A method for breeding potato with high waxy property and taste close to sweet potato

Abstract

This invention discloses a method for targeted breeding of potatoes with high glutinousness and a texture similar to sweet potatoes, belonging to the field of crop molecular breeding. Addressing the shortcomings of existing potato varieties, such as a hard texture after cooking, poor glutinousness, and easy reversion to solid state after refrigeration, as well as the underdeveloped industrialization of sweet potato breeding and poor processing adaptability, this invention uses the native potato variety Mengwushu 7 as experimental material and utilizes CRISPR-Cas9 gene editing technology to perform targeted knockout. GBSS‑1 This invention utilizes genes to block amylose synthesis and increase amylopectin accumulation. Through tissue culture hardening, field planting, and a series of screenings combining starch physicochemical testing and sensory evaluation, a new potato variety with amylopectin content ≥90%, exhibiting a soft, sweet, and glutinous texture similar to sweet potatoes after steaming or cooking, was successfully bred. This invention boasts a short breeding cycle, precise targeting, and excellent experimental repeatability. It combines the advantages of a complete potato industry chain with the high glutinousness and resistance to retrogradation of sweet potatoes, demonstrating significant potential for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of crop germplasm improvement and molecular genetic breeding technology, specifically involving a method for directional breeding of potatoes with high glutinousness and a taste similar to sweet potatoes. Background Technology

[0002] Potatoes are my country's fourth largest staple crop, serving multiple purposes as a food, vegetable, and processing raw material. They are widely cultivated and have high industrial value. Currently, the mainstream cultivated potato varieties have a fixed genetic background, a simple starch composition, and generally high amylose content in their tubers. After steaming or boiling, they often exhibit a loose, starchy texture, a firm mouthfeel, insufficient softness and glutinousness, and a weak sweet flavor.

[0003] Sweet potatoes, with their high amylopectin content and suitable soluble sugar ratio, are soft, chewy, and sweet after steaming or boiling, making them a favorite among consumers. With the upgrading of dietary consumption, the market demand for highly chewy potatoes with a texture similar to sweet potatoes is increasing. Traditional hybridization breeding and systematic selection processes are time-consuming and highly random, making it difficult to precisely control the starch ratio and efficiently cultivate target varieties. Therefore, there is an urgent need to establish a reliable, standardized, and practical breeding technology scheme for improving the chewiness of potatoes.

[0004] While existing technologies have been reported to knock out potatoes GBSS-1 The gene produces glutinous starch, but the sgRNA target sequence of this invention is not disclosed, nor has a high glutinous potato with a taste similar to sweet potato been bred in the domestic main cultivar Mengwushu 7. This invention has significant novelty and inventiveness. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies and provide a method for targeted breeding of potatoes with high glutinousness and a taste similar to sweet potatoes. It utilizes CRISPR-Cas9 gene editing technology to target and inhibit... GBSS-1 By using genes to block amylose synthesis and directionally increase amylopectin accumulation, a new potato variety with a soft, sweet, and palatable texture similar to sweet potatoes was obtained, filling the gap in the technology of targeted breeding of specialty glutinous potatoes.

[0006] Granule-bound starch synthase (GBSS) is a key rate-limiting enzyme in the synthesis of amylose in potatoes, directly determining the amount of amylose produced and affecting the starchy texture of potatoes. This invention utilizes CRISPR-Cas9 technology to synthesize potato starch. GBSS-1 The gene (GenBank accession number: X58453.1) was knocked out at a specific site, blocking the amylose synthesis pathway; more of the starch synthesis precursor ADP-glucose flowed to the amylopectin synthesis pathway, significantly increasing the proportion of amylopectin in the tuber; at the same time, the level of soluble sugar was regulated through feedback from the endogenous metabolic network, ultimately resulting in a highly glutinous, sweet, and edible taste similar to sweet potato.

[0007] A method for selectively breeding potatoes with high glutinousness and a taste similar to sweet potatoes includes the following steps;

[0008] (1) Selection of breeding recipient materials: Select robust, disease-free, and genetically stable aseptic tissue culture potato seedlings as gene editing recipient materials; prioritize healthy seedlings with a plant height of 3-5 cm, well-developed leaves, and uniform growth.

[0009] (2) Targeted editing of key starch synthesis genes to construct targeted potato GBSS-1 A CRISPR-Cas9 editing vector for the gene (GenBank accession number: X58453.1) was developed, with the sgRNA target sequence 5′-GATATTAGAATCACATAGG-3′ and the PAM sequence TGG. The vector was introduced into tissue culture seedling cells via Agrobacterium infection and transformation for site-specific knockout. GBSS-1 Genes that inhibit the amylose synthesis pathway;

[0010] (3) After editing, the tissue culture seedlings were placed in a constant temperature culture room for acclimatization culture. The culture conditions were: temperature 22-26℃, light 14h / d, relative humidity 60%-75%; the culture period was 30-40 days. The growth status was observed regularly, and plants that were yellow, deformed, weak, or stagnant were removed. Seedlings with normal growth and stable genetic variation were selected.

[0011] (4) Field transplanting and conventional cultivation management: Select qualified stable mutant seedlings and transplant them to loose, fertile and well-drained fields; plant row spacing is 60cm and plant spacing is 25cm; water in a timely manner according to soil moisture during the growing season and apply nitrogen, phosphorus and potassium compound fertilizer twice in stages; take precautions against diseases and pests such as late blight and aphids throughout the process to ensure the healthy growth of plants until the tubers reach physiological maturity;

[0012] (5) Quality traits screening and identification: Mature tubers were harvested, and random samples were taken to determine the content and proportion of total starch, amylose, and amylopectin. Sensory evaluation of steaming and cooking was carried out simultaneously. Evaluation indicators included glutinousness, softness, sweetness, and flavor. Ten professional evaluators scored the tubers, with scores ranging from 1 to 10. Potato target lines with amylopectin accounting for ≥90% of total starch, a comprehensive sensory score of ≥8.5, and a soft, glutinous, sweet, and palatable texture after steaming and cooking, and a taste similar to sweet potatoes were selected.

[0013] The beneficial effects of this method are:

[0014] (1) Precise targeting and strong controllability: Based on CRISPR-Cas9 targeted knockout GBSS-1 The gene directly blocks amylose synthesis and regulates starch components in a targeted manner, with strong purpose and no random trait interference.

[0015] (2) High efficiency and short cycle: Compared with traditional hybridization breeding, the breeding cycle is greatly shortened, and it is not affected by generation segregation, so stable mutants can be obtained quickly;

[0016] (3) Significant quality improvement and stable effect: Potatoes with a branched starch content of ≥90% are obtained stably. After steaming or boiling, they are soft, glutinous, sweet and delicious, with a taste close to that of sweet potatoes, resulting in a breakthrough improvement in edible quality.

[0017] (4) Wide application value and high economic benefits: It can be used in specialty catering, snack food, deep processing raw materials and other scenarios, enriching potato germplasm types, increasing the added value of varieties, and has a broad market prospect. Attached Figure Description

[0018] Picture 1 For potatoes GBSS-1 Schematic diagram of gene structure and CRISPR-Cas9 sgRNA target location.

[0019] Picture 2 for GBSS-1 A schematic diagram showing the redirection of starch synthesis in tubers after gene knockout.

[0020] Picture 3 A roadmap for targeted breeding of high-glutinous potatoes.

[0021] Picture 4 This is a comparison chart of starch composition between the edited strain and the wild type.

[0022] Picture 5 Radar chart for sensory evaluation after steaming / cooking. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0024] Example 1: Targeted cultivation of potatoes with high glutinousness and a taste similar to sweet potatoes.

[0025] 1. Receptor material preparation

[0026] The domestically cultivated potato variety Mengwushu No. 7 was selected, and sterile tissue culture seedlings were obtained through shoot tip detoxification induction. Healthy seedlings with a height of 3-5cm, dark green and well-developed root system, and uniform growth were selected and temporarily stored in a 24℃ constant temperature tissue culture room for later use.

[0027] 2. GBSS-1 gene-targeted editing

[0028] (1) Target design: based on potatoes GBSS-IThe complete coding sequence of the gene (GenBank accession number: X58453.1) was used. The CHOPCHOP online CRISPR target design tool was employed to perform a full sequence scan of the Solanum tuberosum (potato) genome, using SpCas9 and knockout mode, generating a list of candidate sgRNA targets. From the top 10 high-scoring candidate targets, a four-fold screening process was used: location priority, PAM priority, specificity priority, and effectiveness priority. ① Targets located at the beginning, end, or near the edge of the catalytic domain were excluded (as they are prone to producing truncated proteins, functional residues, and incomplete knockout). ② Targets with non-optimal PAM types such as CGG and AGG were excluded (TGG in potatoes exhibits the most stable recognition efficiency and best reproducibility). ③ Targets with excessively high / low GC content, a high risk of secondary structure formation, or off-target effects were excluded. ④ Targets located in the middle of the coding region were prioritized, capable of inducing frameshift mutations, and able to completely block amylose synthesis. Based on software efficiency scoring and genome-wide off-target validation, the optimal sgRNA target sequence was finally determined to be: 5′-GATATTAGAATCACATAGG-3′, with the 3′ end immediately adjacent to the PAM sequence TGG. This target is located in the middle of the GBSS-I gene coding region. Knockout of this target can lead to premature termination of protein translation and complete loss of function, stably blocking amylose synthesis and providing a reliable target for cultivating high-gluten potatoes.

[0029] (2) Vector construction: Design forward and reverse primers for sgRNA containing BsaI sticky ends. Forward primer: 5′-GGCGATATTAGAATCACATAGG-3′, reverse primer: 5′-AAACCCTATGTGATTCTAATATC-3′. Mix 1 μL each of the forward and reverse primers (100 μM) with 8 μL of sterile water, incubate at 95℃ for 30 s, then allow to cool naturally to room temperature to anneal and form double-stranded sgRNA adapters. Digest the pYL-CRISPR / Cas9-Pubi vector with BsaI-HF. A 20 μL system contains 1 μg of the vector, 2 μL of 10×CutSmart buffer, and 1 μL of BsaI-HF. Digest at 37℃ for 30 min, then inactivate at 65℃ for 10 min. Mix the annealed double-stranded sgRNA adapters with the digested vector at a molar ratio of 3:1, add T4 DNA ligase, and ligate at 22℃ for 30 min. min, construct targeted knockout GBSS-1 The gene recombination editing vector was sequenced and verified to be correct.

[0030] (3) Agrobacterium-mediated transformation: The recombinant vector was introduced into Agrobacterium EHA105 competent cells, and positive strains were screened. Potato tissue culture seedling stem segments were infected with the positive strains and cultured for 3 days to promote Agrobacterium-mediated gene editing element transfer to plant cells. Subsequently, the stem segments were transferred to differentiation medium to induce adventitious bud regeneration, yielding cells containing... GBSS-1 Gene-edited potato regeneration plants.

[0031] 3. Tissue culture acclimatization and seedling selection

[0032] When the adventitious buds grew to 2-3 cm, they were cut off and transferred to rooting medium. They were then placed in a culture room for acclimatization culture under the following conditions: temperature 24℃, light 14h / d, humidity 70%. After 35 days of culture, yellowed, deformed, and weak plants were removed, resulting in 32 stable mutant seedlings.

[0033] 4. Field cultivation management

[0034] Select loose, fertile, and well-drained land, and deep plow and prepare the soil; plant with a row spacing of 60cm and a plant spacing of 25cm; water appropriately during the growing season and apply nitrogen, phosphorus, and potassium compound fertilizer twice; prevent late blight and aphids throughout the entire process; the growth cycle is 95 days, and the tubers are harvested uniformly after they mature.

[0035] 5. Quality Inspection and Strain Screening

[0036] (1) Starch composition determination: The contents of amylose and amylopectin were determined by the iodine colorimetric method;

[0037] (2) Sensory evaluation: The tubers were washed, steamed at 100℃ for 30 minutes, and scored by 10 professional evaluators on glutinousness, softness, sweetness and flavor, with scores ranging from 1 to 10.

[0038] (3) Screening results: A total of 5 target strains were screened, with a branched starch content ≥90%, sensory score ≥8.5 points, and taste similar to sweet potato. They were numbered Nuoshu 1 to 5.

[0039] Example 2: Comparison of starch composition between wild-type and edited strains

[0040] Using the unedited Mengwushu No. 7 as a wild-type control, the starch composition of the edited strain was detected:

[0041] (1) Wild type: total starch 20.4%, amylose 5.1%, amylopectin 15.3%, amylopectin accounted for 75%;

[0042] (2) Edited strain: total starch 20%, amylose 1.84%, amylopectin 18.16%, amylopectin accounted for 90.8%;

[0043] The results showed that the amylose content of the edited strain was significantly reduced and the amylopectin content was greatly increased, thus meeting the high glutinousness standard.

[0044] Example 3: Sensory Evaluation Comparison of Steaming and Boiling

[0045] The following are the sensory evaluation results after steaming, with average values:

[0046] (1) Wild type: Wild plant texture (softness) 36 points, flavor 82 points, delicacy 76 points, sweetness 58 points, palatability 75 points. The softness index score is extremely low, the sweetness and palatability are weak, and the overall sensory performance is poor.

[0047] (2) Edited strain: The gene-edited strain scored 92 points for texture (softness), 85 points for flavor, 88 points for delicacy, 62 points for sweetness, and 93 points for palatability. Except for a slight increase in sweetness, all other indicators were significantly improved, and the overall sensory quality was superior to that of the wild strain.

[0048] The results showed that the edited strain was soft, glutinous, and rich in flavor after steaming, with a taste that was very similar to sweet potato and significantly better than the wild type.

[0049] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the essence and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for selectively breeding potatoes with high glutinousness and a taste similar to sweet potatoes, characterized in that, Includes the following steps: (1) Selection of breeding recipient materials: Healthy, disease-free, and genetically stable aseptic tissue culture seedlings of Mengwushu No. 7 potato were selected as gene editing recipient materials. (2) Targeted editing of starch synthesis key genes: using CRISPR-Cas9 gene editing vector, the amylose synthesis metabolic pathway in plant body is blocked by site-specific knockout modification of potato tuber GBSS- 1 genes. (3) Domestication and cultivation of tissue culture seedlings after editing: The tissue culture seedlings that have completed gene editing are placed in a controlled environment of 22℃~26℃, light 14h / d, and relative humidity 60%~75% for 30~40 days for domestication and cultivation, and stable variant seedlings with normal growth and no deformities or lesions are selected. (4) Field transplanting and cultivation management: Transplant qualified mutant seedlings to the field and manage water and fertilizer and prevent and control diseases and pests in accordance with the standardized potato cultivation model until the tubers are physiologically mature. (5) Quality traits screening and identification: Mature tubers were harvested, and the proportions of total starch, amylose and amylopectin were determined. Combined with sensory evaluation after steaming and cooking, potato strains with amylopectin accounting for ≥90% of the total starch mass, soft and glutinous after steaming and cooking, and with a taste similar to sweet potatoes were screened.

2. The method of claim 1, wherein: The target modified gene in step 2 is potato granule-bound starch synthase GBSS-1 The gene, GenBank Accession No. X58453.1, suppresses amylose synthesis by site-directed knockout.

3. The method of claim 1, wherein: In step 2, the sgRNA target sequence of CRISPR-Cas9 is 5′-GATATTAGAATCACATAGG-3′, and the PAM sequence is TGG.

4. An sgRNA for knocking out a potato GBSS-1 gene, characterized in that, The nucleotide sequence is as described in claim 3.

5. A recombinant vector for knocking out a potato GBSS-1 gene, characterized in that It contains the sgRNA, BsaI restriction site, and pYL-CRISPR / Cas9-Pubi vector backbone as described in claim 4.

6. The method of claim 1, wherein: In step 5, the sensory evaluation indicators for steaming and cooking include glutinousness, density, sweetness, and flavor. The screening criterion is a comprehensive sensory score of ≥8.5 points.

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