High-quality tomato inbred line breeding method based on wolfberry eccDNAs transfer

Abstract

The invention discloses a high-quality tomato inbred line breeding method based on wolfberry fruit eccDNAs transfer, and belongs to the field of plant biological breeding and traditional breeding, the method comprises the following steps: grafting by using tomato as a scion and wolfberry fruit as a rootstock, carrying out soil burying overwintering cultivation to induce the scion above a graft union to form a regenerated adventitious bud, and carrying out high-quality tomato inbred line breeding. Taking regenerated adventitious buds for asexual propagation, then performing continuous selfing, screening selfing lines with superior characters based on fruit morphology, physiological characteristics and quality indexes, and performing wolfberry eccDNAs verification on second-generation selfing plants. The high-quality flavor tomato inbred line with genetic stability is obtained by optimizing the breeding method, a foundation is created for research and development of new varieties, and the method has good popularization and application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biological breeding and traditional breeding, and particularly relates to a high-quality tomato inbred line breeding method based on eccDNAs (Extrachromosomal circular DNAs) transfer of Lycium barbarum. BACKGROUND

[0002] Tomato (Solanum lycopersicum L.) is an annual or perennial herb of Solanaceae, and is originally from South America and widely cultivated in north and south China, and has become one of the main vegetables in China. The fruit of tomato has rich nutrition and special flavor, and is eaten in various ways, such as raw food, fried food, sauce, etc., and is produced on a large scale in the world, and is an important horticultural crop and economic crop.

[0003] In recent years, people have put forward higher requirements for the green safety, nutritional health and other aspects of vegetables. At the same time, the cultivation mode of vegetables is developing towards mechanization and intelligentization, and new requirements are put forward for the breeding of new germplasm and new varieties of vegetables. The coordinated improvement of yield, quality, resistance and other traits is the trend of breeding new varieties of vegetables in the future.

[0004] The close relative of tomato, Lycium barbarum, is a woody perennial crop with a large number of excellent gene resources related to yield, quality and resistance, and has strong low temperature tolerance, high anthocyanin and vitamin C content. These plant compounds have high antioxidant effect, which can reduce the risk of many chronic diseases, including cancer and cardiovascular disease, by preventing oxidation and peroxidation reactions. Therefore, Lycium barbarum is an excellent parent donor material for genetic improvement of tomato and introduction of excellent gene resources. However, tomato and Lycium barbarum have distant genetic relationship, and traditional distant hybridization and genetic transformation are difficult, and methods such as Agrobacterium-mediated transgenic and gene editing have not been successful. Therefore, establishing a new technology for green, safe and efficient transfer of Lycium barbarum genes to tomato, especially the transfer of genes related to high resistance, high quality and long seasonal growth, will become an important breakthrough for breeding new varieties of high-yield, high-quality and high-resistance tomatoes, and is a relatively advanced research field.

[0005] Horizontal Gene Transfer (HGT) provides a new path for crop genetic improvement. In plants, many studies have shown that grafting can mediate the exchange of genetic material between rootstocks and scions. Although this phenomenon brings the possibility of introducing distant traits (such as improving nutritional quality), its application value in elite breeding still needs to be verified by the stable existence and continuous expression of related traits in offspring.

[0006] Horizontal gene transfer introduces the excellent traits of wolfberry into tomato. The previous grafting technology (see patent CN111213504B) and the horizontal gene transfer technology based on grafting (see patent CN116439059A) developed by the research group successfully transferred the genetic material of wolfberry to the scion tomato. However, the realization of its breeding value depends on the cultivation of new germplasm and new varieties. Continuous selfing and directional screening can gradually retain the transferred wolfberry genetic material, and ultimately obtain a selfing line with stable genetic traits of wolfberry genetic material. In addition, commercial tomato varieties need to meet the requirements of genetic consistency and production stability. After fixing the transferred traits as a selfing line, it can be quickly and large-scale seed production through conventional hybrid breeding, which can significantly reduce production costs. SUMMARY

[0007] The present application aims at the above-mentioned deficiencies of the prior art, and provides a breeding method for a tomato selfing line with high soluble solids content, high soluble sugar content and suitable sugar-acid ratio after wolfberry eccDNAs transfer.

[0008] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows: a high-quality tomato selfing line breeding method based on wolfberry eccDNAs transfer, based on the existing grafting (CN111213504B) and wolfberry eccDNAs transfer to tomato (CN116439059A) induced by grafting, grafting and induction of regenerated adventitious buds, obtaining a selfing line with stable traits through multiple generations of selfing after vegetative propagation, retaining the tomato germplasm resources after wolfberry eccDNAs transfer and providing subsequent breeding materials.

[0009] The method is carried out according to the following steps: (1) the grafting step is performed according to CN111213504B; (2) bury the grafting joint to 3 cm below the soil surface for overwintering cultivation, and induce the scion above the grafting interface to form regenerated adventitious buds. Take the regenerated adventitious buds 1-2 cm above the grafting interface for the next operation; (3) vegetative propagation of the regenerated adventitious buds to obtain regenerated clonal plants; (4) continuously selfing the regenerated clonal plants for 4 generations, and mixing and collecting the seeds of each generation to form a population; (5) In the 4th generation population, based on a plurality of tomato fruit morphological and physiological characteristics measurement indexes of three periods (flowering period, full flowering period, full fruiting period, weight proportion is 30%, 30%, 40% respectively), weighted score screening is carried out, and a plurality of lines with the highest ranking are selected for fruit quality determination; the tomato fruit morphological and physiological characteristics measurement indexes include: single fruit weight (weight 15%), transverse diameter (weight 5%), longitudinal diameter (weight 5%), fruit number (weight 15%), color a value (weight 15%), fruit shape index (weight 5%), SPAD value (Soil Plant Analysis Development, weight 10%), net photosynthetic rate (weight 10%), plant height (weight 5%), stem diameter (weight 10%), leaf length (weight 3%), leaf width (weight 2%); (6) In the lines, at least one of a plurality of tomato quality indexes is further selected to be significantly better than a single plant of a control to be saved for seed; the tomato quality indexes include: Vc, soluble solids, titratable acid, soluble sugar, anthocyanin, polysaccharide, sugar acid ratio, soluble protein content, free amino acid; the control is a line of a tomato synchronous self-crossing 4 generations and screened in the same way without grafting; (7) The selected line is self-crossed for the 5th time, and a genetically stable high-quality flavor tomato inbred line is obtained.

[0010] Further, in the step (1), the tomato scion is a cultivated species "Zheyingfen No. 1" (Solanum lycopersicum), and the wolfberry stock is Lycium barbarum and Lycium ruthenicum.

[0011] Further, in the step (1), the specific grafting method is: selecting a tomato seedling growing to 3 leaves and one heart, cutting the epicotyl of the tomato seedling 0.5 cm from the cotyledon, treating it with 2.0 mg / L NAA for 5 min, cutting two knives symmetrically along the center line from the bottom of the epicotyl to make the cut tomato wedge-shaped with a wide top and a narrow bottom, as a grafted scion; cutting the wolfberry seedling with low lignin content and suitable for grafting from the top end to the 5 cm position, cutting 0.5 cm-1.0 cm downward along the center line of the cross section after cutting, as a stock; inserting the cut tomato scion into the split of the wolfberry 0.5 cm-1.0 cm for grafting.

[0012] Further, the step (3) is asexual propagation: the regenerated adventitious buds are cut in the substrate of peat: vermiculite: perlite = 3:1:1, the humidity is maintained at 80%-85%, and the culture is carried out at 20℃-25℃.

[0013] Further, in the step (4), the second generation of self-pollinated plants are taken to detect the eccDNAs of the wolfberry, and it is confirmed that at least one specific fragment of the eccDNAs of the wolfberry is present in the second generation of the sexual offspring plants.

[0014] Further, in the step (6), the control is the same "Zheyingfen No. 1" as the scion, which is synchronously self-pollinated for four generations and screened in the same way.

[0015] In some specific embodiments, the strain selected in the step (6) has a sugar acid ratio between 10 and 20, and the indexes of soluble sugar, polysaccharide, soluble solids and the like are significantly higher than those of the control.

[0016] The beneficial effects of the present application are as follows: (1) The present application fixes and selects the tomato germplasm resource after the transfer of the wolfberry eccDNAs, thereby providing self-pollinated line materials for subsequent research and new variety preparation.

[0017] (2) The present application selects the self-pollinated line with superior comprehensive traits by using the calculation of 12 weighted scores based on the fruit morphology (single fruit weight, fruit shape index and the like), physiological characteristics (net photosynthetic rate, SPAD value and the like) and quality indexes (soluble solids, sugar acid ratio and the like).

[0018] (3) The mixed seed collection strategy after four generations of continuous self-pollination is adopted to maintain the population genetic diversity and delay the elimination of excellent recessive genes in early screening; and the fifth generation of directional self-pollination is combined to make the target traits (such as sugar acid ratio of 10-20 and significant increase of soluble sugar) stably inherited. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Fig. 4 is a comparison diagram of the fruit quality of the excellent single plants (RZ-n, BZ-n) and the control (ZYF, Zheyingfen) in the fourth generation of seedlings; wherein, Figure 1 (A) of Fig. 4 is a comparison diagram of the sugar acid ratio indexes of each group, Figure 1 (B) of Fig. 4 is a comparison diagram of the soluble sugar content of each group, Figure 1 (C) of Fig. 4 is a comparison diagram of the polysaccharide content of each group, Figure 1 (D) of Fig. 4 is a comparison diagram of the average content of soluble solids of each group. DETAILED DESCRIPTION

[0020] The present application will be further described in detail by specific embodiments, but the present application is not limited to the following embodiments only.

[0021] Example 1 The high-quality tomato self-pollinated line breeding method based on the transfer of wolfberry eccDNAs in the present embodiment has the following specific steps: 1. Take "Zheyingfen No. 1" (Solanum lycopersicum L.) as tomato scion, Lycium barbarum and Lycium ruthenicum as wolfberry rootstock, graft according to the method described in patent CN111213504B: select tomato seedlings growing to 3 leaves and heart, cut the epicotyl of the tomato seedling 0.5 cm from the cotyledon, treat with exogenous auxin (2.0 mg / L NAA) for 5 min, cut two knives symmetrically along the center line from the bottom of the epicotyl, making the cut tomato wide at the top and narrow at the bottom in a wedge shape, as the grafted scion; cut the wolfberry seedling with low lignin content and suitable for grafting from the top to the 5 cm position, cut 0.5 cm-1.0 cm downward along the center line of the cross section after cutting, as the rootstock; insert the cut tomato scion into the split of 0.5 cm-1.0 cm of wolfberry for grafting.

[0022] 2. Buried the grafting joint to 3 cm below the soil surface for winter cultivation, induced the scion above the grafting interface to form regenerative adventitious buds. Take the regenerative adventitious buds 1-2 cm above the grafting interface for the next operation.

[0023] 3. Cut the regenerative adventitious buds in the substrate of peat: vermiculite: perlite = 3:1:1, maintain humidity at 80%-85%, cultivate at 20℃-25℃. Self-pollinate the plants formed by regenerative adventitious buds, mix the seeds, and obtain F1 generation seeds.

[0024] 4. The next season, sow F1 generation seeds, plant 6 plants per material, self-pollinate, mix the seeds, and obtain F2 generation seeds.

[0025] 5. Verify the wolfberry eccDNAs of F2 generation seedlings, identify 12 wolfberry eccDNAs (Table 1) which are the same as the grafting transfer contemporary. Among them, the compared wolfberry genome: https: / / www.ncbi.nlm.nih.gov / datasets / genome / ?taxon=112863.

[0026] Table 1: Record table of 12 wolfberry eccDNAs sequences which are the same as the grafting transfer contemporary

[0027] 6. The next season, sow F2 generation seeds, plant 6 plants per material, self-pollinate, mix the seeds, and obtain F3 generation seeds.

[0028] 7. The next season, sow F3 generation seeds, plant 6 plants per material, self-pollinate, mix the seeds, and obtain F4 generation seeds.

[0029] 8. In the next season, sow F4 generation seeds, planting 6 plants per material. Self-pollinate each plant. Conduct phenotypic surveys at three stages of plant formation (initial flowering, full bloom, and full fruiting, with weights of 30%, 30%, and 40% respectively), including: single fruit weight, transverse diameter, longitudinal diameter, number of fruits, color a value, fruit shape index, SPAD value, net photosynthetic rate, plant height, stem diameter, leaf length, and leaf width. The traits are generally stable. Standardize and weight the measured values ​​of the above traits. The standardization formula is: (measured value - minimum value) / (maximum value - minimum value); the weights are: single fruit weight (weight 15%), transverse diameter (weight 5%), longitudinal diameter (weight 5%), number of fruits (weight 15%), color a value (weight 15%), fruit shape index (weight 5%), SPAD value (weight 10%), net photosynthetic rate (weight 10%), plant height (weight 5%), stem diameter (weight 10%), leaf length (weight 3%), and leaf width (weight 2%). The top three individual plants from each variety were selected for further fruit quality testing. The testing indicators were: vitamin C, soluble solids, titratable acid, soluble sugar, anthocyanins, polysaccharides, sugar-acid ratio, soluble protein content, and free amino acids. A line of "Zheyingfen No. 1" that underwent four generations of synchronous self-pollination and was selected using the same method was used as a control. Individual plants from each variety whose specific nutritional indicators were significantly higher than the control were selected. Figure 1 (Single-plant seed saving) to obtain F5 generation seeds. Figure 1 As shown, the sugar-acid ratios of the strains screened in this embodiment are all between 10 and 20, and several indicators are significantly better than the control.

[0030] 9. The self-pollinated offspring of the control plant “Zheyingfen No. 1” was named ZYF; the offspring of the regenerated adventitious buds of Ningxia wolfberry grafted with “Zheyingfen No. 1” was named RZ-n, where n is the plant number in the single plant selection; the offspring of the regenerated adventitious buds of black wolfberry grafted with “Zheyingfen No. 1” was named BZ-n, where n has the same meaning as above.

[0031] 10. Sow F5 generation seeds in the next season, with 25 plants planted per material sample. Individual plants will be self-pollinated to obtain genetically stable, high-quality flavor tomato inbred lines.

[0032] The above embodiments are only used to illustrate the ideas and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The scope of protection of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design concepts disclosed in the present invention are within the scope of protection of the present invention.

Claims

1. A method for breeding high-quality tomato inbred lines based on Lycium barbarum eccDNAs transfer, characterized in that, Includes the following steps: Tomatoes were used as scions and wolfberries as rootstocks for grafting. The graft union was buried under the soil surface for overwintering cultivation, which induced the scion above the graft union to form regenerated adventitious buds. The regenerated adventitious buds were used for asexual reproduction to obtain regenerated asexual plants; The regenerated clonal plants were continuously self-pollinated for 4 generations, and the seeds of each generation were mixed and collected to form a population. Among them, the second generation of self-pollinated plants were tested for wolfberry eccDNAs. The plants were selected to continue self-pollination based on the criterion of identifying at least one wolfberry eccDNAs specific fragment that is the same as that of the grafted and transferred generation. In the fourth generation population, a weighted scoring system was used to select the top-ranking strains based on various tomato fruit morphology and physiological characteristics. Among the strains, individual plants with tomato quality indicators significantly superior to the control were further selected for seed saving. The control was a strain of ungrafted tomatoes that had been synchronously self-pollinated for 4 generations and screened in the same way. The selected lines were subjected to 5th generation self-pollination to obtain genetically stable, high-quality flavor tomato inbred lines.

2. The method according to claim 1, characterized in that: The grafting process includes the following steps: Select tomato seedlings that have grown to 3 leaves and 1 heart. Cut off the epicotyl of the tomato seedling 0.5 cm from the cotyledon. Treat it with exogenous auxin. Make two symmetrical cuts along the center line from the bottom of the epicotyl to make the cut tomato wedge-shaped, wider at the top and narrower at the bottom. Use this as a grafting scion and insert it into a wolfberry seedling with low lignin content that is suitable for grafting.

3. The method according to claim 2, characterized in that: The grafting scion is inserted into a wolfberry seedling with low lignin content and suitable for grafting. Specifically, the wolfberry seedling is cut horizontally 5 cm down from the top, and then cut 0.5 cm-1.0 cm down along the center line of the horizontal cut cross-section to serve as the rootstock. The cut tomato scion is then inserted into the 0.5 cm-1.0 cm split in the wolfberry seedling for grafting.

4. The method according to claim 1, characterized in that: The regenerated adventitious buds are taken 1-2 cm above the grafting interface.

5. The method according to claim 1, characterized in that: The asexual reproduction method is as follows: regenerated adventitious buds are inserted into a substrate of peat:vermiculite:perlite = 3:1:1, and the humidity is maintained at 80%-85% and cultured at 20℃-25℃.

6. The method according to claim 1, characterized in that: The indicators for measuring the morphology and physiological characteristics of tomato fruits include: single fruit weight, transverse diameter, longitudinal diameter, number of fruits, color a value, fruit shape index, SPAD value, net photosynthetic rate, plant height, stem diameter, leaf length, and leaf width at the initial flowering stage, full flowering stage, and full fruiting stage, respectively.

7. The method according to claim 1, characterized in that: The tomato quality indicators include: vitamin C, soluble solids, titratable acid, soluble sugar, anthocyanins, polysaccharides, sugar-acid ratio, soluble protein content, and free amino acids.

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