence has at least two or more hairpins. In a preferred embodiment, the transcript has two, three, four, or five hairpins. In another embodiment of the invention, the transcript has up to five hairpins. In the hairpin structure, the 5' portion of the final "N" sequence and the upstream of the loop may correspond to the tracr pairing sequence, while the 3' portion of the loop sequence corresponds to the tracr sequence. In the hairpin structure, the 5' portion of the final "N" sequence and the upstream of the loop may alternatively correspond to the tracr sequence, and the 3' portion of the loop sequence corresponds to the tracr pairing sequence. In an alternative embodiment, the CRISPR / Cas system does not require tracrRNA, as is known to those skilled in the art.

[0186] In some embodiments, the guide RNA (capable of guiding Cas to the target locus) may comprise (1) a guide sequence capable of hybridizing with the target locus, and (2) a tracr pairing or positive repeat sequence (oriented 5' to 3', or alternatively 3' to 5', depending on the type of Cas protein, as known to those skilled in the art). In a particular embodiment, the CRISPR / Cas protein is characterized by utilizing a guide RNA comprising a guide sequence capable of hybridizing with the target locus and a positive repeat sequence, and tracr RNA is not required. In a particular embodiment, where the CRISPR / Cas protein is characterized by utilizing tracr RNA, the guide sequence, tracr pairing, and tracr sequence may be present in a single RNA, i.e., sgRNA (oriented 5' to 3', or alternatively 3' to 5'), or the tracr RNA may be a different RNA from the RNA containing the guide and tracr pairing sequences. In these embodiments, tracr hybridizes with the tracr pairing sequence and guides the CRISPR / Cas complex to the target sequence.

[0187] Typically, in the context of endogenous nucleic acid targeting systems, the formation of a nucleic acid targeting complex (comprising a guide RNA that hybridizes to the target sequence and is complexed with one or more nucleic acid targeting effector proteins) results in the modification (e.g., cleavage) of one or both DNA or RNA strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs). As used herein, the term "sequence associated with the target locus of interest" refers to a sequence close to the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence, wherein the target sequence is contained within the target locus of interest). Those skilled in the art will recognize the specific cleavage sites of the selected CRISPR / Cas system relative to the target sequence, which, as is known in the art, may be located within the target sequence or, alternatively, at the 3' or 5' of the target sequence.

[0188] In some embodiments, the unmodified nucleic acid targeting effector protein may have nucleic acid cleavage activity. In some embodiments, the nuclease as described herein may direct the cleavage of one or two nucleic acid (DNA, RNA, or hybrid, which may be single-stranded or double-stranded) strands at or near the target sequence, such as within the target sequence and / or within the complementary sequence of the target sequence or at a sequence associated with the target sequence. In some embodiments, the nucleic acid targeting effector protein may direct the cleavage of one or two DNA or RNA strands within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the cleavage may be blunt-ended (e.g., for Cas9, such as SaCas9 or SpCas9). In some embodiments, the cleavage may be staggered (e.g., for Cpf1), i.e., generating sticky ends. In some embodiments, the cleavage is a staggered cut with 5' overhangs. In some embodiments, the cleavage is a staggered cut with 1 to 5 nucleotides, preferably 4 or 5 nucleotides, at the 5' overhang. In some embodiments, the cleavage site is upstream of the PAM. In some embodiments, the cleavage site is downstream of the PAM. In some embodiments, the nucleic acid targeting effector protein can be mutated relative to the corresponding wild-type enzyme, such that the mutated nucleic acid targeting effector protein lacks the ability to cleave one or both DNA or RNA strands containing the target polynucleotide of the target sequence. As another example, two or more catalytic domains of a Cas protein (e.g., RuvC I, RuvC II, and RuvC III, or the HNH domain of the Cas9 protein) can be mutated to produce a mutant Cas protein that substantially lacks all DNA cleavage activity. In some embodiments, when the cleavage activity of the mutant enzyme is no more than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of the unmutated form of the enzyme, the nucleic acid targeting effector protein can be considered to substantially lack all DNA and / or RNA cleavage activity; examples may be that the nucleic acid cleavage activity of the mutant form is zero or negligible compared to the unmutated form. As used herein, the term "modified" Cas generally refers to a Cas protein that has one or more modifications or mutations (including point mutations, truncations, insertions, deletions, chimeras, fusion proteins, etc.) compared to the wild-type Cas protein from which it is derived. "Derived" means that the derived enzyme is largely based on the wild-type enzyme in the sense of high sequence homology with the wild-type enzyme, but has been mutated (modified) in some manner as known in the art or as described herein.

[0189] In one particular embodiment, a mutant nucleic acid targeting effector protein based on the CRISPR system, as described above, lacking the ability to cleave one or both DNA or RNA strands containing a target polynucleotide containing a target sequence, can be fused to other tools, such as other nucleases, nickases, recombinases, transposases, base editors, or molecular complexes including these tools. As used herein, a “base editor” refers to a protein or fragment thereof having the same catalytic activity as the protein from which it is derived, which, alone or when provided as a molecular complex (referred to herein as a base editing complex), has the ability to mediate targeted base modifications (i.e., the conversion of the base of interest, resulting in a point mutation of interest). Preferably, in the context of this invention, at least one base editor is temporarily or permanently linked to at least one site-specific effector, or optionally linked to a component of at least one site-specific effector complex (e.g., a DNA recognition domain of a CRISPR system, zinc finger, or TAL effector). The linkage can be covalent and / or non-covalent.

[0190] Numerous publications have demonstrated targeted base switching (primarily cytidine (C) to thymine (T) conversion) using CRISPR / Cas9 nickases or nonfunctional nucleases linked to cytidine deaminase domains (i.e., apolipoprotein B mRNA editing catalytic peptide (APOBEC1), such as those derived from rats). Deamination of cytidine (C) is catalyzed by cytidine deaminases and results in uracil (U), which possesses the base-pairing properties of thymine (T). Most known cytidine deaminases act on RNA, and the few known instances that accept DNA require single-stranded (ss) DNA. Studies of the dCas9-target DNA complex have revealed that at least nine nucleotides (nt) of the substituted DNA strand are unpaired during the formation of the Cas9-guide RNA-DNA 'R-loop' complex (Jore et al., Nat. Struct. Mol. Biol., 18, 529-536 (2011)). In fact, the first 11 nt of the pre-interstitial sequence on the substituted DNA strand in the structure of the Cas9 R-loop complex is disordered, indicating that its movement is not highly restricted. It is also speculated that the Cas9 nickase-induced mutation at the cytosine site in the non-template strand may be caused by the accessibility of cytosine deaminases to it. It is inferred that a subset of this ssDNA segment in the R-loop can serve as an effective substrate for dCas9-tethered cytidine deaminases to achieve direct programmable conversion of C to U in DNA (Komor et al., ibid.). Recently, Goudelli et al. ((2017). Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature, 557(7681), 464.) described an adenine base editor (ABE) that mediates the conversion of A·T to G*C in genomic DNA.

[0191] Therefore, any base editing complex according to the present invention may contain at least one cytidine deaminase, or a catalytically active fragment thereof. At least one base editing complex may contain a domain in the form of a cytidine deaminase, or a catalytically active fragment thereof, as a base editor.

[0192] In another embodiment, at least one first targeted base modification is the conversion of any nucleotide C, A, T, or G to any other nucleotide. Any of the C, A, T, or G nucleotides can be exchanged for another nucleotide in a site-specific manner, mediated by a base editor or its catalytically active fragment. Thus, at least one base editing complex can contain any base editor, or its base editor domain or catalytically active fragment, which can convert the nucleotide of interest to any other nucleotide of interest in a targeted manner. In some embodiments, the target sequence should be associated with a PAM (pre-spacer adjacent motif) or a PFS (pre-spacer flanking sequence or site); that is, a short sequence recognized by the CRISPR complex. The precise sequence and length requirements of the PAM vary depending on the CRISPR enzyme used, but a PAM is typically a 2-5 base pair sequence adjacent to the pre-spacer region (i.e., the target sequence). Examples of PAM sequences are given in the Examples section below, and those skilled in the art will be able to identify additional PAM sequences for a given CRISPR enzyme. Furthermore, engineering of the PAM interaction (PI) domain allows for programming of PAM specificity, improving target site recognition fidelity and increasing the versatility of Cas (e.g., Cas9) genome engineering platforms. Cas proteins (such as Cas9) can be engineered to alter their PAM specificity, for example, as described in Kleinstiver BP et al., Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 July 23; 523(7561):481-5. doi:10.1038 / nature14592. In some embodiments, the method includes allowing a CRISPR complex to bind to a target polynucleotide to achieve cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence within the target polynucleotide, wherein the guide sequence is linked to a tracr pairing sequence, which in turn hybridizes to the tracr sequence. Those skilled in the art will understand that other Cas proteins can be modified similarly.

[0193] Cas proteins as mentioned herein, such as but not limited to Cas9, Cpf1 (Cas12a), C2c1 (Cas12b), C2c2 (Cas13a), C2c3, and Cas13b, can be derived from any suitable source and therefore can include different orthologs derived from a variety of (prokaryotic) organisms, as well as well described in the art. In some embodiments, the Cas protein is a (modified) Cas9, preferably a (modified) Staphylococcus aureus Cas9 (SaCas9) or a (modified) Streptococcus pyogenes Cas9 (SpCas9). In some embodiments, the Cas protein is a (modified) Cpf1, preferably an Acidaminococcus sp., such as Acidaminococcus sp. BV3L6Cpf1 (AsCpf1) or a Lachnospiraceae bacterium Cpf1, such as Lachnospiraceae MA2020 or Lachnospiraceae MD2006 (LbCpf1). In some embodiments, the Cas protein is a (modified) C2c2, preferably a Leptotrichia wadei C2c2 (LwC2c2) or a Listeria newyorkensis FSL M6-0635C2c2 (LbFSLC2c2). In some embodiments, the (modified) Cas protein is C2c1. In some embodiments, the (modified) Cas protein is C2c3. In some embodiments, the (modified) Cas protein is Cas13b.

[0194] In some implementations, nucleic acid modification is achieved through random mutagenesis. Cells or organisms can be exposed to mutagens such as UV radiation or mutagenic chemicals (e.g., ethyl methanesulfonate (EMS)) and then mutants with the desired characteristics can be selected. For example, mutants can be identified by TILLING (Targeting Induced Local Lesions in Genomes). This method combines mutagenesis (e.g., using chemical mutagens such as ethyl methanesulfonate (EMS)) with a sensitive DNA screening technique for identifying single-base mutations / point mutations in target genes. The TILLING method relies on the formation of DNA heteroduplexes, which are formed when multiple alleles are amplified by PCR and then heated and slowly cooled. “Bubble-like structures” form at the mismatches of the two DNA strands, which are then cleaved by single-stranded nucleases. The products are then separated by size (e.g., by HPLC). See also McCallum et al., “Targeted screening for induced mutations”; Nat Biotechnol. April 2000; 18(4):455-7 and McCallum et al., “Targeting induced locallesions IN genomes (TILLING) for plant functional genomics”; Plant Physiol. June 2000; 123(2):439-42.

[0195] RNA interference (RNAi) is a biological process in which RNA molecules suppress gene expression or translation by neutralizing targeted mRNA molecules. Two types of small RNA molecules—microRNAs (miRNAs) and small interfering RNAs (siRNAs)—are central to RNAi. RNA is a direct product of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules, increasing or decreasing their activity, for example, by preventing mRNA from being translated into protein. The RNAi pathway is present in many eukaryotes (including animals) and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double-stranded fragments of approximately 21 nucleotides, called siRNAs (small interfering RNAs). Each siRNA unwinds into two single-stranded RNAs (ssRNAs): a lagging strand and a guide strand. The lagging strand is degraded, and the guide strand is introduced into the RNA-induced silencing complex (RISC). Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNAs, but before reaching maturity, miRNAs must first undergo extensive post-transcriptional modifications. miRNAs are expressed as primary transcripts (called miRNA initiators) from a longer RNA-coding gene, which are processed in the cell nucleus by a microprocessor complex into a 70-nucleotide stem-loop structure (called premiRNA). This complex consists of an RNase III enzyme called Drosha and the dsRNA-binding protein DGCR8. The dsRNA portion of this premiRNA is bound and cleaved by Dicer to produce a mature miRNA molecule that can integrate into the RISC complex; thus, miRNAs and siRNAs share the same downstream cellular mechanisms. Short hairpin RNAs or small hairpin RNAs (shRNAs / hairpin vectors) are artificial RNA molecules with tight hairpin loops that can be used to silence target gene expression via RNA interference. The most well-studied results show post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in the messenger RNA molecule and is cleaved by the catalytic component of RISC, Argonaute 2 (Ago2). As used herein, RNAi molecules can be siRNAs, shRNAs, or miRNAs. It should be understood that RNAi molecules can be applied as is in plants, or they can be encoded by appropriate vectors that express RNAi molecules. Delivery and expression systems for RNAi molecules such as siRNA, shRNA, or miRNA are well known in the art.

[0196] As used herein, the term "homozygous" refers to a single cell or plant having the same alleles at one or more loci. When the term is used to refer to a specific locus or gene, it at least means that the locus or gene has the same alleles. As used herein, the term "homozygous" means the genetic condition present when the same alleles are located at corresponding loci on homologous chromosomes. As used herein, the term "heterozygous" refers to a single cell or plant having different alleles at one or more loci. When the term is used to refer to a specific locus or gene, it at least means that the locus or gene has different alleles. As used herein, the term "heterozygous" means the genetic condition present when different alleles are located at corresponding loci on homologous chromosomes. In some embodiments, the QTLs and / or one or more markers as described herein are homozygous. In some embodiments, the QTLs and / or one or more markers as described herein are heterozygous. In some embodiments, the QTL alleles, polynucleotides, and / or one or more marker alleles as described herein are homozygous. In some implementations, the QTL alleles, polynucleotides, and / or one or more marker alleles, as described herein, are heterozygous.

[0197] A “marker” is a genetic or physical map (a means of finding a specific location), or a linkage between a marker and a trait locus (a locus that influences a trait). The location detected by a marker can be determined by detecting polymorphic alleles and their genetic mapping, or by hybridization, sequence matching, or amplification of physically mapped sequences. Markers can be DNA markers (detecting DNA polymorphisms), proteins (detecting variations at the coding polypeptide), or simple genetic phenotypes (such as “waxy” phenotypes). DNA markers can be developed from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., spliced ​​RNA or cDNA). Depending on the DNA marker technology, a marker can consist of complementary primers flanking the locus and / or complementary probes that hybridize to the polymorphic allele at the locus. The term “marker locus” refers to the locus (gene, sequence, or nucleotide) that the marker detects. The terms “marker,” “molecular marker,” or “marker locus” can also be used to refer to nucleic acid or amino acid sequences that are unique enough to characterize a specific locus on the genome. Any detectable polymorphic trait can be used as a marker, as long as it is differentially inherited and shows linkage disequilibrium with the phenotypic trait of interest.

[0198] Markers for detecting genetic polymorphisms among population members are well-established in the field. Markers can be defined by the type of polymorphism they detect and the marking techniques used to detect it. Marker types include, but are not limited to, detection of restriction fragment length polymorphism (RFLP), isoenzyme labeling, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple repeat sequence (SSR), amplified variable sequences in plant genomes, detection of self-continuous sequence replication, or single nucleotide polymorphism (SNP). SNPs can be detected, for example, via DNA sequencing, PCR-based sequence-specific amplification methods, detection of polynucleotide polymorphisms by allele-specific hybridization (ASH), dynamic allele-specific hybridization (DASH), molecular beacons, microarray hybridization, oligonucleotide ligase assays, Flap endonucleases, 5' endonucleases, primer extension, single-strand conformation polymorphism (SSCP), or temperature gradient gel electrophoresis (TGGE). DNA sequencing (such as pyrosequencing) has the advantage of being able to detect a series of linked SNP alleles that make up a haplotype. Haplotypes are often more informative than SNPs (detecting higher levels of polymorphism).

[0199] "(Molecular) marker allele," or "allele of a marker locus," can refer to one of several polymorphic nucleotide sequences found at a marker locus in a population. With respect to SNP markers, an allele refers to a specific nucleotide base present at that SNP locus in a single plant. As used herein, the term (molecular) marker allele may be used interchangeably with "donor allele" or "allele donor," and refers to the (molecular) marker allele associated with improved digestibility according to the invention, unless otherwise explicitly stated.

[0200] "Fine mapping" refers to methods that can more accurately pinpoint (narrow down) the location of a QTL and reduce the size of the introgression fragment containing the QTL. For example, near-isogenic lines of QTLs (QTL-NILs) can be prepared, containing overlapping fragments of different introgression fragments within the originally consistent genetic background of the recurrent parents. Such lines can then be used to map which fragment the QTL is located on and to identify lines with shorter introgression fragments containing the QTL.

[0201] Marker-assisted selection (MAS) is the process of selecting individual plants based on marker genotypes. Marker-assisted anti-selection is the process of using marker genotypes to identify plants that will not be selected, thereby allowing them to be removed from breeding programs or cultivation. Marker-assisted selection uses the presence of molecular markers genetically linked to specific loci or regions of chromosomes (e.g., introgression, transgenes, polymorphisms, mutations, etc.) to select plants that contain those specific loci or regions. For example, molecular markers genetically linked to digestibility QTLs, as defined herein, can be used to detect and / or select plants that contain QTLs on chromosome 9. The closer the molecular marker is genetically linked to the locus (e.g., approximately 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM or less), the less likely the marker is to dissociate from the locus through meiotic recombination. Similarly, the closer two markers are linked to each other (e.g., within 7 cM or 5 cM, 4 cM, 3 cM, 2 cM, 1 cM or less), the less likely the two markers are to separate from each other (and the greater the likelihood that they co-segregate as a unit). The “LOD-score” (log-odds ratio, base 10) is a statistical test commonly used in linkage analysis for animal and plant populations. The LOD score compares the probability of obtaining test data (if two loci (molecular marker loci and / or phenotypic trait loci) are actually linked) to the probability of observing the same data purely by chance. A positive LOD score supports the presence of linkage, and an LOD score greater than 3.0 is considered evidence of linkage. An LOD score of +3 indicates a probability that the observed linkage is not accidental (1 in 1000).

[0202] "Marker haplotype" refers to the combination of alleles at a marker locus.

[0203] A "marker locus" is a specific chromosomal location in a species' genome where a particular marker can be found. Marker loci can be used to track the presence of second-linked loci (e.g., loci that influence the expression of phenotypic traits). For example, marker loci can be used to monitor the segregation of alleles at genetically or physically linked loci.

[0204] A “labeled probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus by nucleic acid hybridization, for example, a nucleic acid probe complementary to a marker locus sequence. A labeled probe containing 30 or more consecutive nucleotides (“all or part” of the marker locus sequence) can be used for nucleic acid hybridization. Alternatively, in some respects, a labeled probe refers to any type of probe capable of distinguishing (i.e., genotyping) the presence of a specific allele at a marker locus.

[0205] The term "molecular marker" can be used to refer to a genetic marker or its encoded product (e.g., a protein) used as a reference point when identifying linked loci. Markers can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., spliced ​​RNA, cDNA, etc.), or encoded polypeptides. The term also refers to nucleic acid sequences complementary to or flanked by the marker sequence, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A "molecular marker probe" is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, for example, a nucleic acid probe complementary to the marker locus sequence. Alternatively, in some respects, a marker probe refers to any type of probe capable of distinguishing (i.e., genotyping) the specific allele present at a marker locus. Nucleic acids are "complementary" when they hybridize specifically in solution, for example, according to the Watson-Crick base pairing rule. Some markers described herein are also called hybridization markers when located on insertion / deletion regions (such as non-collinear regions as described herein). This is because, by definition, insertion regions are polymorphic relative to plants without insertions. Therefore, the marker only needs to indicate the presence or absence of insertion / deletion regions. Such hybridization markers can be identified using any suitable marker detection technique, such as the SNP technique used in the examples provided herein.

[0206] "Genetic markers" are polymorphic nucleic acids in a population, where alleles can be detected and distinguished by one or more analytical methods (e.g., RFLP, AFLP, isoenzymes, SNPs, SSRs, etc.). The terms "molecular marker" and "genetic marker" are used interchangeably herein. The term also refers to nucleic acid sequences complementary to the genome sequence, such as nucleic acids used as probes. Markers corresponding to genetic polymorphism among population members can be detected by methods recognized in the art. These methods include, for example, PCR-based sequence-specific amplification methods, detection of restriction fragment length polymorphism (RFLP), detection of isoenzyme markers, detection of polynucleotide polymorphisms by allele-specific hybridization (ASH), detection of amplified variable sequences in plant genomes, detection of self-continuous sequence replication, detection of simple repeat sequences (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Recognized methods for detecting expressed sequence tags (ESTs) and EST-derived SSR markers, as well as random amplified polymorphic DNA (RAPD), are also known.

[0207] "Polymorphism" is a DNA variation between two or more individuals within a population. Polymorphism preferably has a frequency of at least 1% in the population. Available polymorphisms may include single nucleotide polymorphisms (SNPs), simple repeat sequences (SSRs), or insertion / deletion polymorphisms, also referred to herein as "insertions and deletions." The term "insertion and deletion" refers to an insertion or deletion, wherein one strain may be referred to as having an inserted nucleotide or DNA fragment relative to a second strain, or the second strain may be referred to as having a deleted nucleotide or DNA fragment relative to a first strain.

[0208] The “physical distance” between loci on the same chromosome (e.g., between molecular markers and / or between phenotypic markers) is the actual physical distance expressed in bases or base pairs (bp), kilobases or kilobase pairs (kb), or megabases or megabase pairs (Mb).

[0209] The “genetic distance” between loci on the same chromosome (e.g., between molecular markers and / or phenotypic markers) is measured by the frequency of recombination, or recombination frequency (RF), and is indicated in centimoles (cM). 1 cM corresponds to a 1% recombination frequency. If no recombinants are found, the RF is zero, and the loci are physically very close or they are identical. The farther apart two loci are, the higher the RF.

[0210] A genome's "physical map" is a map showing the linear order of identifiable markers (including genes, markers, etc.) on chromosomal DNA. However, in contrast to a genetic map, the distances between markers are absolute (e.g., measured in base pairs or consecutive segments of separated and overlapping genetic material) and are not based on genetic recombination (which can vary across different populations).

[0211] Alleles are negatively correlated with traits when they are linked, and when the presence of an allele indicates that the desired trait or trait form is not present in plants containing the allele. Alleles are positively correlated with traits when they are linked, and when the presence of an allele indicates that the desired trait or trait form is present in plants containing the allele.

[0212] Centiliter (“cM”) is a unit of measurement for recombination frequency. 1 cM is equal to the 1% probability that a marker at one locus will separate from a marker at another locus due to crossover in a single generation.

[0213] As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA located on a single chromosome in a plant. Genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some respects, genetic elements within a single chromosomal interval are genetically linked, typically having a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively less than or equal to 10 cM. ​​That is, two genetic elements within a single chromosomal interval recombine at a frequency of less than or equal to 20% or 10%.

[0214] In this application, the term "closely linked" means that recombination between two linked loci occurs at a frequency equal to or less than about 10% (i.e., segregation on a genetic map of no more than 10 cM). In other words, closely linked loci co-segregate at least 90% of the time. Marker loci are particularly useful to the subject matter of this disclosure when they exhibit a significant probability of co-segregation (linkage) with a desired trait (e.g., resistance to gray leaf spot). Closely linked loci, such as marker loci and second loci, may exhibit a recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, even more preferably about 7% or less, still more preferably about 6% or less, even more preferably about 5% or less, still more preferably about 4% or less, even more preferably about 3% or less, and still more preferably about 2% or less. In a highly preferred embodiment, the relevant loci exhibit a recombination frequency of about 1% or less, for example, about 0.75% or less, more preferably about 0.5% or less, or even more preferably about 0.25% or less. Two loci are also referred to as "proximately adjacent" to each other: located on the same chromosome, and the distance between the two loci is such that recombination occurs between them at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less). In some cases, two different markers may have the same genetic map coordinates. In this case, the two markers are very close to each other, causing recombination between them to occur at a frequency so low as to be undetectable.

[0215] "Linkage" refers to the tendency for alleles to segregate together more frequently than by chance, if the transmission of alleles is independent. Generally, linkage refers to alleles on the same chromosome. Genetic recombination occurs throughout the genome at a presumed random frequency. Genetic maps are constructed by measuring the frequency of recombination between pairs of traits or markers. The closer the traits or markers on a chromosome are to each other, the lower the recombination frequency and the greater the linkage. If traits or markers are generally co-segregating, they are considered linked in this paper. A recombination probability of 1 / 100 per generation is defined as a genetic map distance of 1.0 centimole (1.0 cM). The term "linkage disequilibrium" refers to the non-random segregation of a genetic locus or trait (or both). In either case, linkage disequilibrium means that the related loci are within sufficient physical proximity along the length of the chromosome such that they segregate together at a frequency greater than random (i.e., non-random). Markers exhibiting linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, for example, from about 51% to about 100%. In other words, two co-segregating markers have a recombination frequency of less than 50% (and, by definition, segregation on the same linkage group is less than 50 cM). As used herein, linkage can be between two markers, or alternatively between a marker and a locus influencing the phenotype. A marker locus can be “associated” (linked) with a trait. The degree of linkage between a marker locus and a locus influencing a phenotypic trait is measured, for example, by the statistical probability of co-segregation of the molecular marker and the phenotype (e.g., F-statistic or LOD score).

[0216] As used herein, the term "sequence identity" refers to the degree of similarity between any given nucleic acid sequence and a target nucleic acid sequence. The percentage of sequence identity is calculated by determining the number of matching positions in the aligned nucleic acid sequences, dividing the number of matching positions by the total number of aligned nucleotides, and multiplying by 100. A matching position is the location where the same nucleotide appears at the same position in the aligned nucleic acid sequence. The percentage of sequence identity for any amino acid sequence can also be determined. To determine the percentage of sequence identity, the BLAST2Sequences (Bl2seq) program, a standalone version of BLASTZ containing BLASTN and BLASTP, is used to compare the target nucleic acid or amino acid sequence with the identified nucleic acid or amino acid sequence. This standalone version of BLASTZ is available from Fish & Richardson's website (World Wide Web, fr.com / blast) or the US government's National Center for Biotechnology Information website (World Wide Web, ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program are available in the Read Me archive accompanying BLASTZ. BI2seq uses the BLASTN or BLASTP algorithm to perform comparisons between two sequences.

[0217] BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, set the options as follows: -i to the file containing the first nucleic acid sequence to be compared (e.g., C:\seql.txt); -j to the file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p to nucleotide sequence alignment; -o to any desired filename (e.g., C:\output.txt); -q to -1; -r to 2; and all other options retain their default settings. The following command will generate an output file containing the comparison between the two sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. If the target sequence shares homology with any part of the identification sequence, the specified output file will present those homologous regions as the alignment sequences. If the target sequence does not share homology with any part of the identification sequence, the specified output file will not present the alignment sequences. Once aligned, the length is determined by counting the number of consecutive nucleotides of the target sequence that appear in the alignment with the identification sequence that begins and ends at any other matching position. A matching position is any location in both the target and identification sequences that presents the same nucleotide. Vacancies in the target sequence are not counted because vacancies are not nucleotides. Similarly, vacancies in the identification sequence are not counted because nucleotides in the target sequence, not the identification sequence, are counted. The percentage of identity at a given length is determined by calculating the number of matching positions at that length, dividing that number by the length, and then multiplying the result by 100. For example, if (i) a 500-base target nucleic acid sequence is compared to a subject nucleic acid sequence, (ii) the Bl2seq program presents 200 bases of the target sequence aligned to a region of the subject sequence, where the first and last bases of that 200-base region match, and (iii) the number of matches on those 200 aligned bases is 180, then the 500-base target nucleic acid sequence contains a length of 200 and 90% sequence identity at that length (i.e., 180 / 200 × 100 = 90). It should be understood that different regions within a single target nucleic acid sequence aligned to the identified sequence can each have their own percentage identity. It should be noted that percentage identity values ​​are rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It should also be noted that the length values ​​will always be integers.

[0218] The term "sequence" as used herein refers to nucleotide sequences, polynucleotides, nucleic acid sequences, nucleic acids, nucleic acid molecules, peptides, polypeptides, and proteins, depending on the context in which the term "sequence" is used. The terms "nucleotide sequence," "polynucleotide," "nucleic acid sequence," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein and refer to any length of polymeric, unbranched nucleotide (ribonucleotide or deoxyribonucleotide, or a combination of both). As will be readily understood by those skilled in the art, nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derived nucleotide bases.

[0219] "Isolated nucleic acid sequence" or "isolated DNA" refers to a nucleic acid sequence that is no longer in the natural environment in which it was isolated (e.g., a nucleic acid sequence in the nuclear or plasmonic genome of a bacterial host cell or plant). When the term "sequence" is used herein, it should be understood to refer to molecules having such sequences, such as nucleic acid molecules. The terms "host cell," "recombinant host cell," or "transformed cell" refer to a new, separate cell (or organism) produced due to the introduction of at least one nucleic acid molecule into said cell. The host cell is preferably a plant cell or a bacterial cell. The host cell may contain nucleic acids as extrachromosomal (free gene) replication molecules, or contain nucleic acids integrated into the nuclear or plasmonic genome of the host cell, or as nucleic acids of an introduced chromosome (e.g., a miniature chromosome).

[0220] When used herein, the terms “polypeptide” or “protein” (these two terms are used interchangeably herein) mean a peptide, protein, or polypeptide encompassing an amino acid chain of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, this invention also covers protein / polypeptide analogs in which the amino acids and / or peptide bonds have been replaced by functional analogs, as well as amino acids other than the 20 genetically encoded amino acids, such as selenocysteine. Peptides, oligopeptides, and proteins may be referred to as polypeptides. The term polypeptide also refers to, but does not exclude, modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well described in the basic text and more detailed monographs and research literature.

[0221] Amino acid substitution encompasses amino acid alterations in which an amino acid is replaced by a different, naturally occurring amino acid residue. Such substitutions can be classified as conserved. <1> In this invention, amino acid residues present in wild-type proteins are replaced by another naturally occurring amino acid with similar characteristics, such as Gly<->Ala, Val<->lle<->Leu, Asp<->Glu, Lys<->Arg, Asn<->Gln, or Phe<->Trp<->Tyr. The substitutions covered by this invention can also be “non-conservative,” in which amino acid residues present in wild-type proteins are replaced by amino acids with different characteristics, such as naturally occurring amino acids from different classes (e.g., replacing charged or hydrophobic amino acids with alanine). As used herein, “similar amino acid” refers to an amino acid having similar amino acid side chains, i.e., amino acids with polar, nonpolar, or nearly neutral side chains. As used herein, “dissimilar amino acid” refers to an amino acid with different amino acid side chains; for example, amino acids with polar side chains are dissimilar to amino acids with nonpolar side chains. Polar side chains generally tend to be present on the surface of proteins, where they can interact with the aqueous environment present in the cell (“hydrophilic” amino acids). On the other hand, "nonpolar" amino acids tend to be located at the center of proteins, where they can interact with similar nonpolar neighbors ("hydrophobic" amino acids). Examples of amino acids with polar side chains are arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, and threonine (all hydrophilic except for hydrophobic cysteine). Examples of amino acids with nonpolar side chains are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan (all hydrophobic except for neutral glycine).

[0222] The term "gene," as used herein, refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. The term includes double- and single-stranded DNA and RNA. It also includes modifications of known types, such as methylation, "capping," and substitution of one or more naturally occurring nucleotides with analogs. Preferably, a gene contains a coding sequence encoding a polypeptide as defined herein. A "coding sequence" is a nucleotide sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into mRNA and / or translated into a polypeptide. The boundaries of a coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Coding sequences may include, but are not limited to, mRNA, cDNA, recombinant nucleic acid sequences, or genomic DNA, and in some cases, introns may be present.

[0223] As used herein, the term "endogenous" refers to a gene or allele present at its natural genomic location. The term "endogenous" may be used interchangeably with "natural" or "wild-type." However, this does not preclude the presence of one or more nucleic acid differences from the wild-type allele. In a particular embodiment, the differences from the wild-type allele may be limited to fewer than nine, preferably fewer than six, more particularly fewer than three nucleotide differences, such as zero nucleotide differences. More particularly, the difference from the wild-type sequence may be only one nucleotide. Preferably, the endogenous allele encodes a modified protein with fewer than nine, preferably fewer than six, more particularly fewer than three, and even more preferably only one or no amino acid differences compared to the wild-type protein.

[0224] As used herein, the term "exogenous polynucleotide" refers to a polynucleotide, such as a gene (or cDNA) or allele, that has been introduced or has been introduced into a cell (or plant) through recombination. Exogenous polynucleotides can be free or integrated into the genome. Integration can be random or site-directed. Integration may include the substitution of the corresponding endogenous polynucleotide. It should be understood that exogenous polynucleotides do not naturally exist in cells or plants.

[0225] When referring to a nucleic acid sequence (e.g., DNA or genomic DNA) that has “significant sequence identity” with a reference sequence or at least 60% sequence identity with a reference sequence, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% nucleic acid sequence identity, in one embodiment, the nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using stringent hybridization conditions. In another embodiment, the nucleic acid sequence contains one or more mutations compared to the given nucleotide sequence, but can still be identified using stringent hybridization conditions. “Stringent hybridization conditions” can be used to identify nucleotide sequences that are substantially identical to a given nucleotide sequence. Stringent conditions are sequence-dependent and vary under different conditions. Typically, stringent conditions are chosen to be about 5°C lower than the thermal melting temperature (Tm) of the specific sequence at defined ionic strengths and pH. Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at defined ionic strengths and pH). Typically, stringent conditions are chosen where the salt concentration is about 0.02 mol at pH 7 and the temperature is at least 60°C. Decreasing the salt concentration and / or increasing the temperature increases the stringency. Stringent conditions for RNA-DNA hybridization (using, for example, a 100 nt probe, Northern blotting) include, for example, those involving at least one wash for 20 minutes at 63°C in 0.2X SSC, or equivalent conditions. Stringent conditions for DNA-DNA hybridization (using, for example, a 100 nt probe, Southern blotting) include, for example, those involving at least one wash (usually two) for 20 minutes at 50°C (typically about 55°C) in 0.2X SSC, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001). Examples of highly stringent hybridization conditions are those that primarily involve hybridization only of nucleic acid molecules having at least 90% or at least 95% sequence identity. Such highly stringent hybridization conditions are, for example, 4X SSC at 65°C, followed by multiple washes in 0.1X SSC at 65°C for approximately 1 hour. As used herein, the term "highly stringent hybridization conditions" can also mean hybridization at 68°C for 16 hours in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA, followed by two washes at 68°C with 2x SSC and 0.1% SDS. Preferably, hybridization is performed under stringent conditions. Less stringent hybridization conditions include, for example, hybridization at 37°C in 4x SSC, followed by multiple washes at room temperature in 1x SSC.

[0226] As used in this article, degenerate nucleotides refer to those based on the IUPAC nucleotide code standard.

[0227] As used in this article, F35H (ExPASy enzyme entry EC 1.14.13.88) refers to the gene or protein of flavonoid 3',5'-hydroxylase. F35H is also known as F3'5'H, F3',5'H, cytochrome P450 flavonoid 3',5'-hydroxylase, or flavanone, NADPH: oxygen oxidoreductase. F35H catalyzes the following reaction: flavanone + 2NADPH + 2O(2) <=> 3',5'-dihydroxyflavanone + 2NADP(+) + 2H(2)O.

[0228] This invention relates to maize plants or plant parts, such as straw, characterized by having specific (molecular) markers (alleles) particularly associated with improved (straw) digestibility. The invention also relates to the use of such markers for generating and identifying or selecting maize plants or plant parts. Furthermore, the invention relates to (isolated) polynucleotides containing such markers or suitable for identifying or detecting such markers.

[0229] Suitable markings for use according to certain embodiments of the invention are provided in Table A below.

[0230] Table A

[0231]

[0232]

[0233]

[0234]

[0235]

[0236] Preferred and suitable markings for use in certain embodiments of the invention are provided in Table B below.

[0237] Table B

[0238]

[0239]

[0240] The most preferred and appropriate designations used in certain embodiments of the invention are provided in Table C below.

[0241] Table C

[0242]

[0243] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of a QTL, or a fragment thereof, on chromosome 9 of the maize plant or plant part (the genome, such as isolated genetic material from the plant or plant part), the QTL being particularly associated with improved (straw) digestibility, side-linked with SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or contained in a polynucleotide or QTL (allele) containing and / or side-linked with (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01.

[0244] As mentioned herein, a polynucleotide (such as a QTL (allele) as described herein) is referred to as having certain molecular markers or marker alleles attached to it if it is contained within a polynucleotide in which a first marker (allele) is located upstream (i.e., 5') and a second marker (allele) is located downstream (i.e., 3'). Such first and second markers (alleles) can border polynucleotides. Nucleic acids can also contain such first and second markers (alleles), such as at or near the 5' and 3' ends, for example, within 50 kb of the 5' and 3' ends, preferably within 10 kb of the 5' and 3' ends, such as within 5 kb of the 5' and 3' ends, within 1 kb of the 5' and 3' ends, or less.

[0245] Technicians should understand that detecting any (molecular) marker (allele) in Tables A, B, or C is equivalent to detecting any other (molecular) marker (allele), provided they are closely linked. For example, screening for polynucleotides containing ma61134d16 or ma61134d15 can be performed by screening for these markers (alleles) themselves, or alternatively by screening Tables A or B, preferably any other markers (alleles) in Table B.

[0246] In some embodiments, neither ma61134d16 nor ma61134d15 is present in the polynucleotides or QTLs (alleles) of the present invention as described elsewhere herein.

[0247] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of a QTL, or a fragment thereof, on chromosome 9 of the maize plant or plant part (the genome, such as the isolated genetic material from the plant or plant part), the QTL being particularly associated with improved (straw) digestibility, side-linked with SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or contained in a polynucleotide or QTL (allele) containing and / or side-linked with the (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01.

[0248] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of a QTL, or a fragment thereof, on chromosome 9 of the maize plant or plant part (the genome, such as isolated genetic material from the plant or plant part), the QTL being particularly associated with improved (straw) digestibility, side-linked with SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or contained in a polynucleotide or QTL (allele) containing and / or side-linked with (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01; and selecting plants or plant parts in which the QTL is present.

[0249] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of a QTL, or a fragment thereof, on chromosome 9 of the maize plant or plant part (the genome, such as the isolated genetic material from the plant or plant part), the QTL being particularly associated with improved (straw) digestibility, side-linked with SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or contained in a polynucleotide or QTL (allele) containing and / or side-linked with (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01; and selecting plants or plant parts in which the QTL is present.

[0250] Preferably, the QTL contains one or more (molecular) markers (alleles) from Table A. More preferably, the QTL contains one or more (molecular) markers (alleles) from Table B. Most preferably, the QTL contains one or more (molecular) markers (alleles) from Table C.

[0251] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more (molecular) markers (alleles) of Table A.

[0252] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part) particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more (molecular) markers (alleles) of Table A.

[0253] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more (molecular) markers (alleles) of Table A; and selecting plants or plant parts in which the polynucleotide is present.

[0254] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide containing one or more (molecular) markers (alleles) of Table A; and selecting plants or plant parts in which the polynucleotide is present.

[0255] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more (molecular) markers (alleles) of Table B.

[0256] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part) particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more (molecular) markers (alleles) of Table B.

[0257] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more (molecular) markers (alleles) of Table B; and selecting plants or plant parts in which the polynucleotide is present.

[0258] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more (molecular) markers (alleles) of Table B; and selecting plants or plant parts in which the polynucleotide is present.

[0259] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more (molecular) markers (alleles) of Table C.

[0260] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part) particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more (molecular) markers (alleles) of Table C.

[0261] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more (molecular) markers (alleles) of Table C; and selecting plants or plant parts in which the polynucleotide is present.

[0262] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide containing one or more (molecular) markers (alleles) of Table C; and selecting plants or plant parts in which the polynucleotide is present.

[0263] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more of SEQ ID NO:13-195 (with a marker (donor) allele at the specified position in Table A).

[0264] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part) particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more of SEQ ID NO:13-195 (with a marker (donor) allele at the position specified in Table A).

[0265] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one of SEQ ID NO:13-195 (with a marker (donor) allele at the specified position in Table A); and selecting plants or plant parts in which the polynucleotide is present.

[0266] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more of SEQ ID NO:13-195 (with a marker (donor) allele at the specified position in Table A); and selecting plants or plant parts in which the polynucleotide is present.

[0267] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more of SEQ ID NO:109-154 (with a marker (donor) allele at the specified position in Table A).

[0268] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part) particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more of SEQ ID NO:109-154 (with a marker (donor) allele at the specified position in Table A).

[0269] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more of SEQ ID NO:109-154 (with a marker (donor) allele at the specified position in Table A); and selecting plants or plant parts in which the polynucleotide is present.

[0270] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more of SEQ ID NO:109-154 (with a marker (donor) allele at the specified position in Table A); and selecting plants or plant parts in which the polynucleotide is present.

[0271] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more of SEQ ID NO:124-125 (with a marker (donor) allele at the position specified in Table A).

[0272] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part) particularly associated with improved (straw) digestibility, the polynucleotides comprising one or more of SEQ ID NO:124-125 (with a marker (donor) allele at the specified position in Table A).

[0273] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more of SEQ ID NO:124-125 (with a marker (donor) allele at the specified position in Table A); and selecting plants or plant parts in which the polynucleotide is present.

[0274] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising one or more of SEQ ID NO:124-125 (with a marker (donor) allele at the specified position in Table A); and selecting plants or plant parts in which the polynucleotide is present.

[0275] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising (molecular) markers (alleles) ma61134d15 and / or ma61134d16.

[0276] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotides comprising (molecular) markers (alleles) ma61134d15 and / or ma61134d16.

[0277] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising (molecular) markers (alleles) ma61134d15 and / or ma61134d16; and selecting plants or plant parts in which the polynucleotide is present.

[0278] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of polynucleotides, such as QTLs or fragments thereof, in the genome of the maize plant or plant part (e.g., the isolated genetic material from the plant or plant part), particularly associated with improved (straw) digestibility, the polynucleotide comprising (molecular) markers (alleles) ma61134d15 and / or ma61134d16; and selecting plants or plant parts in which the polynucleotide is present.

[0279] In some of the above embodiments, identifying / detecting or screening for the presence of polynucleotides includes identifying / detecting or screening for the presence of any designated (molecular) marker (allele) or SEQ ID No.

[0280] In some embodiments, the polynucleotide or QTL (allele) is located on chromosome 9 and contains and / or is flanked by the (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or is contained in a polynucleotide or QTL (allele) containing and / or flanked by the (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01.

[0281] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more (molecular) markers (alleles) of Table A in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part).

[0282] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of one or more (molecular) markers (alleles) of Table A in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part).

[0283] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more (molecular) markers (alleles) of Table A in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0284] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of one or more (molecular) markers (alleles) of Table A in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0285] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more (molecular) markers (alleles) of Table B in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part).

[0286] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of one or more (molecular) markers (alleles) of Table B in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part).

[0287] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more (molecular) markers (alleles) of Table B in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0288] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of one or more (molecular) markers (alleles) of Table B in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0289] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more (molecular) markers (alleles) of Table C in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part).

[0290] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of one or more (molecular) markers (alleles) of Table C in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part).

[0291] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more (molecular) markers (alleles) of Table C in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0292] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening for the presence of one or more (molecular) markers (alleles) of Table C in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0293] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more of the following in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part): SEQ ID NO:13-195 (with a marker (donor) allele at the specified position in Table A).

[0294] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening the presence of one or more of the following in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part): SEQ ID NO:13-195 (with a marker (donor) allele at the specified position in Table A).

[0295] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening the presence of one of SEQ ID NO:13-195 (with a marker (donor) allele at the specified position in Table A) in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the SEQ ID NO is present.

[0296] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening the presence of one or more of SEQ ID NO:13-195 (with a marker (donor) allele at the specified position in Table A) in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the SEQ ID NO is present.

[0297] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more of the following in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part): SEQ ID NO:109-154 (with a marker (donor) allele at the specified position in Table A).

[0298] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of one or more of the following in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part): SEQ ID NO:109-154 (with a marker (donor) allele at the specified position in Table A).

[0299] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening the presence of one or more of SEQ ID NO: 109-154 (with a marker (donor) allele at the specified position in Table A) in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the SEQ ID NO is present.

[0300] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening the presence of one or more of SEQ ID NO: 109-154 (with a marker (donor) allele at the specified position in Table A) in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the SEQ ID NO is present.

[0301] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of one or more of the following in the genome of the maize plant or plant part (e.g., isolated genetic material from the plant or plant part): SEQ ID NO:124-125 (with a marker (donor) allele at the specified position in Table A).

[0302] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening the presence of one or more of the following in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part): SEQ ID NO:124-125 (with a marker (donor) allele at the specified position in Table A).

[0303] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening the presence of one or more of SEQ ID NO: 124-125 (with a marker (donor) allele at the specified position in Table A) in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the SEQ ID NO is present.

[0304] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening the presence of one or more of SEQ ID NO: 124-125 (with a marker (donor) allele at the specified position in Table A) in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the SEQ ID NO is present.

[0305] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of (molecular) markers (alleles) ma61134d15 and / or ma61134d16 in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part).

[0306] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening for the presence of (molecular) markers (alleles) ma61134d15 and / or ma61134d16 in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part).

[0307] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of (molecular) markers (alleles) ma61134d15 and / or ma61134d16 in the genome of the maize plant or plant part (such as isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0308] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; identifying or screening the presence of (molecular) markers (alleles) ma61134d15 and / or ma61134d16 in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part); and selecting plants or plant parts in which the (molecular) markers (alleles) are present.

[0309] In some embodiments of the identification method of the present invention, the maize plant or plant part contains a polynucleotide containing one or more (molecular) markers (alleles) from Table A. In some embodiments of the identification method of the present invention, the maize plant or plant part contains a polynucleotide containing one or more (molecular) markers (alleles) from Table B. In some embodiments of the identification method of the present invention, the maize plant or plant part contains a polynucleotide containing one or more (molecular) markers (alleles) from Table C.

[0310] In some embodiments of the identification method of the present invention, the maize plant or plant part contains a polynucleotide having a sequence as shown in SEQ ID NO: 1 or 4. In some embodiments of the identification method of the present invention, the maize plant or plant part contains a polynucleotide having a coding sequence or encoding a polynucleotide having a sequence as shown in SEQ ID NO: 2 or 5. In some embodiments of the identification method of the present invention, the maize plant or plant part contains a polynucleotide encoding a polypeptide having a sequence as shown in SEQ ID NO: 3 or 6.

[0311] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotide sequences selected from the following in the genome (e.g., isolated genetic material from the plant or plant part):

[0312] a) The nucleotide sequence of SEQ ID NO:7;

[0313] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0314] c) A nucleotide sequence that is at least 90%, preferably at least 95%, identical to SEQ ID NO:7 or 8;

[0315] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or having at least 90%, preferably at least 95%, the same amino acid sequence as the sequence of SEQ ID NO:9;

[0316] The nucleotide sequence contains one or more nucleotides at the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or at the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably resulting in a frameshift, and most preferably an insertion of 8 or 7 nucleotides.

[0317] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening the presence of polynucleotides in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part) containing sequences selected from:

[0318] a) The nucleotide sequence of SEQ ID NO:7;

[0319] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0320] c) A nucleotide sequence that is at least 90%, preferably at least 95%, identical to SEQ ID NO:7 or 8;

[0321] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or having at least 90%, preferably at least 95%, the same amino acid sequence as the sequence of SEQ ID NO:9;

[0322] The nucleotide sequence contains one or more nucleotides at the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or at the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably resulting in a frameshift, and most preferably an insertion of 8 or 7 nucleotides.

[0323] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotide sequences selected from the following in the genome (e.g., isolated genetic material from the plant or plant part):

[0324] a) The nucleotide sequence of SEQ ID NO:7;

[0325] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0326] c) A nucleotide sequence that is at least 90%, preferably at least 95%, identical to SEQ ID NO:7 or 8;

[0327] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or having at least 90%, preferably at least 95%, the same amino acid sequence as the sequence of SEQ ID NO:9;

[0328] The nucleotide sequence contains one or more nucleotides at the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or at the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably resulting in a frameshift, and most preferably an insertion of 8 or 7 nucleotides; and the plant or plant part in which the polynucleotide is present is selected.

[0329] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening the presence of polynucleotides in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part) containing sequences selected from:

[0330] a) The nucleotide sequence of SEQ ID NO:7;

[0331] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0332] c) A nucleotide sequence that is at least 90%, preferably at least 95%, identical to SEQ ID NO:7 or 8;

[0333] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or having at least 90%, preferably at least 95%, the same amino acid sequence as the sequence of SEQ ID NO:9;

[0334] The nucleotide sequence contains one or more nucleotides at the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or at the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably resulting in a frameshift, and most preferably an insertion of 8 or 7 nucleotides; and the plant or plant part in which the polynucleotide is present is selected.

[0335] Those skilled in the art will understand that the corresponding nucleotide positions can be determined by appropriate comparison, as is known in the art.

[0336] In some implementations, the insertion of 7 nucleotides has the sequence gcggtct.

[0337] In some implementations, the 8-nucleotide insertion has the sequence gcggttct.

[0338] In some implementations, the polynucleotide has a sequence selected from the following:

[0339] a) The nucleotide sequence of SEQ ID NO:7;

[0340] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0341] c) The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9;

[0342] The nucleotide sequence has an insertion of 8 or 7 nucleotides at the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7.

[0343] In some implementations, the polynucleotide has a sequence selected from the following:

[0344] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0345] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0346] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0347] In some implementations, the polynucleotide has a sequence selected from the following:

[0348] a) The nucleotide sequence of SEQ ID NO: 1 or 4;

[0349] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5;

[0350] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6.

[0351] In one aspect, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotide sequences selected from the following in the genome (e.g., isolated genetic material from the plant or plant part):

[0352] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0353] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0354] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0355] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening the presence of polynucleotides in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part) containing sequences selected from:

[0356] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0357] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0358] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0359] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising identifying or screening for the presence of polynucleotide sequences selected from the following in the genome (e.g., isolated genetic material from the plant or plant part):

[0360] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0361] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0362] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1; and

[0363] Select plants or plant parts that contain the polynucleotide.

[0364] In one embodiment, the present invention relates to a method for identifying or selecting maize plants or plant parts, particularly maize plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising isolating genetic material from at least one cell of the plant or plant part; and identifying or screening the presence of polynucleotides in the genome of the maize plant or plant part (such as the isolated genetic material from the plant or plant part) containing sequences selected from:

[0365] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0366] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0367] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1; and

[0368] Select plants or plant parts that contain the polynucleotide.

[0369] In some implementations, the identification or screening of the presence of a 7bp insertion sequence includes identifying or screening the presence of a sequence with gcggtct (corresponding to positions 103-109 of SEQ ID NO:4).

[0370] In some implementations, the identification or screening of the presence of an 8bp insertion sequence including the identification or screening of a sequence having gcggttct (corresponding to positions 103-110 of SEQ ID NO:1) is included.

[0371] SEQ ID NO:1 and 4 correspond to the genomic sequences of the mutant F35H gene according to certain embodiments of the present invention, and respectively contain an 8bp insertion sequence or a 7bp insertion sequence as described elsewhere herein.

[0372] SEQ ID NO:2 and 5 correspond to the coding sequences of the mutant F35H gene according to certain embodiments of the invention, and respectively contain an 8 bp insert sequence or a 7 bp insert sequence as described elsewhere herein (i.e., at the position corresponding to the specified genomic sequence).

[0373] SEQ ID NO:3 and 6 correspond to polypeptide sequences encoded by the mutant F35H gene according to certain embodiments of the invention, and respectively contain an 8bp insert sequence or a 7bp insert sequence as described elsewhere herein.

[0374] It should be understood that in the methods described above, when a polynucleotide, QTL allele, (molecular) marker allele, or SEQ ID NO is present or identified / detected, the plant or plant part is identified as having improved digestibility, particularly improved straw digestibility. Therefore, in some embodiments, the methods described above for identifying plants or plant parts are methods for identifying plants or plant parts having improved digestibility, particularly improved straw digestibility. Alternatively, in the methods described above, when a polynucleotide, QTL allele, (molecular) marker allele, or SEQ ID NO is not present or identified / detected, the plant or plant part is identified as not having improved digestibility, particularly not improved straw digestibility.

[0375] Methods for screening or detecting the presence of polynucleotides, QTL alleles, (molecular) marker alleles, or SEQ ID NO as described herein are known in the art. Without limitation, screening or detection may encompass or include sequencing, hybridization-based methods (such as (dynamic) allele-specific hybridization, molecular beacons, SNP microarrays), enzyme-based methods (such as PCR, KASP (competitive allele-specific PCR), RFLP, ALFP, RAPD, Flap endonuclease, primer extension, 5'-nuclease, oligonucleotide ligation assays), and post-amplification methods based on the physical properties of DNA (such as single-strand conformational polymorphism, temperature gradient gel electrophoresis, denaturing high-performance liquid chromatography, high-resolution melting of the entire amplicon, use of DNA mismatch-binding proteins, SNPLEx, Surveyor nuclease assays), etc. Preferably, one or more polynucleotides, QTL alleles, (molecular) marker alleles, or SEQ ID NO can be detected by polynucleotides, such as allele-specific polynucleotides (molecular markers), which are suitable as forward or reverse primers for hybridization with loci with improved digestibility that are co-separated in chromosomal regions.

[0376] In one aspect, the present invention relates to the identification or selection of maize plants or plant parts by the methods of the present invention as described elsewhere herein.

[0377] In one aspect, the present invention relates to methods, such as methods for generating or producing maize plants or plant parts, particularly plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising introducing into the genome of the maize plant or plant part a polynucleotide, (molecular) marker (allele), QTL (allele), or mutated F35H gene as described elsewhere herein.

[0378] In one aspect, the present invention relates to a method for generating or producing maize plants or plant parts, particularly plants or plant parts having improved digestibility, preferably improved straw digestibility, the method comprising introducing into the genome of the maize plant or plant part a polynucleotide, (molecular) marker (allele), QTL (allele) or mutated F35H gene as described elsewhere herein.

[0379] In one aspect, the present invention relates to a method for improving the digestibility of maize plants or plant parts, preferably straw digestibility, the method comprising introducing into the genome of the maize plant or plant part a polynucleotide, (molecular) marker (allele), QTL (allele) or mutated F35H gene as described elsewhere herein.

[0380] In one aspect, the present invention relates to methods, such as methods for generating plants or plant parts and / or improving the digestibility of plants or plant parts, preferably straw digestibility, the method comprising introducing a QTL (allele) into the genome of a plant, the QTL (allele) being particularly associated with improved (straw) digestibility and containing a mutated nucleotide sequence encoding a cytochrome P450 flavonoid 3',5'-hydroxylase.

[0381] In one aspect, the present invention relates to methods, such as methods for generating plants or plant parts and / or for improving the digestibility of plants or plant parts, preferably straw digestibility, the method comprising introducing a QTL (allele) into the genome of a plant, the QTL (allele) containing a mutated nucleotide sequence encoding a cytochrome P450 flavonoid 3',5'-hydroxylase.

[0382] In some embodiments, the QTL (allele) is located on chromosome 9 and contains and / or is flanked by the (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or is contained in a polynucleotide or QTL (allele) containing and / or flanked by the (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01.

[0383] In some implementations, the QTL (allele) is located on chromosome 9 and includes one or more of the (molecular) markers (alleles) selected from Table A.

[0384] In some implementations, the QTL (allele) is located on chromosome 9 and includes one or more of the (molecular) markers (alleles) selected from Table B.

[0385] In some implementations, the QTL (allele) is located on chromosome 9 and includes one or more (molecular) markers (alleles) selected from Table C.

[0386] In one aspect, the present invention relates to methods, such as methods for generating plants or plant parts and / or improving the digestibility of plants or plant parts, preferably straw digestibility, the method comprising introducing into the genome of a plant a polynucleotide containing a (molecular) marker (allele) selected from Table A, preferably all (optionally except one of ma61134d15 or ma61134d16).

[0387] In one aspect, the present invention relates to methods, such as methods for generating plants or plant parts and / or improving the digestibility of plants or plant parts, preferably straw digestibility, the method comprising introducing into the genome of a plant a polynucleotide containing a (molecular) marker (allele) selected from Table B, preferably all (optionally except one of ma61134d15 or ma61134d16).

[0388] In one aspect, the present invention relates to methods, such as methods for generating plants or plant parts and / or improving the digestibility of plants or plant parts, preferably straw digestibility, the method comprising introducing into the genome of a plant a polynucleotide comprising a (molecular) marker (allele) selected from Table C.

[0389] In one aspect, the present invention relates to methods, such as methods for generating plants or plant parts and / or improving the digestibility of plants or plant parts, preferably straw digestibility, the method comprising introducing into the genome of a maize plant or plant part a polynucleotide containing the (molecular) marker (allele) ma61134d15 and / or ma61134d16.

[0390] In one aspect, the present invention relates to methods, such as methods for generating plants or plant parts and / or improving the digestibility of plants or plant parts, preferably straw digestibility, the method comprising introducing polynucleotides having sequences selected from the following into the genome of a plant:

[0391] a) The nucleotide sequence of SEQ ID NO:7;

[0392] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0393] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0394] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0395] Wherein the nucleotide sequences a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0396] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0397] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0398] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0399] In some implementations, digestibility is improved compared to that of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0400] In some embodiments, the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0401] In some embodiments, the polynucleotide or QTL (allele) comprises one or more of the marker alleles of the present invention as described elsewhere herein.

[0402] In some implementations, the polynucleotide or QTL (allele) contains a mutated F35H gene as described elsewhere herein.

[0403] In some embodiments, the present invention relates to a method for obtaining or generating maize plants or plant parts, the method comprising (a) providing a first plant having a polynucleotide, such as a QTL (allele), as described elsewhere herein, a QTL (allele) associated with improved (straw) digestibility, optionally wherein said QTL (allele) is located on a chromosomal region, preferably on chromosome 9, and comprising and / or side-linked with (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or comprising in a polynucleotide or QTL (allele) comprising and / or side-linked with (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or by means of, as per NCIMB accession number NCIMB. (a) the first plant (or its offspring) obtained from the corn seeds preserved in 43997, (b) the first plant is hybridized with a second plant, such as a second plant that does not have the polynucleotide, such as the QTL (allele), (c) offspring plants that have the polynucleotide, such as the QTL (allele), and optionally (d) parts of the plant are harvested from the offspring.

[0404] In some embodiments, the present invention relates to a method for obtaining or generating maize plants or plant parts, the method comprising (a) providing a first plant having a (molecular) marker allele, such as a (molecular) marker allele selected from Table A associated with improved (straw) digestibility; (b) hybridizing the first plant with a second plant, such as a second plant not having the (molecular) marker allele; (c) selecting progeny plants having the (molecular) marker allele; and optionally (d) harvesting the plant parts from the progeny.

[0405] In some embodiments, the present invention relates to a method for obtaining or generating maize plants or plant parts, the method comprising (a) providing a first plant having a (molecular) marker allele, such as a (molecular) marker allele selected from Table B associated with improved (straw) digestibility; (b) hybridizing the first plant with a second plant, such as a second plant not having the (molecular) marker allele; (c) selecting progeny plants having the (molecular) marker allele; and optionally (d) harvesting the plant parts from the progeny.

[0406] In some embodiments, the present invention relates to a method for obtaining or generating maize plants or plant parts, the method comprising (a) providing a first plant having a (molecular) marker allele, such as a (molecular) marker allele selected from Table C associated with improved (straw) digestibility; (b) hybridizing the first plant with a second plant, such as a second plant not having the (molecular) marker allele; (c) selecting progeny plants having the (molecular) marker allele; and optionally (d) harvesting the plant parts from the progeny.

[0407] In some embodiments, the polynucleotide, QTL (allele), (molecular) marker (allele), and / or F35H mutation in the first plant are homozygous. In some embodiments, the polynucleotide, QTL (allele), (molecular) marker (allele), and / or F35H mutation in the first plant are heterozygous. In some embodiments, the polynucleotide, QTL (allele), (molecular) marker (allele), and / or F35H mutation in the second plant are heterozygous. In some embodiments, the polynucleotide, QTL (allele), (molecular) marker (allele), and / or F35H mutation in the second plant are absent.

[0408] In some embodiments, offspring in which the polynucleotides, QTLs (alleles), (molecular) markers (alleles), and / or mutated F35H of the present invention are selected in a homozygous state.

[0409] In some embodiments, offspring in which the polynucleotides, QTLs (alleles), (molecular) markers (alleles), and / or mutated F35H of the present invention are selected in a heterozygous state.

[0410] In some embodiments, the present invention relates to a method for obtaining or generating maize plants or plant parts, the method comprising transforming maize plants or plant parts, preferably plant cells, more preferably immature or mature embryos, inflorescences, protoplasts or callus tissues, with the polynucleotide, one or more (molecular) markers (alleles), or the QTL (allele); and optionally regenerating plants from the plant cells, preferably immature or mature embryos, inflorescences, protoplasts or callus tissues.

[0411] As described elsewhere in this document, in some embodiments, the maize plant or plant part does not endogenously contain the described polynucleotides, (molecular) markers (alleles), QTLs (alleles), and / or mutated F35H.

[0412] In some embodiments, the methods for obtaining plants or plant parts according to the invention as described herein, such as methods for obtaining plants or plant parts with improved digestibility, involve or include transgenic and / or gene editing and / or base editing, such as including CRISPR / Cas, TALEN, ZFN, and a wide range of nucleases; (induced) mutagenesis, which may or may not be random mutagenesis, such as tilling. In some embodiments, the methods for obtaining plants or plant parts according to the invention as described herein, such as methods for obtaining plants or plant parts with improved digestibility, involve or include RNAi application, which may or may not be, include, or involve transgenic application. For example, non-transgenic application may involve, for example, applying RNAi components (such as double-stranded siRNA) to the plant or plant surface, for example, as a spray. Stable integration into the plant genome is not required.

[0413] In some embodiments, the methods for obtaining plants or plant parts according to the present invention, such as methods for obtaining plants or plant parts with improved digestibility, do not involve or include transgenic processes, gene / base editing, and / or mutagenesis.

[0414] In some embodiments, the methods for obtaining plants or plant parts according to the present invention, as described herein, such as methods for obtaining plants or plant parts with improved digestibility, involve, comprise, or consist of breeding and selection.

[0415] In some embodiments, the methods for obtaining plants or plant parts according to the present invention, as described herein, such as methods for obtaining plants or plant parts with improved digestibility, do not involve, comprise, or consist of breeding and selection.

[0416] Knockdown or knockout of F35H can be achieved through any of the mutagenesis methods described herein.

[0417] Those skilled in the art will understand that the introduction or mutation of two or more sequences, genes, markers, or alleles as described herein can be simultaneous or sequential, and the introduction or mutation of two or more sequences, genes, markers, or alleles as described herein can be simultaneously recombinant (e.g., by transformation) or by breeding techniques (e.g., introgression), or the introduction of one of two or more sequences, genes, markers, or alleles can be recombinant while the other is introduced by breeding techniques (e.g., introgression).

[0418] In one aspect, the present invention relates to a corn plant or plant part obtained by or through the methods of the invention as described herein, such as methods for generating, producing, or obtaining a plant or plant part, particularly a plant or plant part with improved (straw) digestibility. The invention also relates to the offspring of such plants.

[0419] In one aspect, the present invention relates to a plant or plant part comprising a polynucleotide, such as a QTL (allele), particularly associated with improved (straw) digestibility, said polynucleotide, such as a QTL (allele), comprising a nucleotide sequence having a mutated gene encoding a gene encoding cytochrome P450 flavonoid 3',5'-hydroxylase (F35H).

[0420] In some embodiments, the QTL (allele) is located on chromosome 9 and contains and / or is flanked by the (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or is contained in a polynucleotide or QTL (allele) containing and / or flanked by the (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01.

[0421] In some implementations, the QTL (allele) is located on chromosome 9 and includes one or more of the (molecular) markers (alleles) in Table A8, preferably all of them (optionally except for one of ma61134d15 or ma61134d16).

[0422] In some implementations, the QTL (allele) is located on chromosome 9 and includes one or more of the (molecular) markers (alleles) in Table B, preferably all of them (optionally except one of ma61134d15 or ma61134d16).

[0423] In some implementations, the QTL (allele) is located on chromosome 9 and includes one or more of the (molecular) markers (alleles) in Table C.

[0424] In one aspect, the present invention relates to a plant or plant part comprising a nucleotide sequence having a mutated gene encoding a cytochrome P450 flavonoid 3',5'-hydroxylase (F35H).

[0425] In one aspect, the present invention relates to a plant or plant part comprising one or more (molecular) markers (alleles) selected from Table A, preferably all of them (optionally except one of ma61134d15 or ma61134d16).

[0426] In one aspect, the present invention relates to a plant or plant part comprising one or more (molecular) markers (alleles) selected from Table B, preferably all of them (optionally except one of ma61134d15 or ma61134d16).

[0427] In one aspect, the present invention relates to a plant or plant part comprising one or more (molecular) markers (alleles) selected from Table C, preferably all of them.

[0428] In one aspect, the present invention relates to a plant or plant part comprising:

[0429] a) The nucleotide sequence of SEQ ID NO:7;

[0430] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0431] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0432] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0433] Wherein the nucleotide sequences a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0434] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0435] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0436] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0437] Those skilled in the art will understand that the described polynucleotide can be operatively linked to one or more regulatory sequences, particularly regulatory sequences affecting the transcription of the polynucleotide, especially the coding region of the polynucleotide. Such regulatory sequences may include suitable (plant) promoters, transcription start sites, transcription termination sites, etc., as known in the art.

[0438] In some implementations, the plant is not a plant variety.

[0439] In some implementations, the wild-type or unmutated F35H gene contains

[0440] (i) A nucleotide sequence containing the sequence of SEQ ID NO:7;

[0441] (ii) a cDNA or nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0442] (iii) A nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:9;

[0443] (iv) A nucleotide sequence having at least 60% identity with the sequence of SEQ ID NO: 7 or 8; such as at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity or at least 95% sequence identity;

[0444] (v) A nucleotide sequence encoding a polypeptide having at least 60% identity with the sequence of SEQ ID NO:9; such as at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity or at least 95% sequence identity;

[0445] (vi) A nucleotide sequence that hybridizes under strict hybridization conditions to the reverse complementary sequence of a nucleotide sequence as defined in (i), (ii) or (iii); and

[0446] (vii) A nucleotide sequence that encodes a polypeptide-derived protein encoded by a nucleotide sequence from (i) to (vi) by substitution, deletion and / or addition of one or more amino acids.

[0447] In some implementations, the wild-type or unmutated F35H gene contains

[0448] (i) A nucleotide sequence containing the sequence of SEQ ID NO:7;

[0449] (ii) a cDNA or nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0450] (iii) A nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:9;

[0451] In some implementations, the wild-type or unmutated F35H gene contains

[0452] (i) A nucleotide sequence having at least 60% identity with the sequence of SEQ ID NO: 7 or 8; such as at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity or at least 95% sequence identity; or

[0453] (ii) A nucleotide sequence encoding a polypeptide having at least 60% identity with the sequence of SEQ ID NO:9; such as at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity or at least 95% sequence identity.

[0454] In some implementations, the wild-type or unmutated F35H gene contains

[0455] (i) A nucleotide sequence having at least 60% identity with the sequence of SEQ ID NO:8; such as at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity or at least 95% sequence identity; or

[0456] (ii) A nucleotide sequence encoding a polypeptide having at least 60% identity with the sequence of SEQ ID NO:9; such as at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity or at least 95% sequence identity.

[0457] In some implementations, the wild-type or unmutated F35H gene contains

[0458] (i) A nucleotide sequence that hybridizes with the reverse complementary sequence of the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8 under strict hybridization conditions.

[0459] In some implementations, the wild-type or unmutated F35H gene is included; or

[0460] (ii) A nucleotide sequence encoding a polypeptide-derived protein encoded by the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8 by substitution, deletion and / or addition of one or more amino acids.

[0461] Those skilled in the art will understand that the wild-type or unmutated F35H gene product is a functional gene product with enzymatic activity, as defined elsewhere in this document.

[0462] Technicians will further understand that the sequence variations described above for wild-type F35H do not include frameshift or nonsense mutations.

[0463] As used herein, a mutated F35H or a mutation in F35H may include or refer to any type of F35H mutation. In some embodiments, the mutation alters the expression of wild-type or native F35H protein and / or mRNA. In some embodiments, the mutation reduces or eliminates the expression of (wild-type or native) F35H protein and / or mRNA, as described elsewhere herein. The mutation may affect transcription and / or translation. The mutation may occur in exons or introns. The mutation may occur in regulatory elements, such as promoters, enhancers, terminators, insulators, etc. The mutation may occur in coding sequences. The mutation may occur at splice signal sites, such as splice donor or splice acceptor sites. The mutation may be a frameshift mutation. The mutation may be a nonsense mutation. The mutation may be an insertion or deletion of one or more nucleotides. The mutation may be a non-conserved mutation (in which one or more wild-type amino acids are substituted by one or more non-wild-type amino acids). The mutation may affect or alter the function of the F35H protein, such as enzyme activity. The mutation can reduce or (substantially) eliminate the function of the F35H protein, such as enzyme activity. A reduction in function, such as reduced enzyme activity, can mean a reduction of at least 10%, preferably at least 30%, more preferably at least 50%, such as at least 20%, 40%, 60%, 80% or more, such as at least 85%, at least 90%, at least 95%, or more. A (substantially) eliminated function, such as (substantially) eliminated enzyme activity, can mean a reduction of at least 80%, preferably at least 90%, more preferably at least 95%. The mutation can be a dominant-negative mutation. In some embodiments, the mutation is evaluated with reference to the maize inbred line PH207 as defined elsewhere herein.

[0464] In some embodiments, the F35H mutation is an insertion of one or more nucleotides in the coding sequence. In some embodiments, the F35H mutation is a nonsense mutation. In some embodiments, the F35H mutation results in altered expression of the F35H gene. In some embodiments, the F35H mutation results in F35H gene knockout or F35H mRNA and / or protein knockdown. In some embodiments, the mutation results in a frameshift of the coding sequence of F35H. In some embodiments, the mutation results in an altered sequence of the protein encoded by the F35H gene.

[0465] In some embodiments, the F35H mutation is an insertion of one or more nucleotides, preferably in an exon, preferably in the first exon, preferably a frameshift insertion. In some embodiments, the insertion is 7 or 8 nucleotides. In some embodiments, the insertion is between positions 102 and 103 of the F35H gene represented by the nucleotide sequence of SEQ ID NO:7. A person skilled in the art can determine the corresponding position in F35H homologs or orthologs. In some embodiments, the insertion comprises or consists of the nucleotide sequence gcsgtct, such as gcggtct. In some embodiments, the insertion comprises or consists of the nucleotide sequence gcsgttct, such as gcggttct. In some embodiments, the mutated F35H comprises the nucleotide sequence of SEQ ID NO:1 or 4, or has the cDNA or coding sequence of SEQ ID NO:2 or 5, or encodes a protein having the sequence of SEQ ID NO:3 or 6. Alternatively, the mutation is a substitution, preferably a substitution of at least one nucleic acid, resulting in the exchange of at least one amino acid or causing an amino acid-coding codon to become a stop codon.

[0466] F35H mRNA and / or protein expression can be reduced or eliminated by mutating the F35H gene itself (including coding, non-coding, and regulatory elements). Methods for introducing mutations are described elsewhere in this document. Alternatively, F35H mRNA and / or protein expression can be reduced or eliminated by (specifically) interfering with transcription and / or translation, such as to reduce or eliminate mRNA and / or protein transcription or translation. Alternatively, F35H mRNA and / or protein expression can be reduced or eliminated by (specifically) interfering with mRNA and / or protein stability, such as to reduce mRNA and / or protein stability. For example, mRNA (stability) can be reduced by means of RNAi, as described elsewhere in this document. miRNAs can also be used to affect mRNA (stability). In some embodiments, the term "mutated" F35H also covers F35H expression reduction achieved by reducing mRNA or protein stability. In some embodiments, the term "mutated" F35H does not cover F35H expression reduction achieved by reducing mRNA or protein stability.

[0467] In one aspect, the present invention relates to the use of one or more of the polynucleotides or QTLs (alleles) according to the invention described elsewhere herein for producing or generating plants or plant parts, particularly those with improved (straw) digestibility.

[0468] In one aspect, the present invention relates to the use of one or more of the (molecular) markers described herein for identifying plants or plant parts, particularly those with improved (straw) digestibility. In another aspect, the present invention relates to the use of one or more of the (molecular) markers described herein for identifying plants or plant parts, particularly those with improved (straw) digestibility, said (molecular) markers being capable of detecting at least one (diagnostic) marker allele. In another aspect, the present invention relates to the detection of one or more of the (molecular) markers (alleles) described herein, said (molecular) markers (alleles) for identifying plants or plant parts, particularly those with improved (straw) digestibility, or for distinguishing plants or plant parts, particularly those with improved (straw) digestibility, from those without improved (straw) digestibility.

[0469] The marker alleles of the present invention as described herein can be diagnostic marker alleles, which can be used to identify and / or select plants or plant parts with improved digestibility, preferably improved straw digestibility.

[0470] In one aspect, the present invention relates to an isolated polynucleotide comprising the (molecular) marker allele of the present invention, or a (unique) fragment thereof, or a complementary or reverse complementary sequence of the (molecular) marker allele of the present invention, or a (unique) fragment thereof. In some embodiments, the present invention relates to a polynucleotide comprising at least 10 consecutive nucleotides of the (molecular) marker (allele) of the present invention, preferably at least 15 consecutive nucleotides, more preferably at least 17 or 18 consecutive nucleotides, such as at least 20 consecutive nucleotides, or a complementary or reverse complementary sequence of the (molecular) marker (allele) of the present invention. In some preferred embodiments, the fragment comprises at least one nucleotide of the polymorphism of the corresponding (molecular) marker (allele), or its complementary or reverse complementary sequence.

[0471] In one aspect, the present invention relates to an isolated polynucleotide comprising a polynucleotide having a sequence selected from:

[0472] a) The nucleotide sequence of SEQ ID NO:7;

[0473] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0474] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0475] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0476] Wherein the nucleotide sequences in a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0477] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0478] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5 or a nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0479] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6;

[0480] Or (unique) segments of the sequence from a) to g);

[0481] Or the complementary sequence or the reverse complementary sequence or a (unique) fragment of the sequence from a) to g).

[0482] In some embodiments, the present invention relates to a polynucleotide comprising at least 10 consecutive nucleotides, preferably at least 15 consecutive nucleotides, more preferably at least 17 or 18 consecutive nucleotides, such as at least 20 consecutive nucleotides, or their complementary or reverse complementary sequences, and preferably comprising each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to the 5' or 3' of the polymorphism, as in the case of insertion or deletion), preferably wherein said polymorphism (or at least one nucleotide of the polymorphism) is the nucleotide closest to the 3', the second nucleotide closest to the 3', or the third nucleotide closest to the 3', preferably the nucleotide closest to the 3'.

[0483] In some embodiments, the present invention relates to a polynucleotide comprising at least 10 consecutive nucleotides, preferably at least 15 consecutive nucleotides, more preferably at least 17 or 18 consecutive nucleotides, such as at least 20 consecutive nucleotides, or their complementary or reverse complementary sequences, and preferably comprising each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to the 5' or 3' of the polymorphism, as in the case of insertion or deletion), preferably wherein said polymorphism (or at least one nucleotide of the polymorphism) is the nucleotide closest to the 3', the second nucleotide closest to the 3', or the third nucleotide closest to the 3', preferably the nucleotide closest to the 3'.

[0484] In some embodiments, the present invention relates to a polynucleotide comprising at least 10 consecutive nucleotides, preferably at least 15 consecutive nucleotides, more preferably at least 17 or 18 consecutive nucleotides, such as at least 20 consecutive nucleotides, or their complementary or reverse complementary sequences, and preferably comprising each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to 5' or closest to 3' of the polymorphism, as in the case of insertion or deletion), preferably wherein said polymorphism (or at least one nucleotide of the polymorphism) is the nucleotide closest to 3', the second nucleotide closest to 3', or the third nucleotide closest to 3', preferably the nucleotide closest to 3'.

[0485] In some embodiments, the polynucleotides can distinguish between the (molecular) markers (alleles) of the present invention and non-molecular marker alleles, such as being able to specifically hybridize with the (molecular) marker alleles of the present invention.

[0486] In some embodiments, the polynucleotide is capable of hybridizing with a unique nucleotide fragment or region of any of SEQ ID NO: 1, 2, 4, 5, 13-195, or any of SEQ ID NO: 1, 2, 4, 5, 13-195, preferably SEQ ID NO: 1, 2, 4, 5, or 109-154, more preferably the complementary or reverse complementary sequence of any of SEQ ID NO: 1, 2, 4, 5, 124, or 125, and preferably contains each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to 5' or closest to 3' of the polymorphism, as in the case of insertion or deletion), preferably said polymorphism (or at least one nucleotide thereof) is the nucleotide closest to 3', the second nucleotide closest to 3', or the third nucleotide closest to 3', preferably the nucleotide closest to 3'.

[0487] It should be understood that the (unique) segment or fragment preferably refers to a segment or fragment containing at least one unique nucleotide of an SNP (or polymorphism, e.g., in the case of insertion or deletion) or the corresponding marker allele of the present invention (as indicated, for example, in Tables A, B, or C), or a segment or fragment containing a 5' or 3' linker of an insertion sequence of a marker allele of the present invention, or a segment or portion contained within an insertion sequence of a marker allele of the present invention, or a segment or fragment containing a linker of a deletion marker allele of the present invention. In some embodiments, the polynucleotide or its complementary or reverse complementary sequence does not (substantially) hybridize or bind to (genomic) DNA derived from the maize inbred line PH207. In some embodiments, the sequence of the polynucleotide or its complementary or reverse complementary sequence is not present or is absent in the maize inbred line PH207.

[0488] In one aspect, the present invention relates to a (isolated) polynucleotide comprising or contained in a QTL (allele) (on chromosome 9) particularly associated with improved digestibility and comprising and / or side-attached (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or comprising in a polynucleotide or QTL (allele) comprising and / or side-attached (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01; its complementary or reverse complementary sequence; or a (unique) fragment thereof, its complementary or reverse complementary sequence.

[0489] In one aspect, the present invention relates to a polynucleotide capable of specifically hybridizing with the (molecular) marker allele of the present invention, or its complementary sequence, or its reverse complementary sequence, and preferably comprising each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to the 5' or 3' of the polymorphism, as in the case of insertion or deletion), preferably wherein said polymorphism (or at least one nucleotide thereof) is the nucleotide closest to the 3', the second nucleotide closest to the 3', or the third nucleotide closest to the 3', preferably the nucleotide closest to the 3'.

[0490] In one aspect, the present invention relates to a polynucleotide capable of specifically hybridizing with a polynucleotide having a sequence as shown in any of SEQ ID NO:13-195, or a complementary sequence thereof, or an inverse complementary sequence thereof, and preferably comprising each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to the 5' or closest to the 3' of the polymorphism, as in the case of insertion or deletion), preferably wherein said polymorphism (or at least one nucleotide thereof) is the nucleotide closest to the 3', the second nucleotide closest to the 3', or the third nucleotide closest to the 3', preferably the nucleotide closest to the 3'.

[0491] In one aspect, the present invention relates to a polynucleotide capable of specifically hybridizing with a polynucleotide having a sequence as shown in any of SEQ ID NO:109-154, or a complementary sequence thereof, or an inverse complementary sequence thereof, and preferably comprising each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to the 5' or closest to the 3' of the polymorphism, as in the case of insertion or deletion), preferably wherein said polymorphism (or at least one nucleotide thereof) is the nucleotide closest to the 3', the second nucleotide closest to the 3', or the third nucleotide closest to the 3', preferably the nucleotide closest to the 3'.

[0492] In one aspect, the present invention relates to a polynucleotide capable of specifically hybridizing with a polynucleotide having a sequence as shown in any of SEQ ID NO:124-125, or a complementary sequence thereof, or an inverse complementary sequence thereof, and preferably comprising each of the respective polymorphisms listed in Table A (or at least the nucleotide closest to the 5' or closest to the 3' of the polymorphism, as in the case of insertion or deletion), preferably wherein said polymorphism (or at least one nucleotide thereof) is the nucleotide closest to the 3', the second nucleotide closest to the 3', or the third nucleotide closest to the 3', preferably the nucleotide closest to the 3'.

[0493] In some embodiments, the polynucleotide is a primer. In some embodiments, the polynucleotide is a probe. In some embodiments, the polynucleotide is an allele-specific primer. In some embodiments, the polynucleotide is a KASP primer.

[0494] In some implementations, the polynucleotide is an allele-specific polynucleotide, such as an allele-specific primer or probe.

[0495] In some implementations, the polynucleotide contains at least 15 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, such as at least 30, 35, 40, 45, or 50 nucleotides, such as at least 100, 200, 300, or 500 nucleotides.

[0496] In some embodiments, the polynucleotide contains 15 to 500 nucleotides, preferably 17 to 100 nucleotides, more preferably 17 to 50 nucleotides, and most preferably 17 to 30 nucleotides.

[0497] Technicians will understand that polynucleotides, such as primers or probes, may further contain (5') additional nucleotides, which may serve as, for example, tags, tails, or barcodes.

[0498] It should be understood that "specific hybridization" means hybridization of a polynucleotide with a marker allele (as defined elsewhere herein under strict hybridization conditions), but (substantially) not hybridizing with a polynucleotide that does not contain a marker allele or (substantially) cannot be used as a PCR primer. For example, in a suitable readout, the hybridization signal with a marker allele or the PCR amplification of the marker allele is at least 5-fold stronger, preferably at least 10-fold or more, than the hybridization signal with an unmarked allele or any other sequence.

[0499] In one aspect, the present invention relates to a kit comprising such polynucleotides, such as primers (comprising forward and / or reverse primers) and / or probes. The kit may also include instructions for use.

[0500] It should be understood that in embodiments involving a set of forward and reverse primers, only one of the two primers (forward or reverse) may be required to distinguish between the (molecular) marker alleles and unmarked alleles of the present invention, and therefore may be unique. The other primer may or may not be able to distinguish between the (molecular) marker alleles and unmarked alleles of the present invention, and therefore may or may not be unique.

[0501] With the aid of further guidance, but not limited thereto, the present invention is embodied in the following aspects and embodiments.

[0502] In one aspect, the present invention relates to a method for identifying maize plants or plant parts, the method comprising screening for the presence of polynucleotides containing the (molecular) markers (alleles) ma61134d15 and / or ma61134d16 in the genome of maize plants or plant parts.

[0503] In some embodiments, the polynucleotide is contained in or included in a QTL (allele) that is specifically associated with improved digestibility on chromosome 9 and contains and / or is flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or is contained in a polynucleotide or QTL (allele) containing and / or flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01.

[0504] In one aspect, the present invention relates to a method for identifying maize plants or plant parts, the method comprising screening for the presence of a QTL (allele) specifically associated with enhanced digestibility on chromosome 9 and comprising and / or flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01, or comprising a polynucleotide or QTL (allele) comprising and / or flanked by (molecular) markers (alleles) SYN38529 and PZE-109103504, preferably PZE-109076467 and ma61161s01.

[0505] In some implementations, the QTL (allele) includes the (molecular) marker (allele) ma61134d15 and / or ma61134d16.

[0506] In some implementations, the method includes screening for the presence of one or more (molecular) markers (alleles) selected from Table A.

[0507] In some implementations, the method includes screening for the presence of one or more (molecular) markers (alleles) selected from Table B.

[0508] In some implementations, the method includes screening for the presence of one or more (molecular) markers (alleles) selected from Table C.

[0509] In some implementations, the polynucleotide contains one or more (molecular) markers (alleles) as defined in Statement 5.

[0510] In some implementations, QTLs (alleles) comprise one or more (molecular) markers (alleles) selected from Table A, Table B, or Table C.

[0511] In one aspect, the present invention relates to a method for identifying maize plants or plant parts, the method comprising screening or detecting the presence of one or more (molecular) markers (alleles) selected from Table A, Table B, or Table C in the genome of a maize plant or plant part.

[0512] In some implementations, the markers are contained on chromosome 9, particularly in a QTL associated with improved digestibility, which is flanked by and / or contains the (molecular) markers (alleles) SYN38529 and PZE-109103504.

[0513] In one aspect, the present invention relates to a method for identifying maize plants or plant parts, the method comprising screening for the presence of polynucleotides in the genome of maize plants or plant parts containing sequences selected from:

[0514] a) The nucleotide sequence of SEQ ID NO:7;

[0515] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0516] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0517] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0518] The nucleotide sequence contains one or more nucleotides at the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or at the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides.

[0519] In some implementations, the insertion of 8 nucleotides has the sequence gcggttct, or the insertion of 7 nucleotides has the sequence gcggtct.

[0520] In some implementations, the nucleotide sequence is selected from:

[0521] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0522] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0523] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0524] In one aspect, the present invention relates to a method for identifying maize plants or plant parts, the method comprising screening for the presence of polynucleotides in the genome of maize plants or plant parts containing sequences selected from:

[0525] a) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0526] b) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0527] c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0528] In some implementations, the method is used to identify plants or plant parts that have improved digestibility.

[0529] In some implementations, the method is used to identify plants or plant parts that have improved straw digestibility.

[0530] In some implementations, digestibility is improved compared to that of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0531] In some embodiments, the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0532] In some embodiments, the method further includes isolating genomic DNA from a plant or plant part.

[0533] In some embodiments, the method further includes selecting a plant or plant part containing one or more of a polynucleotide, a (molecular) marker (allele), or a QTL (allele).

[0534] In some implementations, the plant portion is not used as propagation material.

[0535] In some implementations, the plant part is straw.

[0536] In some embodiments, the plant or plant part contains a polynucleotide having a sequence selected from:

[0537] a) The nucleotide sequence of SEQ ID NO:7;

[0538] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0539] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0540] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0541] Wherein the nucleotide sequences a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0542] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0543] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0544] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0545] In some implementations, a plant or plant part is identified as having improved digestibility if one or more of a polynucleotide, a (molecular) marker (allele), or a QTL (allele) is present in the genome of the plant or plant part.

[0546] In some implementations, a plant or plant part is identified as having improved straw digestibility if one or more of a polynucleotide, a (molecular) marker allele, or a QTL (allele) is present in the genome of the plant or plant part.

[0547] In one aspect, the present invention relates to maize plants or plant parts comprising one or more (molecular) markers (alleles) as defined in Table A.

[0548] In one aspect, the present invention relates to maize plants or plant parts comprising one or more (molecular) markers (alleles) as defined in Table B.

[0549] In one aspect, the present invention relates to maize plants or plant parts comprising one or more (molecular) markers (alleles) as defined in Table C.

[0550] In one aspect, the present invention relates to maize plants or plant parts comprising the polynucleotides or QTLs (alleles) as described above.

[0551] In some embodiments, the plant or part of the plant is derived from a plant containing the polynucleotide, the (molecular) marker (allele), or the QTL (allele) obtained by introduction or introgression.

[0552] In some implementations, the plant or plant part is the result of mutagenesis mediated by transposons or transposon factors.

[0553] In some implementations, the plant or plant part is genetically modified or gene-edited.

[0554] In one aspect, the present invention relates to a method for generating or producing maize plants or plant parts and / or for improving (straw) digestibility, the method comprising introducing, as described above, a polynucleotide, one or more (molecular) markers (alleles), or QTLs (alleles) into the genome of the maize plant or plant part.

[0555] In one aspect, the present invention relates to a method for generating or producing maize plants or plant parts and / or for improving (straw) digestibility, the method comprising introducing polynucleotides having sequences selected from the following into the genome of the maize plant or plant part:

[0556] a) The nucleotide sequence of SEQ ID NO:7;

[0557] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0558] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0559] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0560] Wherein the nucleotide sequences a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0561] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0562] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0563] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0564] In some implementations, the introduction into the genome involves introgression.

[0565] In some embodiments, the method includes the following steps: (a) providing a first maize plant having a polynucleotide, one or more (molecular) markers (alleles), or QTLs (alleles) as defined in any one of statements 1 to 9, or a first maize plant (or its offspring) obtained from maize seeds such as those deposited with NCIMB accession number NCIMB 43997; (b) hybridizing the first maize plant with a second maize plant; and (c) selecting offspring plants having a polynucleotide, one or more (molecular) markers (alleles), or QTLs (alleles) as defined in any one of statements 1 to 9.

[0566] In some implementations, the method further includes (d) harvesting plant portions from the offspring.

[0567] In some implementations, the introduction into the genome involves mutagenesis mediated by transposons or transposon factors.

[0568] In some implementations, the introduction into the genome involves transgenesis or gene editing.

[0569] In some implementations, the plant part is a plant cell, tissue, organ, or seed.

[0570] In some implementations, the plant part is an immature or mature embryo, inflorescence, protoplast, or callus.

[0571] In some embodiments, the method includes the steps of: transforming a plant or plant part, preferably plant cells, more preferably immature or mature embryos, inflorescences, protoplasts or callus tissue, with the polynucleotide, one or more (molecular) markers (alleles), or the QTL (allele); and optionally regenerating a plant from the plant cells, preferably immature or mature embryos, inflorescences, protoplasts or callus tissue.

[0572] In some implementations, the method is a method for improving the digestibility of plants or plant parts.

[0573] In some implementations, this method is used to improve the digestibility of straw.

[0574] In some implementations, the plant part is straw.

[0575] In one aspect, the present invention relates to a corn plant or plant part that can be obtained by the method described above.

[0576] In some implementations, the polynucleotide, one or more of the (molecular) markers (alleles), or the QTL (allele) are homozygous.

[0577] In some implementations, the polynucleotide, one or more of the (molecular) markers (alleles), or the QTL (allele) are heterozygous.

[0578] In one aspect, the present invention relates to the use of molecular markers as defined in Table A for the identification or selection of maize plants or plant parts.

[0579] In one aspect, the present invention relates to the use of molecular markers as defined in Table B for the identification or selection of maize plants or plant parts.

[0580] In one aspect, the present invention relates to the use of molecular markers as defined in Table C for the identification or selection of maize plants or plant parts.

[0581] In some implementations, the label is used to identify or select maize plants or plant parts that have improved digestibility.

[0582] In some implementations, the label is used to identify or select corn plants or plant parts that have improved straw digestibility.

[0583] In some implementations, digestibility is improved compared to that of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0584] In some embodiments, the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0585] In some implementations, the plant part is straw.

[0586] In one aspect, the present invention relates to the use of polynucleotides or QTLs (alleles) as defined herein according to the present invention for generating or producing maize plants or plant parts.

[0587] In some embodiments, the present invention relates to the use of polynucleotides or QTLs (alleles) as defined herein according to the invention for generating or producing maize plants or plant parts with improved digestibility.

[0588] In some embodiments, the present invention relates to the use of polynucleotides or QTLs (alleles) as defined herein according to the invention for generating or producing maize plants or plant parts having improved straw digestibility.

[0589] In some implementations, digestibility is improved compared to that of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0590] In some embodiments, the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0591] In some implementations, the plant part is the seed or straw.

[0592] In one aspect, the present invention relates to an isolated polynucleotide comprising a polynucleotide having a sequence selected from:

[0593] a) The nucleotide sequence of SEQ ID NO:7;

[0594] b) A nucleotide sequence having the coding sequence of SEQ ID NO:8;

[0595] c) A nucleotide sequence that is at least 90% identical to SEQ ID NO:7 or 8;

[0596] d) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:9;

[0597] Wherein the nucleotide sequences a) to d) contain one or more nucleotides at the position corresponding to the position between thymine at position 102 and guanine at position 103 of SEQ ID NO:7 or the corresponding position in SEQ ID NO:8, preferably not a multiple of three, more preferably an insertion of 8 or 7 nucleotides; or

[0598] e) The nucleotide sequence of SEQ ID NO:1 or 4 or the nucleotide sequence that is at least 90% identical to SEQ ID NO:1 or 4, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0599] f) A nucleotide sequence having the coding sequence of SEQ ID NO:2 or 5, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 5, preferably containing gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or containing gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1;

[0600] g) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 6, or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:3 or 6, wherein the nucleotide sequence preferably contains gcsgtct, preferably gcggtct, at positions 103-109 corresponding to SEQ ID NO:4, or gcsgttct, preferably gcggttct, at positions 103-110 corresponding to SEQ ID NO:1.

[0601] In one aspect, the present invention relates to the use of (isolated) polynucleotides for generating or producing maize plants or plant parts.

[0602] In some embodiments, the present invention relates to the use of (isolated) polynucleotides for generating or producing maize plants or plant parts with improved digestibility.

[0603] In some embodiments, the present invention relates to the use of (isolated) polynucleotides for generating or producing maize plants or plant parts with improved straw digestibility.

[0604] In some implementations, digestibility is improved compared to that of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0605] In some embodiments, the digestibility is improved by at least 5%, preferably at least 10%, compared to the digestibility of the maize line PH207 or a (near) isotropic line that does not contain any of the polynucleotides, QTLs (alleles), or one or more (molecular) markers (alleles) mentioned above.

[0606] In some implementations, the plant part is straw.

[0607] In one aspect, the present invention relates to an isolated polynucleotide comprising a molecular marker (allele) selected from Table A, its complementary or reverse complementary sequence, or a fragment thereof.

[0608] In one aspect, the present invention relates to an isolated polynucleotide comprising a molecular marker (allele) selected from Table B, its complementary or reverse complementary sequence, or a fragment thereof.

[0609] In one aspect, the present invention relates to an isolated polynucleotide comprising a molecular marker (allele) selected from Table C, its complementary or reverse complementary sequence, or a fragment thereof.

[0610] In some embodiments, the (isolated) polynucleotide contains 10 to 500 nucleotides, preferably 15 to 250 nucleotides, more preferably 18 to 250 nucleotides, and most preferably 20 to 250 nucleotides.

[0611] In some embodiments, the (isolated) polynucleotide particularly suitable as a molecular marker comprises at least 15, preferably at least 18, more preferably at least 20 consecutive nucleotides of any of SEQ ID NO:13 to 195, or complementary to, or anticomplemental to, consecutive nucleotides of any of SEQ ID NO:13 to 195, and preferably comprises at least one nucleotide of the corresponding polymorphism (donor allele) as provided in Table A.

[0612] In some embodiments, the (isolated) polynucleotide particularly suitable as a molecular marker comprises at least 15, preferably at least 18, more preferably at least 20 consecutive nucleotides of any of SEQ ID NO: 109 to 154, or complementary to, or anticomplemental to, consecutive nucleotides of any of SEQ ID NO: 109 to 154, and preferably comprises at least one nucleotide of the corresponding polymorphism (donor allele) as provided in Table A.

[0613] In some embodiments, the (isolated) polynucleotide particularly suitable as a molecular marker comprises at least 15, preferably at least 18, more preferably at least 20 consecutive nucleotides of any of SEQ ID NO: 124 to 125, or complementary to, or anticomplemental to, consecutive nucleotides of any of SEQ ID NO: 124 to 125, and preferably comprises at least one nucleotide of the corresponding polymorphism (donor allele) as provided in Table A.

[0614] In some implementations, the (isolated) polynucleotides are specific to plants or plant parts that have improved straw digestibility.

[0615] In one aspect, the present invention relates to (isolated) polynucleotides that specifically hybridize with molecular markers, their complementary sequences or reverse complementary sequences of Table A.

[0616] In one aspect, the present invention relates to (isolated) polynucleotides that specifically hybridize with molecular markers in Table B, their complementary sequences or reverse complementary sequences.

[0617] In one aspect, the present invention relates to (isolated) polynucleotides that specifically hybridize with molecular markers in Table C, their complementary sequences or reverse complementary sequences.

[0618] In some implementations, the (isolated) polynucleotides are primers or probes.

[0619] In some implementations, the (isolated) polynucleotides are allele-specific primers.

[0620] In some implementations, the isolated polynucleotides are KASP primers.

[0621] In one aspect, the present invention relates to primers or probes capable of specifically detecting polynucleotides, one or more (molecular) markers (alleles), or QTLs (alleles) as described elsewhere herein.

[0622] In one aspect, the present invention relates to primer sets capable of specifically detecting polynucleotides, one or more (molecular) markers (alleles), or QTLs (alleles) as described elsewhere herein.

[0623] In one aspect, the present invention relates to corn seeds named K0001, a representative sample of which was deposited on July 6, 2022, with NCIMB (National Collection of Industrial Food and Marine Bacteria; Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland) accession number NCIMB 43997, or to plants or plant parts, preferably straw, or derived thereof, grown or obtained therefrom. In another aspect, the present invention relates to corn seeds such as those deposited with NCIMB accession number NCIMB 43997, or to plants or plant parts, preferably straw, grown or obtained therefrom, or derived thereof. Seeds (or plants grown from them, plant parts obtained from them, or their progeny) deposited with NCIMB accession number NCIMB 43997 contain ma61134d16 as defined in Table A or Table 3, detectable by the molecular marker SEQ ID NO:124. Seeds (or plants grown from them, plant parts obtained from them, or their progeny) deposited with NCIMB accession number NCIMB 43997 contain the F35H gene (on chromosome 9) having the sequence shown in SEQ ID NO:4 (containing a 7 bp insertion). Plants grown from or obtained from seeds deposited with NCIMB accession number NCIMB 43997, or their progeny, exhibit increased or improved straw digestibility compared to corn plants without the aforementioned molecular marker or sequence. In one aspect, the invention relates to plant parts obtained or available from plants grown from corn seeds such as those deposited with NCIMB accession number NCIMB 43997, preferably straw, or their offspring. In another aspect, the invention relates to plants or plant parts, preferably straw, or their offspring, containing the F35H gene, as present in corn seeds such as those deposited with NCIMB accession number NCIMB 43997. In yet another aspect, the invention relates to plants or plant parts, preferably straw, or their offspring, containing the F35H gene (on chromosome 9) having the sequence shown in SEQ ID NO:4 (containing a 7 bp insertion), as present in corn seeds such as those deposited with NCIMB accession number NCIMB 43997.In one aspect, the present invention relates to plants or plant parts, such as preferably straw, or their offspring, which contain ma61134d16 (on chromosome 9) – detectable by the molecular marker SEQ ID NO: 124, as present in corn seeds such as those deposited with NCIMB accession number NCIMB 43997. It should be understood that, when referring to offspring derived from seeds deposited with NCIMB accession number NCIMB 43997 (the obtained plants), the molecular marker (ma61134d16), polynucleotide, sequence, QTL, SEQ ID NO (4 or 124), or F35H gene (i.e., present in the deposited seed NCIMB 43997 and resulting in increased straw digestibility) described according to the invention is present, i.e., at least heterozygous. Aspects and embodiments of the invention are further supported by the following non-limiting examples.

[0624] Example

[0625] Example 1

[0626] The gene F35H (wild-type sequence: SEQ ID NO: 7-9) was previously identified as a major factor in the digestibility of silage maize (WO 2019 / 206927). This patent application describes a knockout allele of this gene containing a long insertion in exon 1 of the f35h gene (SEQ ID NO: 10-12). Furthermore, this first patent application describes new favorable allele gains for TILLING-identified functional alleles. Dominant and co-dominant KASP and KPE markers (KPE markers represent capillary electrophoresis and show length polymorphism. This technique has been (and still occasionally is) used in SSR) are provided to detect natural insertions and TILLING mutants. These markers are routinely used in marker-assisted selection in breeding and trait integration, as well as in diagnostic analysis of the presence of favorable alleles in new double haploid (DH) lines. Surprisingly, a large number of lines were identified—these markers show conflicting data (see Table 1).

[0627] Table 1

[0628]

[0629] These newly developed lines were sequenced using 40X Illumina, and their gene spaces were assembled. Comparisons of these lines with PH207 (a non-SILO reference line) and with SILO lines carrying the initially identified Silo-09-02 allele revealed approximately 200 bp insertion loss. However, a 7–8 bp insertion was found in exon 1 of the f35h gene. This was not ruled out because blasting of the original approximately 200 bp insertion (long insertion) against the maize repetitive sequence database (http: / / maize.jcvi.org / repeat_db.shtml) did not reveal an annotated transposon. Markers were designed for the 8 bp and 7 bp insertions (ma61134d15; SEQ ID NO:125 and ma61134d16; SEQ ID NO:124).

[0630] Digestible neutral detergent fiber (DNDF, cell wall digestibility) was tested in six DH populations using NIRS calibration. Segregated populations were used for wild-type F35H and 8bp insertion mutants, or mutants with both long and short insertions. Digestibility differences were significant in the first case but not in the latter, suggesting that the 8bp insertion has the same effect on DNDF as the original transposon-like insertion. All tested lines showed a positive phenotype of increased straw digestibility (Table 2).

[0631] Table 2: Phenotypic analysis of six DH populations with 8bp inserts

[0632]

[0633] Maize lines containing a 7bp insertion (SEQ ID NO:4-6) were extensively tested as inbred lines and hybrid combinations, demonstrating high performance and high digestibility. Figure 1 ).

[0634] Although WO 2019 / 206927 only lists markers showing polymorphism between the SILO-09-02 line and Ku 5 maize, this invention provides more markers showing polymorphism in Ku 1, Ku 4 or Ku 9 (the new target Ku for this trait) in the range of + / -20 cM (10 cM) (Table 3).

[0635] Table 3. List of selected markers in the + / -20 cM genomic regions associated with Library 5. Markers with the highest MAF (Minor Allele Frequency) value were selected for use in as many other breeding libraries as possible.

[0636]

[0637]

[0638]

[0639]

[0640]

Claims

1. A method for identifying maize plants or maize plant parts, comprising screening for the presence or detection of polynucleotides containing the molecular marker alleles ma61134d15 and / or ma61134d16 in the genome of maize plants or maize plant parts; wherein ma61134d15 is an 8-nucleotide insertion between positions 134254381 and 134254382 on chromosome 9 of reference strain PH207 or at position 76.19 cM on chromosome 9 of reference strain PH207, and detectable by the molecular marker SEQ ID NO: 125; and ma61134d16 is an 8-nucleotide insertion between positions 134254381 and 134254382 on chromosome 9 of reference strain PH207 or at position 76.19 cM on chromosome 9 of reference strain PH207. The insertion of 7 nucleotides at cM can be detected by the molecular marker SEQ ID NO:

124.

2. The method of claim 1, wherein ma61134d15 is an insertion as shown in SEQ ID NO:

1.

3. The method of claim 1, wherein ma61134d16 is an insertion as shown in SEQ ID NO:

4.

4. A maize plant or a part of a maize plant, wherein the genome of the maize plant or the part of a maize plant contains a polynucleotide having a sequence selected from: a) A nucleotide sequence of SEQ ID NO: 1 or 4, wherein gcsgtct is contained at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttct is contained at positions 103-110 corresponding to SEQ ID NO: 1; b) A nucleotide sequence having the coding sequence of SEQ ID NO: 2 or 5, which contains gcsgtct at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttct at positions 103-110 corresponding to SEQ ID NO:

1. c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or 6, wherein the nucleotide sequence contains gcsgtct at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttct at positions 103-110 corresponding to SEQ ID NO:

1.

5. The corn plant or corn plant portion according to claim 4, wherein gcsgtct is gcggtct and gcsgttct is gcggttct.

6. The maize plant or maize plant portion according to claim 4, wherein the maize plant or maize plant portion is derived from a maize plant containing the polynucleotide obtained by introduction or introgression, or wherein the maize plant or maize plant portion is mutated by transposon or transposon factor, is transgenic, or is gene-edited.

7. A method for generating or producing maize plants or maize plant parts and / or for improving the digestibility of maize stalks, comprising introducing the polynucleotide as defined in any one of claims 1 to 6 into the genome of the maize plant or maize plant parts.

8. The method of claim 7, further comprising introducing a polynucleotide having a sequence selected from the group consisting of a maize plant or a portion thereof into the genome of the maize plant: a) A nucleotide sequence of SEQ ID NO: 1 or 4, wherein gcsgtct is contained at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttct is contained at positions 103-110 corresponding to SEQ ID NO: 1; b) A nucleotide sequence having the coding sequence of SEQ ID NO: 2 or 5, which contains gcsgtct at positions 103-109 of SEQ ID NO: 4, or gcsgttct at positions 103-110 of SEQ ID NO: 1, respectively. c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or 6, wherein the nucleotide sequence contains gcsgtct at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttct at positions 103-110 corresponding to SEQ ID NO:

1.

9. The method according to claim 8, wherein gcsgtct is gcggtct and gcsgttct is gcggttct.

10. The method according to any one of claims 7-9, comprising (a) providing a first maize plant having a polynucleotide as defined in any one of claims 1 to 6, or one or more molecular marker alleles as defined in any one of claims 1 to 6, or a first maize plant obtained from a seed of Zea mays deposited with NCIMB accession number NCIMB 43997 or its offspring, (b) hybridizing the first maize plant with a second maize plant, and (c) selecting progeny plants having a polynucleotide as defined in any one of claims 1 to 6, or one or more molecular marker alleles as defined in any one of claims 1 to 6.

11. The method according to any one of claims 7-9, wherein the following is introduced: Stable or transient integration can be achieved through transformation, transfection, microinjection, and gene gun bombardment. Using gene editing technology; Homologous recombination; Endogenous genes are modified using targeted mutagenesis or gene editing techniques.

12. The method of claim 11, wherein the gene editing technology is an insertion using a CRISPR system, TALEN, zinc finger nuclease, or a wide range of nucleases.

13. The method of claim 12, wherein the CRISPR system is CRISPR / Cas9, CRISPR / Cas12, CRISPR / CasX, or CRISPR / CasY.

14. The method of claim 11, wherein the homologous recombination is performed using one of the gene editing techniques and includes a repair template.

15. The method of claim 11, wherein targeted mutagenesis is tilling.

16. A maize plant or a portion of a maize plant that can be obtained by the method according to any one of claims 7 to 15.

17. The use of molecular markers as defined in any one of claims 1-3 for the identification or selection of maize plants or maize plant parts.

18. Use of the polynucleotide as defined in any one of claims 8-9 for the generation or production of maize plants or maize plant parts.

19. An isolated polynucleotide comprising a polynucleotide having a sequence selected from: a) A nucleotide sequence of SEQ ID NO: 1 or 4, wherein gcsgtct is contained at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttct is contained at positions 103-110 corresponding to SEQ ID NO: 1; b) A nucleotide sequence having the coding sequence of SEQ ID NO: 2 or 5, which contains gcsgtct at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttct at positions 103-110 corresponding to SEQ ID NO:

1. c) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or 6, wherein the nucleotide sequence contains gcsgtct at positions 103-109 corresponding to SEQ ID NO: 4, or gcsgttc at positions 103-110 corresponding to SEQ ID NO:

1.

20. The isolated polynucleotide according to claim 19, wherein gcsgtct is gcggtct and gcsgttct is gcggttct.

21. An isolated polynucleotide comprising SEQ ID NO:124 or SEQ ID NO:125, or its complementary or reverse complementary sequence.

22. The isolated polynucleotide according to claim 21, wherein it is a primer or probe.

23. The isolated polynucleotide according to claim 22, wherein it is an allele-specific primer.

24. The isolated polynucleotide according to claim 22, wherein it is a KASP primer.

Images