Method for breeding new variety of four-season strawberry by using SSR (Simple Sequence Repeat) molecular marker

Abstract

The invention relates to the technical field of strawberry breeding, and relates to a method for breeding a new variety of four-season strawberry by using an SSR molecular marker. The method mainly comprises the following steps: carrying out hybrid F1 generation seedling stage screening by adopting SSR molecular markers, carrying out field planting in March in spring of the next year after F1 generation single plants left after molecular marker screening are maintained and overwintering in a seedling raising shed under normal conditions, carrying out single plant field evaluation screening of superior plants and superior plant line propagation, and carrying out field evaluation screening. According to the method, a set of method is researched, developed and summarized in the aspects of resource evaluation, four-season genetic research, parent selection and matching, hybrid seed treatment, F1-generation single plant molecular marker screening, field evaluation screening and the like, and the efficiency of four-season strawberry breeding is remarkably improved.

Description

Technical Field

[0001] This application relates to the field of strawberry breeding technology, and in particular to a method for breeding new varieties of seasonal strawberries using SSR molecular markers. Background Technology

[0002] strawberry( Fragaria x ananassa Duch., also known as pineapple strawberry, belongs to the genus Fructus Strawberry in the family Rosaceae. Fragaria Strawberries are brightly colored, soft and juicy, fragrant, sweet and sour, and rich in nutrients, making them popular with consumers both domestically and internationally. Everbose strawberries can also complete flower bud differentiation and fruiting under long-day conditions in summer (at suitable growing temperatures), thus extending the harvest period. The complementary flowering habits of seasonal and everbose strawberries make year-round production of fresh strawberries possible. Currently, everbose strawberries are cultivated on a large scale globally, ensuring the supply of fresh strawberries in summer and autumn. In the central Yunnan region (a cool area at an altitude of 2300-2600 meters), the planting area of ​​everbose strawberry varieties exceeds 80,000 mu / year, accounting for more than 70% of the national summer and autumn strawberry production area and more than 80% of the yield, making it a major guarantee for the year-round supply of fresh strawberries nationwide.

[0003] New variety breeding is the foundation for the sustainable and healthy development of the strawberry industry. Currently, Yunnan's ever-blooming strawberry varieties mainly rely on imports. Therefore, it is necessary to conduct research and development on ever-blooming strawberry breeding technologies, promote the breeding of local ever-blooming strawberry varieties, cultivate varieties with independent intellectual property rights, reduce the industry's dependence on foreign varieties in the summer strawberry sector, and improve the quality and sustainability of the industry's development. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, this application provides a method for breeding new perennial strawberry varieties in central Yunnan, which can significantly improve the efficiency of breeding new perennial strawberry varieties.

[0005] On the one hand, this application provides an application of SSR molecular markers in the breeding of new strawberry varieties, wherein the primers for the SSR molecular markers are Bx125-F and Bx125-R; The sequence of Bx125-F is shown in SEQ ID NO: 1; the sequence of Bx125-R is shown in SEQ ID NO: 2.

[0006] On the other hand, this application also provides a method for breeding new strawberry varieties using SSR molecular markers, the method comprising the following steps: S1. Select varieties with the target trait as parents, and pre-treat the F1 generation seeds obtained by hybridization to obtain pre-treated hybrid F1 generation seeds. S2, the pretreated parent seeds obtained in S1 are hybridized to obtain the hybrid F1 generation population. The hybrid F1 generation is screened using SSR molecular markers, wherein the primers for the SSR molecular markers are Bx125-F and Bx125-R. The sequence of Bx125-F is shown in SEQ ID NO: 1; the sequence of Bx125-R is shown in SEQ ID NO: 2; S3 involves sequentially cultivating and propagating the F1 generation individual plants selected from S2 to obtain new strawberry varieties.

[0007] Furthermore, the selection of varieties for the target traits in S1 includes: for processing varieties, American everbearing strawberry varieties are selected as parent materials; for fresh-eating varieties, European and Japanese everbearing strawberry varieties are selected as parent materials; and one of the two parents of the hybrid combination is a seasonal strawberry variety.

[0008] Furthermore, the pretreatment in S1 involves treating the seeds with 98% sulfuric acid for 7-10 minutes, then washing and drying them. In early September, the seeds are sown in 128-cell trays filled with substrate. After germination, the seeds are transplanted to 50-cell trays filled with the substrate and then maintained.

[0009] Furthermore, the composition and mass ratio of the matrix are peat:perlite 7:3.

[0010] Furthermore, the maintenance and propagation in S3 are carried out in an area with an altitude of 1700-1900 m.

[0011] Furthermore, the weight ratio of each element N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B and Mo in the strawberry elevated cultivation fertilizer water maintained in S3 is 100:25:110:80:24:2.5:0.5:0.02:0.05:0.5:0.01.

[0012] Beneficial effects 1. The molecular markers provided in this application can identify and remove approximately 50% of seasonal strawberry plants 70-80 days after hybrid seed sowing, reducing the cost and time spent on field screening by 50%.

[0013] 2. This application emphasizes that the single plants selected through molecular marker screening need to be overwintered and then transplanted into elevated substrate cultivation troughs in March of the following year for the evaluation of traits such as flowering. This measure avoids the erroneous elimination of nearly 50% of the ever-blooming F1 single plants. During the research and development process, a gene that inhibits summer flowering in strawberries was discovered (…). FaDSFThe gene exists, but it only inhibits flowering in the year of sowing, causing half of the four-season plants to not flower in the year of sowing (similar to seasonality). However, after overwintering and being exposed to low temperatures, the inhibitory effect of this gene disappears. If the hybrid F1 seeds are sown in the same year and the flowering and other traits are evaluated, misjudgment is likely to occur, which will lead to the elimination of nearly 50% of the four-season F1 plants.

[0014] 3. The breeding method provided in this application utilizes the microclimate advantages of different altitude areas in central Yunnan at each stage of the breeding process, and arranges field operations in a timely manner, thereby improving the overall breeding efficiency. In the stages of breeding resource evaluation (S1) and superior line screening (S5), the plants are planted in areas at an altitude of 2200 to 2600 meters in central Yunnan. The highest summer temperature in this area is generally below 30℃, and the yield and quality of fresh fruit are best in this area, making the quantitative traits such as fresh fruit yield and fruit sugar and acid content representative. Hybridization (S2), seedling raising (S3), and F1 generation single plant screening (S4) are arranged in areas at an altitude of 1700 to 1900 meters around Kunming, Yuxi, and Qujing. This area has warmer winters than the high-altitude areas in step S1, and the perennial strawberries do not enter deep dormancy in winter. After spring, the plants grow rapidly and produce a large number of flowers, which facilitates the arrangement of hybridization, fruit development, seed collection, sowing and seedling raising, and sampling. Choosing to pollinate in mid-March can significantly improve the fruit setting rate and the rate of large fruits (due to more seeds). Sowing in September allows the plants to mature before winter (December), making it easier to sample leaves for molecular marker screening. It also reduces the time spent on greenhouse maintenance (compared to sowing in May-August) and the cost of materials.

[0015] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0016] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments of this application taken in conjunction with the accompanying drawings (tables), wherein the same reference numerals generally represent the same components in the exemplary embodiments of this application.

[0017] Figure 1 Gel electrophoresis image of F1 generation selection for the SSR molecular marker Bx125. Detailed Implementation

[0018] Unless otherwise specified, all materials used in the following examples are purchased from the market and are available to the public.

[0019] Example 1 This application provides a method for breeding new perennial strawberry varieties in central Yunnan, comprising the following steps: S1. Evaluation of Breeding Resources: The main economic traits of everbearing strawberry breeding resources (varieties) were evaluated in the cool summer zone (altitude 2200-2600 meters, daytime maximum temperature not exceeding 30℃) of the central Yunnan region, a low-latitude plateau area (around 23-26 degrees north latitude). The evaluation indicators mainly included the number of inflorescences, fruit yield, fruit firmness, and soluble solids content of the fruit. Seedlings of each breeding resource (variety) were transplanted into elevated substrate cultivation troughs in mid-March. Plant management followed conventional techniques (Wang Guoxian, Sun Jianli, Jiang Haiyu, Yang Chunmei, Wu Lifang, Shan Qinli, Yu Rongpei, Ruan Jiwei, Determination and Analysis of Main Economic Traits of Day-Neutral Strawberry Varieties, Southwest China Journal of Agricultural Sciences, 2018, 10: 2179-2184). The evaluation period was from late April to late November. The number of inflorescences and fruit yield were the cumulative numbers for the entire evaluation period, while fruit firmness and soluble solids content were the average values ​​of 3-5 sampling data. The specific determination methods were based on commonly used methods (Ruan JW, YH Lee, and YR, Yeoung, Flowering and fruiting of day-neutral and ever-bearing strawberry cultivars in high-elevation for summer and autumn fruit production in Korea, Horticulture, Environment, and Biotechnology, 2013a, 54(2):109-120. and Ruan JW, YH Lee, SJ Hong, and Y.R. Yeoung, Sugar and organic acid contents of day-neutral and ever-bearing strawberry cultivars in high-elevation for summer and autumn fruit production in Korea, Horticulture, Environment, and Biotechnology, 2013b, 54(3):214-222.), which will not be elaborated here.

[0020] The weight ratio of each element in the fertilizer and water formula for plant maintenance—N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B, and Mo—is 100:25:110:80:24:2.5:0.5:0.02:0.05:0.5:0.01, where NO3- - and NH4+ The molecular ratio is 85:15. The formula significantly increases the N / K fertilizer ratio and NH4+. + The ratio promotes the vegetative growth of plants and avoids flowering and fruiting fatigue in seasonal varieties, which can lead to stagnation of vegetative growth and subsequent yield loss.

[0021] S2, Selection and Hybridization of Breeding Parents: Select breeding resources (varieties) possessing some of the target breeding traits as parent materials. For processing varieties, prioritize American everbearing strawberry (also known as Japanese neutral strawberry) varieties as parent materials. This type of variety has large, firm fruit, red flesh, and high organic acid content. For fresh-eating varieties, prioritize European and Japanese everbearing strawberry (also known as long-day strawberry) varieties as parent materials. This type of variety has high sugar content, low organic acid content, and good taste (Table 1-6). One of the two parents in a hybrid combination can be a seasonal strawberry variety. The selected parental breeding materials (seedlings) were planted in elevated substrate cultivation troughs. In mid-March, reciprocal crosses were performed within the selected parental combinations. Hybrid pollination was carried out under artificial isolation conditions to avoid contamination and infection by non-target pollen. The fruits were fully mature about 45 days after pollination. The seeds were removed from the mature fruits, washed, and dried in an oven (Shanghai Boxun BGZ-240) at 40℃ for 72 hours before being stored for later use.

[0022] The hybrid parent plants are planted in areas with an altitude of 1,700 to 1,900 meters around Kunming, Yuxi and Qujing. These areas are warmer in winter than the high-altitude areas in step S1, and the ever-blooming strawberries do not enter deep dormancy in winter. After spring, the plants grow rapidly and produce a large number of flowers, which is convenient for hybridization, fruit development and seed collection.

[0023] The hybridization pollination time is arranged in mid-March, when the temperature rises and the sunshine is stronger. Seasonal varieties grow vigorously and have a large number of flowers. Even if one of the hybrid parents is a seasonal variety, it will also bloom in large quantities at this time. Select the first-level flowers (1 flower / inflorescence) and second-level flowers (2 flowers / inflorescence) with strong inflorescences of the mother plant as pollen recipients. After successful pollination, the number of seeds per fruit can reach 400-600. The number of hybrid seeds of each parent combination should be maintained at more than 2,000.

[0024] S3, Seed Treatment and Seedling Raising: Treat seeds with 98% sulfuric acid for 7-10 minutes to corrode part of the seed coat, then wash with clean water and dry. Sow the treated seeds in early September in 128-cell trays filled with a substrate of peat (70%) and perlite (30%). Sowing and seedling management follow conventional management techniques. 40-60 days after germination, when the seedling roots have filled the 128-cell trays, they can be transplanted to 50-cell trays. Fill the substrate as above and maintain them in a regular greenhouse. Management techniques follow conventional management techniques.

[0025] Sowing, seedling raising, and F1 single plant selection were carried out in areas with an altitude of 1700 to 1900 meters around Kunming, Yuxi, and Qujing. These areas are warmer in winter than the high-altitude areas in step S1, and the perennial strawberries do not enter deep dormancy in winter, which facilitates the arrangement of sowing, seedling raising, sampling for DNA extraction and molecular markers.

[0026] S4. Screening of F1 Hybrid Plants: Summer flowering is a fundamental trait of everbearing strawberry varieties. Accurate and rapid screening of F1 plants with summer flowering habits within the hybrid F1 population is a core issue in improving the breeding efficiency of everbearing strawberries. Molecular marker screening of F1 plants is one of the more efficient methods. During the F1 seedling stage, DNA was extracted from leaves taken from the seedling shed in mid-to-late November. SSR molecular markers were used in the laboratory for screening the F1 hybrids. Primer series information is shown in Table 7. F1 plants with a band at 300bp on primer amplification gel electrophoresis (approximately 50% differing between parental pairs) are seasonal strawberry varieties. Figure 1 Seedlings should be removed promptly during the seedling stage to avoid the cost and time spent on screening during later field planting.

[0027] The F1 generation plants selected after molecular marker screening were overwintered under normal conditions in a seedling shed and then transplanted into the aforementioned elevated substrate cultivation troughs in March of the following year, each plant being individually numbered. During this period, they were cared for using conventional methods. In the summer and autumn of the same year, the F1 generation plants underwent their first screening for traits such as flowering quantity, fresh fruit yield, and quality. During the flowering and fruiting period from May to November, 3-4 field evaluations and screenings were conducted. For each selected plant, a plastic stake was inserted into the substrate next to it. Finally, based on the number of stakes, the number of superior F1 plants, accounting for no more than 3% of the total, was selected. During this period, runners were removed to conserve plant nutrients for flowering and fruiting.

[0028] F1 individual plant selection was conducted in areas surrounding Kunming, Yuxi, and Qujing cities at altitudes of 1700 to 1900 meters, for the same reasons as S3. F1 seedlings obtained from seed sowing needed to overwinter before being transplanted to elevated substrate cultivation conditions for evaluation and selection. This was because a gene inhibiting summer flowering of strawberries was discovered in the research. FaDSF The gene exists, but it only inhibits flowering in the year of sowing, resulting in only half of the plants with the summer-flowering gene exhibiting summer flowering (all-season) in the year of sowing. The inhibitory effect of this gene disappears after overwintering (Table 8). If evaluation is conducted in the year of sowing, nearly 50% of the all-season F1 plants will be eliminated, resulting in a small number of selectable populations.

[0029] S5, Evaluation and Screening of Superior Strains: The superior individual plants selected from the field fruiting stage above are propagated vegetatively in the following summer, with each individual plant propagating to more than 10 plants to become superior strains. Superior strains are then screened in areas at the same altitude as in step S1. In the third spring, these superior strains are planted in the aforementioned elevated substrate cultivation troughs. Under conventional management conditions (the same as in S1), in-depth evaluations of traits including yield and fruit quality are conducted on the superior strains to screen out new varieties with promising prospects for promotion.

[0030] Table 1: Comparison of flowering quantity among 6 day-neutral varieties and 5 long-day varieties Note: Duncan's multi-range test significance test; different letters indicate significant differences at the 0.05 level. Evaluation period: April to November 2011; evaluation location: altitude 2340 meters.

[0031] Table 2: Comparison of fruit shape and firmness among 6 day-neutral varieties and 5 long-day varieties Note: Duncan's multi-range test significance test; different letters indicate significant differences at the 0.05 level. Evaluation period: April to November 2011; evaluation location: altitude 2340 meters.

[0032] Table 3: Comparison of fruit yield among 6 day-neutral varieties and 5 long-day varieties Note: Commercial fruit refers to fruit weighing more than 10 grams per fruit of a certain variety and with a regular shape; small fruit refers to fruit weighing less than or equal to 10 grams per fruit of a certain variety and with a regular shape; deformed fruit refers to fruit with an irregular shape or disease spots. Duncan's multi-range significance test was used, with different letters indicating significant differences at the 0.05 level; the evaluation period was from April to November 2011, and the evaluation location was at an altitude of 2340 meters.

[0033] Table 4: Comparison of soluble sugar content among 6 day-neutral varieties and 5 long-day varieties Note: The significance test of the Duncan multi-range test is used. Different letters indicate that the difference is significant at the 0.05 level. The evaluation period was from April to November 2011, and the evaluation location was at an altitude of 2340 meters.

[0034] Table 5: Comparison of organic acid content among 6 day-neutral varieties and 5 long-day varieties Table 6: Comparison of sugar-acid ratio, soluble solids content, and fruit firmness among 6 day-neutral varieties and 5 long-day varieties Table 7: Primer sequence information for screening seasonal single plants in F1 generation Table 8: Segregation of seasonal and seasonal plants in the F1 population of 'Zhangji' × 'Albion' 2nd crosses Note: y The segregation ratio (X) of a trait-segregating population 2 0.05,1=3.84) This application presents a set of methods for resource evaluation, seasonality genetic research, parental selection, hybrid seed treatment, F1 generation single-plant molecular marker screening, and field evaluation. These methods significantly improve the efficiency of seasonal strawberry breeding and provide a methodological guide and reference for the localization of seasonal strawberry breeding. Based on population genetic research, a major gene ( FaPFRU The dominant gene controls the seasonal (summer flowering) trait, and corresponding molecular markers have been developed for identification during the seedling stage, allowing for timely culling of non-seasonal F1 generation plants; there is another gene ( FaDSF The gene suppresses flowering in perennial plants during the sowing year, resulting in only about half (approximately 1 / 4 of the total F1 plants) of plants with the perennial gene exhibiting summer flowering (perennial). However, the suppressive effect of this gene disappears after overwintering due to low temperatures. Therefore, F1 seedlings must be overwintered and then transplanted the following year for phenotypic evaluation and selection, such as flowering. Otherwise, perennial plants in the F1 generation will be incorrectly eliminated. Based on this genetic information, appropriate hybridization and sowing times were set to avoid excessively long seedling maintenance time, which would lead to additional costs and quality degradation. Combined with parental selection and other techniques, the probability of selecting plants with the desired trait in the F1 generation was increased, thus improving the overall efficiency of perennial strawberry breeding.

Claims

1. The application of an SSR molecular marker in the breeding of new strawberry varieties, characterized in that, The primers for the SSR molecular markers are Bx125-F and Bx125-R; The sequence of Bx125-F is shown in SEQ ID NO: 1; the sequence of Bx125-R is shown in SEQ ID NO:

2.

2. A method for breeding new strawberry varieties using SSR molecular markers, characterized in that, The method includes the following steps: S1. Select varieties with the target trait as parents, and pre-treat the F1 generation seeds obtained by hybridization to obtain pre-treated hybrid F1 generation seeds. S2, the pretreated parental seeds obtained in S1 are hybridized to obtain the hybrid F1 generation population. The hybrid F1 generation is screened using SSR molecular markers, wherein the primers for the SSR molecular markers are Bx125-F and Bx125-R. The sequence of Bx125-F is shown in SEQ ID NO: 1; the sequence of Bx125-R is shown in SEQ ID NO: 2; S3 involves sequentially cultivating and propagating the F1 generation individual plants selected from S2 to obtain new strawberry varieties.

3. The method according to claim 2, characterized in that, The selection of varieties for the target traits in S1 includes: for processing varieties, American everbearing strawberry varieties are selected as parent materials; for fresh-eating varieties, European and Japanese everbearing strawberry varieties are selected as parent materials; and one of the two parents of the hybrid combination is a seasonal strawberry variety.

4. The method according to claim 2, characterized in that, The pretreatment in S1 involves treating the seeds with 98% sulfuric acid for 7-10 minutes, then washing and drying them. In early September, the seeds are sown in 128-cell trays, filled with substrate, and transplanted to 50-cell trays 40-60 days after germination. The seeds are then filled with the substrate and maintained.

5. The method according to claim 4, characterized in that, The matrix composition and mass ratio is peat:perlite 7:

3.

6. The method according to claim 2, characterized in that, The maintenance and propagation in S3 are carried out in an area with an altitude of 1700-1900 m.

7. The method according to claim 2, characterized in that, The weight ratio of each element N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B and Mo in the fertilizer water for elevated strawberry cultivation in S3 is 100:25:110:80:24:2.5:0.5:0.02:0.05:0.5:0.01.

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