Molecular marker related to soybean recurrent selection group physical property and application thereof

Abstract

The invention provides a molecular marker related to soybean recurrent selection group physical property and application of the molecular marker, and relates to the technical field of biology, and the molecular marker is a 38759bp base sequence deleted in an interval of Chr13: 22776273bp to 222815032bp on a soybean MS1 gene. Molecular markers ms1A4 and ms1B1 and molecular markers ms1A6 and ms1B4 are designed and developed according to a soybean ms1 gene large fragment deletion region, and can be used for detecting genotypes of recurrent selection populations to determine fertility, and a new thought for screening specific genotypes or field phenotypes is provided. And the molecular markers of three genotypes of homozygous dominant (MS1MS1), heterozygous (MS1ms1) and homozygous recessive (ms1ms1) in a soybean ms1 recurrent selection group can be detected at one time. The technical problem that in the prior art, all genotypes of soybean recurrent selection groups cannot be detected at a time is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a molecular marker related to the sports characteristics of a soybean recurrent selection group and an application thereof. Background Art

[0002] my country's modern soybean breeding work began in the early 20th century, and 6,180 soybean varieties have been bred so far. The soybean yield per hectare increased from 611 kg / hectare in 1949 to 1990.6 kg / hectare in 2023. However, compared with the average soybean yield per hectare in the United States, Brazil and the world, my country's soybean yield per hectare is relatively low, with an average annual yield growth rate of only 0.6%. The fundamental solution to increase soybean production is to increase soybean yield per hectare and increase soybean planting area, but it is difficult for my country's soybean planting area to increase significantly in the short term. Therefore, tapping the potential for increased yield of improved varieties is an important way to improve soybean competitiveness.

[0003] Traditional conventional soybean breeding has problems such as relatively insufficient utilization of germplasm resources, close relationship between breeding parents and increasingly narrow genetic basis, which has led to the lack of improvement in yield of varieties bred in my country in recent years. Using recurrent selection to improve breeding populations can enrich genetic diversity and create excellent new materials with multi-gene aggregation for the selection and breeding of new soybean varieties.

[0004] In 1973, Brim and Stuber first proposed the use of soybean male sterile lines for recurrent selection breeding. The discovery and use of soybean nuclear male sterile lines provided the possibility for soybean recurrent selection breeding and a new method for traditional breeding. Currently, 27 nuclear sterile mutants have been found, all of which are recessive nuclear sterile, and are respectively ms0 , ms1-ms9 , ms12 , msp , msMOS , mst-M and oeLh Among them, 15 gene loci control ms1 Nuclear sterile lines are widely used in soybean recurrent selection hybrid breeding. Kenworthy, Brim and others conducted three rounds of population selection on soybean seed yield, protein content, oil content and other traits, and achieved significant positive effects, with yield increasing linearly. Werner and others optimized soybean yield using recurrent selection populations, and Igor and others also screened soybean yield-related agronomic traits through recurrent selection. Prohaska and Fehr selected for soybean resistance to iron deficiency chlorosis, and the average genetic progress of chlorosis increased by 0.2 units per round of selection. Walker and Schmitthenner selected for soybean resistance to Phytophthora root rot, and there was a significant linear improvement in resistance from C0 to C3. Domestic researchers also subsequently carried out related research, and Song Qijian and others used ms1The male sterile line and 40 parents constructed the first soybean ms1 Male sterile recurrent population. Zhu Chengsong et al. conducted two rounds of yield recurrent selection on a high-oil population introduced from the United States and found that the yield increased by 9.13% per round on average, and the protein content, 100-grain weight, and number of grains per pod were significantly improved. Zhao Shuangjin et al. ms1 The recurrent population used 24 local materials and carried out gene enrichment and recurrent selection for 6 years to form an LD basic population suitable for selection in local ecological types. Zhao Zhigang et al. conducted genetic variation, correlation, principal component and cluster analysis on 11 main agronomic traits of 40 new spring soybean lines obtained based on ms1 recurrent selection to make a comprehensive evaluation of their agronomic traits and screened out excellent lines 13LD-222 and 12LD-89. Zhao Qingsong et al. used ms1 Genes were used to establish an efficient glyphosate-resistant transgenic breeding population and conduct recurrent selection, rapidly broadening the genetic basis of transgenic materials for use in glyphosate-resistant transgenic soybean breeding. ms1 Based on the genetic diversity of the reincarnation population and the relationship between the material groups within the population, the influence of directional selection on the genetic basis of the population was explored. ms1 A number of new varieties with broad genetic basis and stable excellent traits have been cultivated from nuclear sterile materials. For example, the Institute of Cereals and Oil Crops of Hebei Academy of Agricultural and Forestry Sciences has successfully cultivated and approved new varieties of high-oil soybean "Jidou 19", high-protein soybean varieties "Jidou 20", "Jidou 21", "Jidou 22", "Jidou 24", "Jidou 1258", "Jidou 33" and other new varieties. Cangzhou Academy of Agricultural and Forestry Sciences has cultivated "Cangheidou No. 2" and "Cangheidou No. 3", and Ningxia Academy of Agricultural and Forestry Sciences has cultivated "Ningdou No. 8", "Ningdou No. 9" and "Ningdou No. 10".

[0005] The cultivation of the above varieties is mainly based on ms1 The sterile material is used as the female parent, and other excellent materials are used as the mixed male parent. Through cross-pollination, multiple genetic backgrounds are aggregated for breeding. ms1 The gene was first discovered as a natural mutation in North Carolina, USA, and was named ms1-1 (North Carolina). Currently, ms1 Contains 7 allelic mutations, except ms1-1 (North Carolina), also includes ms1-2 (Ames1), ms1-3 (Urbana) ms1-4 (Tonica), Shenyang Agricultural University male sterile L-78-387 mutant (SMS), T287 mutant ms1 (Ames2), T290 mutant ms1 (Danbury). ms1-2 (Ames), ms1-3 (Urbana) ms1-4 (Tonica) has been cloned and identified. Among them, Fang et al. ms1-2 exist Glyma.13G114200 A nucleotide G was missing at 1470 bp, resulting in premature termination of translation at amino acid 539; ms1-3 In Glyma.13G114200 There is a nucleotide C to T at 787bp of the structure, which causes an important amino acid in the domain to change from Arg to Cys, thus confirming Glyma.13G114200 For soybean MS1 gene, and confirmed by biochemical analysis that it encodes a kinesin. ms1-4 It was reported by two teams at the same time. Jiang et al. used soybean resequencing data as a public reference pool and located the ms1-4 loci, and used CRISPR / Cas9 gene knockout experiments to verify Glyma.13G114200 for MS1 Nadeem et al. ms1-4 The mutant was genetically mapped and a large deletion of about 38.7 Kb was found in the mutant region, which also confirmed Glyma.13G114200 Nuclear sterility gene MS1 .

[0006] At present, the MS1 recurrent selection population widely used in breeding is based on ms1-4 Allelic mutant material is constructed, which is used in the offspring of the reincarnation selection population ms1 It provides the possibility to identify genotypes and assist in the selection of excellent strains. ms1-4 Single genotype identification markers have been developed, but they cannot detect all genotypes at once and have not been widely used in breeding. Most breeders still use conventional methods to randomly select fertile plants in the recurrent population, plant rows the following year, and then select the next generation, resulting in the separation of sterile plants from the previous generation of heterozygous fertile plants in the plant rows, which increases the workload and breeding costs.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] One of the objects of the present invention is to provide a substance for detecting molecular markers for use in identifying or assisting in identifying the sports characteristics of a soybean recurrent selection group or in preparing products for identifying or assisting in identifying the sports characteristics of a soybean recurrent selection group, or in identifying or assisting in identifying the genotype of a soybean recurrent selection group or in preparing products for identifying or assisting in identifying the genotype of a soybean recurrent selection group, or in soybean recurrent selection breeding or in preparing products for soybean recurrent selection breeding, so as to solve the technical problem in the prior art that all genotypes of a soybean recurrent selection group cannot be detected at one time.

[0009] The second object of the present invention is to provide a kit for identifying or assisting in identifying the sports characteristics and / or genotype of a soybean recurrent selection group.

[0010] The third object of the present invention is to provide a method for identifying or assisting in identifying the sports characteristics of a soybean recurrent selection population.

[0011] A fourth object of the present invention is to provide a method for identifying or assisting in identifying the genotype of a soybean recurrent selection population.

[0012] A fifth object of the present invention is to provide a method for soybean recurrent selection breeding.

[0013] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: In the first aspect, the present invention provides the use of a substance for detecting a molecular marker in any one of A1 to A3, wherein the molecular marker is soybean MS1 The 38759bp base sequence is missing in the range of Chr13: 22776273bp~22815032bp on the gene; A1. Identifying or assisting in identifying the fertility of a soybean recurrent selection population or preparing a product for identifying or assisting in identifying the fertility of a soybean recurrent selection population; A2. Identifying or assisting in identifying the genotype of a soybean recurrent selection population or preparing a product for identifying or assisting in identifying the genotype of a soybean recurrent selection population; A3. Soybean recurrent selection breeding or preparation of soybean recurrent selection breeding products.

[0014] Furthermore, the soybean recurrent selection population genotype includes ms1ms1 , MS1MS1 or MS1ms1 At least one of; The soybean recurrent selection population traits include fertile or sterile.

[0015] Further, the product includes detecting soybean MS1 A substance with a base sequence of 38759 bp missing in the range of Chr13:22776273bp~22815032bp on the gene.

[0016] Further, the detection of soybean MS1 The substance of the base sequence of 38759 bp deleted in the interval of Chr13:22776273 bp~22815032 bp on the gene is any of the following: C1, the substance contains PCR primers for amplifying soybean genomic DNA fragments including the molecular marker; C2, the substance is a PCR reagent containing the PCR primers described in C1; C3. The substance is a kit containing the PCR primers described in C1 or the PCR reagents described in C2.

[0017] Further, the PCR primers include those for amplifying soybean MS1 Primer set ms1AB1 or primer set ms1AB2 for the 38759 bp base sequence missing in the range of 22776273 bp to 22815032 bp on the gene Chr13; The primer set ms1AB1 includes a primer pair ms1A4 and a primer pair ms1B1, the nucleotide sequence of the primer pair ms1A4 is shown in SEQ ID NO.11 and SEQ ID NO.12; the nucleotide sequence of the primer pair ms1B1 is shown in SEQ ID NO.17 and SEQ ID NO.18; The primer set ms1AB1 includes a primer pair ms1A6 and a primer pair ms1B4, the nucleotide sequence of the primer pair ms1A6 is shown in SEQ ID NO.15 and SEQ ID NO.16; the nucleotide sequence of the primer pair ms1B4 is shown in SEQ ID NO.23 and SEQ ID NO.24.

[0018] In a second aspect, the present invention provides a kit for identifying or assisting in identifying the sports characteristics and / or genotype of a soybean recurrent selection population, including a kit for amplifying a soybean MS1 Primer set ms1AB1 or primer set ms1AB2 for the 38759 bp base sequence missing in the range of Chr13: 22776273 bp to 22815032 bp on the gene; The primer set ms1AB1 includes a primer pair ms1A4 and a primer pair ms1B1, the nucleotide sequence of the primer pair ms1A4 is shown in SEQ ID NO.11 and SEQ ID NO.12; the nucleotide sequence of the primer pair ms1B1 is shown in SEQ ID NO.17 and SEQ ID NO.18; The primer set ms1AB1 includes a primer pair ms1A6 and a primer pair ms1B4, the nucleotide sequence of the primer pair ms1A6 is shown in SEQ ID NO.15 and SEQ ID NO.16; the nucleotide sequence of the primer pair ms1B4 is shown in SEQ ID NO.23 and SEQ ID NO.24.

[0019] In the third aspect, the present invention provides a method for identifying or assisting in identifying the fertility of a soybean recurrent selection group, comprising detecting whether there is a 38759bp deletion in the Chr13:22776273bp~22815032bp interval in the soybean genome to be tested. If so, the soybean to be tested is fertile, otherwise it is infertile.

[0020] Further, the detection of whether there is a 38759 bp deletion in the Chr13:22776273 bp~22815032 bp interval of the soybean genome to be tested includes using the soybean genome DNA to be tested as a template and performing PCR amplification with the above-mentioned primer pair ms1F or primer pair ms1S; If there is an amplification product for primer pair ms1F, it is sterile, otherwise it is fertile; If the primer pair ms1S produces an amplification product, the cell is fertile, otherwise it is infertile.

[0021] Further, the detection of whether there is a 38759 bp deletion in the Chr13:22776273 bp~22815032 bp interval of the soybean genome to be tested includes using the soybean genome DNA to be tested as a template and performing PCR amplification with the above-mentioned primer set ms1AB1 or primer set ms1AB2; If the amplification product of primer set ms1AB1 only includes the amplification product of 972 bp, it is sterile, otherwise it is fertile; If the amplification product of primer set ms1AB2 only includes the amplification product of 1825bp, it is sterile, otherwise it is fertile.

[0022] In a fourth aspect, the present invention provides a method for identifying or assisting in identifying the genotype of a soybean recurrent selection population, comprising using the soybean genomic DNA to be tested as a template and performing PCR amplification using the primer set ms1AB1 or the primer set ms1AB2 described above; If the amplification product of primer set ms1AB1 only includes the amplification product of 972 bp, the genotype is ms1ms1 ; If the amplification product of primer set ms1AB1 only includes the amplification product of 1295 bp, the genotype is MS1MS1 ; If the amplification products of primer set ms1AB1 include a 972 bp amplification product and a 1295 bp amplification product, the genotype is MS1ms1 ; If the amplification product of primer set ms1AB2 only includes the amplification product of 1825 bp, the genotype is ms1ms1 ; If the amplification product of primer set ms1AB2 only includes the amplification product of 1010bp, the genotype is MS1MS1 ; If the amplification products of primer set ms1AB2 include a 1825 bp amplification product and a 1010 bp amplification product, the genotype is MS1ms1 .

[0023] In a fifth aspect, the present invention provides a method for soybean recurrent selection breeding, comprising using the above method to screen out the soybean recurrent selection population. MS1ms1 Heterozygous genotype, homozygous genotype soybean recurrent selection population is obtained for breeding.

[0024] The use of the detection molecular marker substance provided by the present invention in identifying or assisting in identifying the sports characteristics of a soybean recurrent selection group or preparing a product for identifying or assisting in identifying the sports characteristics of a soybean recurrent selection group, or identifying or assisting in identifying the genotype of a soybean recurrent selection group or preparing a product for identifying or assisting in identifying the genotype of a soybean recurrent selection group, or in soybean recurrent selection breeding or preparing a product for soybean recurrent selection breeding, according to soybean ms1 Large gene deletion region (soybean MS1 The molecular marker combinations ms1A1~ms1A6 and ms1B1~ms1B6 were designed and developed based on the 38759bp base sequence missing in the interval of Chr13:22776273bp~22815032bp on the gene. It was found that the molecular markers ms1A4 and ms1B1 as well as the molecular markers ms1A6 and ms1B4 have obvious polymorphism, which can be used to detect the genotype of the recurrent selection population and determine the fertility, providing a new idea for screening specific genotypes or field phenotypes for soybean recurrent selection breeding, and can detect soybeans at one time. ms1 Homozygous dominant in recurrently selected populations ( MS1MS1 ), heterozygous ( MS1ms1 ) and homozygous recessive ( ms1ms1 ) molecular markers of three genotypes, thereby achieving accurate identification of individual plants in the recurrent selection population in the contemporary era and accelerating the recurrent selection breeding process. This solves the technical problem that the existing technology cannot detect all genotypes of the soybean recurrent selection population at one time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The ms1S and ms1F molecular marker electrophoresis detection results provided in Example 1 of the present invention, wherein A is the ms1S PCR electrophoresis detection result, and B is the ms1F PCR electrophoresis detection result; Figure 2 The positions and amplified fragments of ms1F and ms1S on the Williams 82 reference genome provided in Example 1 of the present invention; Figure 3 The electrophoresis detection results of the molecular markers ms1A1-ms1A6 provided in Example 2 of the present invention, wherein A is a homozygous sterile plant and B is a homozygous fertile plant; Figure 4 The electrophoresis detection results of the molecular markers ms1B1-ms1B6 provided in Example 2 of the present invention, wherein A is a homozygous fertile plant and B is a homozygous sterile plant; Figure 5 The one-step genotype identification molecular marker screening result provided in Example 3 of the present invention, wherein A is the PCR amplification band of ms1A4 / ms1B1 to ms1A4 / ms1B6; B is the PCR amplification band of ms1A6 / ms1B1 to ms1A6 / ms1B6; Figure 6 The one-step genotype identification molecular marker verification result provided in Example 3 of the present invention, wherein A is the PCR amplification band of ms1AB1; B is the PCR amplification band of ms1AB2; Figure 7 The reliability verification of the one-step genotype identification molecular marker provided in Example 4 of the present invention, wherein A is the PCR amplification band of ms1AB1; B is the PCR amplification band of ms1AB2; Figure 8 The one-step genotyping molecular marker electrophoresis detection result provided in Example 5 of the present invention, wherein A is the PCR amplification band of molecular marker ms1AB1 for individual strains 1 to 24, and B is the PCR amplification band of molecular marker ms1AB2 for individual strains 1 to 24; Fig. 9 Provided for Example 5 of the present invention ms1 Field performance of fertile individual plant offspring in the recurrent selection population, where A is MS1MS1 The genotype is fertile and the fertility of the offspring is not separated; B is MS1ms1 Genotype fertile offspring fertility segregant lines. DETAILED DESCRIPTION

[0027] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0028] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0029] In one aspect, the present invention provides an application of a substance for detecting a molecular marker in any one of A1 to A3, wherein the molecular marker is soybean MS1 The 38759bp base sequence is missing in the range of Chr13: 22776273bp~22815032bp on the gene; A1. Identifying or assisting in identifying the fertility of a soybean recurrent selection population or preparing a product for identifying or assisting in identifying the fertility of a soybean recurrent selection population; A2. Identifying or assisting in identifying the genotype of a soybean recurrent selection population or preparing a product for identifying or assisting in identifying the genotype of a soybean recurrent selection population; A3. Soybean recurrent selection breeding or preparation of soybean recurrent selection breeding products.

[0030] Based on soybean ms1 Large gene deletion region (soybean MS1 The molecular marker combinations ms1A1~ms1A6 and ms1B1~ms1B6 were designed and developed based on the 38759bp base sequence missing in the interval of Chr13:22776273bp~22815032bp on the gene. It was found that the molecular markers ms1A4 and ms1B1 as well as the molecular markers ms1A6 and ms1B4 have obvious polymorphism, which can be used to detect the genotype of the recurrent selection population and determine the fertility, providing a new idea for screening specific genotypes or field phenotypes for soybean recurrent selection breeding, and can detect homozygous dominant genotypes in soybean ms1 recurrent selection populations at one time ( MS1MS1 ), heterozygous ( MS1ms1 ) and homozygous recessive ( ms1ms1 ) molecular markers of three genotypes, thereby achieving accurate identification of individual plants in the recurrent selection population in the contemporary era and accelerating the recurrent selection breeding process. This solves the technical problem that the existing technology cannot detect all genotypes of the soybean recurrent selection population at one time.

[0031] In some specific embodiments, the soybean recurrent selection population genotype includes ms1ms1 , MS1MS1 or MS1ms1 At least one of the following; the soybean recurrent selection group sports characteristics include fertile or sterile.

[0032] In some specific embodiments, the product includes a detection soybean MS1 A substance with a base sequence of 38759 bp missing in the range of Chr13:22776273bp~22815032bp on the gene.

[0033] In some specific embodiments, the detection of soybean MS1The substance of the base sequence of 38759 bp deleted in the interval of Chr13:22776273 bp~22815032 bp on the gene is any of the following: C1, the substance contains PCR primers for amplifying soybean genomic DNA fragments including the molecular marker; C2, the substance is a PCR reagent containing the PCR primers described in C1; C3. The substance is a kit containing the PCR primers described in C1 or the PCR reagents described in C2.

[0034] In some specific embodiments, the PCR primers include those for amplifying soybean MS1 A primer set ms1AB1 or primer set ms1AB2 for a base sequence of 38759 bp deleted in the interval of Chr13: 22776273bp~22815032bp on the gene; the primer set ms1AB1 includes a primer pair ms1A4 and a primer pair ms1B1, and the nucleotide sequence of the primer pair ms1A4 is shown in SEQ ID NO.11 and SEQ ID NO.12; the nucleotide sequence of the primer pair ms1B1 is shown in SEQ ID NO.17 and SEQ ID NO.18; the primer set ms1AB1 includes a primer pair ms1A6 and a primer pair ms1B4, and the nucleotide sequence of the primer pair ms1A6 is shown in SEQ ID NO.15 and SEQ ID NO.16; the nucleotide sequence of the primer pair ms1B4 is shown in SEQ ID NO.23 and SEQ ID NO.24.

[0035] According to another aspect of the present invention, a kit for identifying or assisting in identifying the sports characteristics and / or genotype of a soybean recurrent selection population is provided, including a kit for amplifying a soybean MS1 Primer set ms1AB1 or primer set ms1AB2 for the 38759 bp base sequence missing in the range of 22776273 bp to 22815032 bp on the gene Chr13; The primer set ms1AB1 includes primer pair ms1A4 and primer pair ms1B1, the nucleotide sequence of the primer pair ms1A4 is shown in SEQ ID NO.11 and SEQ ID NO.12; the nucleotide sequence of the primer pair ms1B1 is shown in SEQ ID NO.17 and SEQ ID NO.18; the primer set ms1AB1 includes primer pair ms1A6 and primer pair ms1B4, the nucleotide sequence of the primer pair ms1A6 is shown in SEQ ID NO.15 and SEQ ID NO.16; the nucleotide sequence of the primer pair ms1B4 is shown in SEQ ID NO.23 and SEQ ID NO.24.

[0036] The kit may also include reagents for PCR amplification or reagents for detecting amplification products.

[0037] According to another aspect of the present invention, a method for identifying or assisting in identifying the fertility of a soybean recurrent selection group is provided, comprising detecting whether there is a 38759 bp deletion in the Chr13:22776273bp~22815032bp interval in the soybean genome to be tested. If so, the soybean to be tested is fertile, otherwise it is infertile.

[0038] In some specific embodiments, the detecting whether there is a 38759 bp deletion in the Chr13:22776273 bp to 22815032 bp interval of the soybean genome to be tested comprises using the soybean genome DNA to be tested as a template and performing PCR amplification with the above-mentioned primer pair ms1F or primer pair ms1S; If the primer pair ms1F produces an amplification product, the strain is sterile, otherwise it is fertile; if the primer pair ms1S produces an amplification product, the strain is fertile, otherwise it is sterile.

[0039] In some specific embodiments, the detecting whether there is a 38759 bp deletion in the Chr13:22776273 bp~22815032 bp interval of the soybean genome to be tested comprises using the soybean genome DNA to be tested as a template and performing PCR amplification with the above-mentioned primer set ms1AB1 or primer set ms1AB2; If the amplification product of the primer set ms1AB1 only includes the amplification product of 972 bp, it is sterile, otherwise it is fertile; if the amplification product of the primer set ms1AB2 only includes the amplification product of 1825 bp, it is sterile, otherwise it is fertile.

[0040] According to another aspect of the present invention, a method for identifying or assisting in identifying the genotype of a soybean recurrent selection population is provided, comprising using the soybean genomic DNA to be tested as a template and performing PCR amplification using the primer set ms1AB1 or the primer set ms1AB2; If the amplification product of primer set ms1AB1 only includes the amplification product of 972 bp, the genotype is ms1ms1 ; If the amplification product of primer set ms1AB1 only includes the amplification product of 1295bp, the genotype is MS1MS1 ; If the amplification products of primer set ms1AB1 include a 972 bp amplification product and a 1295 bp amplification product, the genotype is MS1ms1 ; If the amplification product of primer set ms1AB2 only includes the amplification product of 1825 bp, the genotype is ms1ms1; If the amplification product of primer set ms1AB2 only includes the amplification product of 1010bp, the genotype is MS1MS1 ; If the amplification products of primer set ms1AB2 include amplification products of 1825 bp and 1010 bp, the genotype is MS1ms1 .

[0041] According to another aspect of the present invention, a method for soybean recurrent selection breeding is also provided, comprising using the above method to screen out the soybean recurrent selection population. MS1ms1 Heterozygous genotype, homozygous genotype soybean recurrent selection population is obtained for breeding.

[0042] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] 1. Test materials: Soybean ms1 The offspring of the recurrent selection population were separated into individual plants as materials. The above basic population was constructed by the Huanghuaihai Northern Variety Improvement Post Team of the National Soybean Industry Technology System, and the derived population was provided by the Northeast Early-maturing Variety Improvement Post Team.

[0044] 2. Population DNA extraction: Soybean DNA was extracted using a plant genomic DNA extraction kit (Jiangsu Kangwei Century Biotechnology Co., Ltd.) ms1 The genomic DNA of individual strains in the recurrent selection population was tested for DNA quality using 1% agarose gel electrophoresis (80 mL TAE, 0.8 g agarose, 8 μL Super Gel Red).

[0045] Example 1 ms1-4 Verification of large deletion mutation sites The complete genome sequence of soybean variety Williams 82 (Glycine_max_v2.1) was obtained through NCBI (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000004515.6 / ); MS1 Gene (Chr13:22,791,534-22,798,417) ms1-4 The sequence characteristics of large fragment deletion mutations were analyzed using PrimerPremier 5.0 software. ms1-4 Specific markers ms1S and ms1F were designed before and after the mutant deletion fragment, and the primer sequences and characteristics are shown in Table 1.

[0046] Table 1 Specific molecular markers used to amplify the deletion fragment of ms1 mutant

[0047] The control single plant Williams 82, ms1 Homozygous fertile plants in the recurrent selection population ( MS1MS1 ) and sterile plants ( ms1ms1 ) for PCR amplification. The PCR amplification system was 25 μL, including 12.5 μL 2×Es Taq Master mix (Dye), 1 μL upstream primer, 1 μL downstream primer, 2 μL template DNA, and 8.5 μL ddH2O. The PCR amplification program was 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, 30 cycles, and finally 72°C for final extension for 5 min. The PCR product of the specific target fragment was sent to Beijing BGI Genomics Technology Co., Ltd. for bidirectional sequencing by 1% agarose gel electrophoresis and gel imaging system.

[0048] Primers ms1S and ms1F were designed to detect the control variety Williams 82 ( MS1MS1 )、 ms1 Homozygous fertile plants in the recurrent selection population ( MS1MS1 ) and sterile plants ( ms1ms1 ) The electrophoresis results of PCR amplification are as follows Figure 1 As shown, the ms1S PCR results are as follows Figure 1 As shown in A, 1 to 4 are sterile plants ( ms1ms1 ), 5 and 6 are fertile plants ( MS1MS1 ), 7 and 8 are the control varieties Williams82 ( MS1MS1 ), the control plant and the fertile plant had no deletion site and could not be amplified, showing no band, and the sterile plant showed a 1066bp band; the ms1F PCR results were as follows Figure 1 As shown in B, 1 to 4 are sterile plants ( ms1ms1 ), 5 and 6 are fertile plants ( MS1MS1 ), 7 and 8 are the control varieties Williams82 ( MS1MS1 ); M: DNA maker, the control plant and the fertile plant showed an 882bp band, and the sterile plant had no band. The single target fragments obtained from different genotypes were sequenced for the first generation, among which the sterile plant ( ms1ms1 ) 38759bp bases are missing at 22776273bp~22815032bp, fertile single plant ( MS1MS1 ) and sterile plants ( ms1ms1) and the soybean variety Williams 82 reference genome sequence (Glycine_max_v2.1) are compared as follows Figure 2 shown.

[0049] Example 2 Development of specific molecular markers exist ms1-4 and MS1 Primer Premier 5.0 software was used to develop specific molecular markers based on gene sequences. A total of 12 molecular markers were developed, including ms1-4 Six primers were designed before and after the mutant deletion sequence, named ms1A1~ms1A6; six primers were designed on MS1, named ms1B1~ms1B6, and the primer sequences and characteristics are shown in Table 2. The development principle of markers is that the primer length is 20~30bp, the number of mismatched bases does not exceed 3, and the target band length is 1000bp~2000bp to reduce mismatched bands.

[0050] Table 2 Molecular markers used to detect the genotypes of individual plants in the recurrent selection population

[0051] Based on soybean ms1 The mutant sequence of male sterile gene was obtained, and molecular markers were designed to perform PCR amplification on the leaf DNA of homozygous sterile and homozygous fertile plants. The results were as follows Figure 3 As shown, A is a homozygous sterile plant; B is a homozygous fertile plant; M is a DNA maker. It can be seen that ms1A2, ms1A3, and ms1A5 have bands in both homozygous sterile plants and homozygous fertile plants. ms1A1, ms1A4, and ms1A6 only have bands in homozygous sterile plants, and no bands in homozygous fertile plants. The bands of ms1A4 and ms1A6 are bright and specific. Therefore, ms1A4 and ms1A6 were selected.

[0052] exist MS1 The molecular markers designed above were used to amplify the DNA of leaves of homozygous sterile and homozygous fertile plants by PCR. The results are as follows Figure 4 As shown, A is a homozygous fertile plant, B is a homozygous sterile plant; M is a DNA maker. It can be seen that ms1B1~ms1B6 can amplify obvious bands in homozygous fertile plants, but no bands can be amplified in homozygous sterile plants. Therefore, ms1B1~ms1B6 can be used for detection MS1MS1 genotype.

[0053] Example 3 Single genotype primer molecular marker screening The markers ms1A4, ms1A6 and ms1B1-ms1B6 selected in Example 2 were mixed separately to identify heterozygous fertile plants ( MS1ms1) DNA was amplified by PCR, and the results were as follows Figure 5 As shown in the figure, A is the PCR amplification band of ms1A4 / ms1B1~ ms1A4 / ms1B6; B is the PCR amplification band of ms1A6 / ms1B1~ ms1A6 / ms1B6; M is DNA maker. Two pairs of primer combinations (ms1A4 / ms1B1) and (ms1A6 / ms1B4) with obvious polymorphism were screened out and named ms1AB1 and ms1AB2 respectively for one-step method. MS1 / ms1 genotype.

[0054] Using ms1AB1 and ms1AB2 ms1ms1 Genotype individual MS1ms1 Genotype individual MS1MS1 The genotyped individual strains were amplified by PCR, and the results were as follows Figure 6 As shown, A is the PCR amplification band of ms1AB1; B is the PCR amplification band of ms1AB2; 1 and 2 are ms1ms1 PCR amplification bands of genotype individual strains; 3 and 4 are MS1ms1 PCR amplification bands of genotype individual strains; 5 and 6 are MS1MS1 PCR amplification bands of genotyped individual plants; M is DNA maker. ms1ms1 A 972 bp band was amplified; MS1ms1 Two bands were amplified, one of 972 bp and one of 1295 bp; MS1MS1 A 1295 bp band was amplified. ms1ms1 A 1825 bp band was amplified; MS1ms1 Two bands were amplified, one 1825 bp and one 1010 bp; MS1MS1 A 1010 bp band was amplified.

[0055] Among them, the PCR reaction system is: the total volume is 20μL, including 10μL of 2×Super Pfx enzyme, 2μL of primers, 2μL of template DNA, and 6μL of ddH2O. The PCR amplification program is: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 58℃ annealing for 30s, 72℃ extension for 30s, 30 cycles, and 72℃ final extension for 10min. The sequences of each labeled primer, annealing temperature and target fragment length are shown in Table 1. Electrophoresis detection: 1% agarose gel, 120V electrophoresis for 40min to detect whether the PCR amplified fragment meets the expected size and is specific and single.

[0056] Example 4 One-step molecular marker screening and reliability verification The selected specific molecular markers ms1A and ms1B were mixed, and PCR amplification was performed on the representative single-strain DNA samples. The PCR reaction system was the same as that described in Example 3. By electrophoresis detection, obvious polymorphism was detected. 12 single strains were randomly selected from the isolated population, and PCR amplification was performed using the selected material DNA using the selected one-step molecular markers, and 1% agarose gel electrophoresis was used for detection, and repeated three times to verify the stability and repeatability of the markers.

[0057] The results are as follows Figure 7 As shown, A is the PCR amplification band of ms1AB1; B is the PCR amplification band of ms1AB2; M is DNAmaker; 1-4: PCR amplification bands of homozygous sterile plants; 5-8: PCR amplification bands of heterozygous fertile plants; 9-12: PCR amplification bands of homozygous fertile plants. It can be seen that the ms1AB1 and ms1AB2 markers selected were used to amplify the 12 ms1 The genotypes of the offspring of the recurrent selection population were tested, and the polymorphic bands obtained were clear and consistent with the expected results, confirming that the two sets of markers were stable and reproducible, and could accurately detect the presence of different MS1 Genotypes of soybean materials.

[0058] Example 5 One-step molecular labeling ms1 Application in the Reincarnation Selection Group The screened one-step molecular markers were used to select 24 rice varieties planted in the Fanjiatun Base of Jilin Academy of Agricultural Sciences (124°83′E, 43°43′N) in 2023. ms1 The genotypes of the offspring of the recurrent selection population were identified.

[0059] The identification results are as follows Figure 8 As shown, A is the PCR amplification band of molecular marker ms1AB1 for individual strains 1 to 24, B is the PCR amplification band of molecular marker ms1AB2 for individual strains 1 to 24; M is DNA maker. It can be seen that the individual strains numbered 3, 6, 13, 15, 16, 19, 20, and 24 were all amplified. MS1MS1 Genotype bands, the individual plants in the field showed fertility. The individual plants numbered 7, 9-12, 14, 17, and 23 all amplified ms1ms1 Genotype bands, individual plants in the field showed sterility. Individual plants numbered 1, 2, 4, 5, 8, 18, 21, and 22 amplified MS1ms1 Genotype bands, the field performance of the single plant is fertile. Further, the field performance is fertile, and the genotypes are identified as MS1MS1 and MS1ms1 The seeds of the individual plants with the same genotype will continue to be planted in the Fanjiatun base in 2024, and the phenotypic segregation of their offspring will be observed. Fig. 9As shown, where A is MS1MS1 The genotype is fertile and the fertility of the offspring is not separated; B is MS1ms1 The fertility of the genotype fertile offspring is isolated. MS1MS1 There was no fertility segregation in the genotype materials, and the plants were all fertile; MS1ms1 The above results show that the two sets of molecular markers are suitable for ms1 The one-step identification of the offspring genotypes of the recurrent selection population can quickly and easily screen individual plants with good trait performance and non-segregating fertility in offspring for the selection of excellent varieties and accelerate the breeding process.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance for detecting molecular markers in any one of A1 to A3, characterized in that: The molecular marker is soybean MS1 The 38759bp base sequence is missing in the range of Chr13: 22776273bp~22815032bp on the gene; A1. Identifying or assisting in identifying the fertility of a soybean recurrent selection population or preparing a product for identifying or assisting in identifying the fertility of a soybean recurrent selection population; A2. Identifying or assisting in identifying the genotype of a soybean recurrent selection population or preparing a product for identifying or assisting in identifying the genotype of a soybean recurrent selection population; A3. Soybean recurrent selection breeding or preparation of soybean recurrent selection breeding products.

2. The use according to claim 1, characterized in that: The soybean recurrent selection population genotype includes ms1ms1 , MS1MS1 or MS1ms1 At least one of; The soybean recurrent selection population traits include fertile or sterile.

3. The use according to claim 1, characterized in that: The product includes detection of soybean MS1 A substance with a base sequence of 38759 bp missing in the range of Chr13:22776273bp~22815032bp on the gene.

4. The use according to claim 3, characterized in that: The detection soybean MS1 The substance of the base sequence of 38759 bp deleted in the interval of Chr13:22776273 bp~22815032 bp on the gene is any of the following: C1, the substance contains PCR primers for amplifying soybean genomic DNA fragments including the molecular marker; C2, the substance is a PCR reagent containing the PCR primers described in C1; C3. The substance is a kit containing the PCR primers described in C1 or the PCR reagents described in C2.

5. The use according to claim 4, characterized in that: The PCR primers include those for amplifying soybean MS1 Primer set ms1AB1 or primer set ms1AB2 for the 38759 bp base sequence missing in the range of 22776273 bp to 22815032 bp on the gene Chr13; The primer set ms1AB1 includes a primer pair ms1A4 and a primer pair ms1B1, the nucleotide sequence of the primer pair ms1A4 is shown in SEQ ID NO.11 and SEQ ID NO.12; the nucleotide sequence of the primer pair ms1B1 is shown in SEQ ID NO.17 and SEQ ID NO.18; The primer set ms1AB1 includes a primer pair ms1A6 and a primer pair ms1B4, the nucleotide sequence of the primer pair ms1A6 is shown in SEQ ID NO.15 and SEQ ID NO.16; the nucleotide sequence of the primer pair ms1B4 is shown in SEQ ID NO.23 and SEQ ID NO.

24.

6. A kit for identifying or assisting in identifying the sports characteristics and / or genotype of a soybean recurrent selection population, characterized in that: Including soybeans for augmentation MS1 Primer set ms1AB1 or primer set ms1AB2 for the 38759 bp base sequence missing in the range of 22776273 bp to 22815032 bp on the gene Chr13; The primer set ms1AB1 includes a primer pair ms1A4 and a primer pair ms1B1, the nucleotide sequence of the primer pair ms1A4 is shown in SEQ ID NO.11 and SEQ ID NO.12; the nucleotide sequence of the primer pair ms1B1 is shown in SEQ ID NO.17 and SEQ ID NO.18; The primer set ms1AB1 includes a primer pair ms1A6 and a primer pair ms1B4, the nucleotide sequence of the primer pair ms1A6 is shown in SEQ ID NO.15 and SEQ ID NO.16; the nucleotide sequence of the primer pair ms1B4 is shown in SEQ ID NO.23 and SEQ ID NO.

24.

7. A method for identifying or assisting in identifying the fertility of a soybean recurrent selection population, characterized in that: The method comprises detecting whether there is a 38759 bp deletion in the Chr13: 22776273 bp to 22815032 bp interval of the soybean genome to be tested. If so, the soybean to be tested is fertile, otherwise it is infertile.

8. The method according to claim 7, characterized in that The method of detecting whether there is a 38759 bp deletion in the Chr13:22776273 bp to 22815032 bp interval in the soybean genome to be tested comprises using the soybean genome DNA to be tested as a template and performing PCR amplification using the primer set ms1AB1 or the primer set ms1AB2 of claim 5; If the amplification product of primer set ms1AB1 only includes the amplification product of 972 bp, it is sterile, otherwise it is fertile; If the amplification product of primer set ms1AB2 only includes the amplification product of 1825bp, it is sterile, otherwise it is fertile.

9. A method for identifying or assisting in identifying the genotype of a soybean recurrent selection population, characterized in that: The method comprises using the soybean genomic DNA to be tested as a template and performing PCR amplification using the primer set ms1AB1 or the primer set ms1AB2 of claim 5; If the amplification product of primer set ms1AB1 only includes the amplification product of 972 bp, the genotype is ms1ms1 ; If the amplification product of primer set ms1AB1 only includes the amplification product of 1295 bp, the genotype is MS1MS1 ; If the amplification products of primer set ms1AB1 include a 972 bp amplification product and a 1295 bp amplification product, the genotype is MS1ms1 ; If the amplification product of primer set ms1AB2 only includes the amplification product of 1825 bp, the genotype is ms1ms1 ; If the amplification product of primer set ms1AB2 only includes the amplification product of 1010bp, the genotype is MS1MS1 ; If the amplification products of primer set ms1AB2 include a 1825 bp amplification product and a 1010 bp amplification product, the genotype is MS1ms1 .

10. A method for soybean recurrent selection breeding, characterized in that: The method of claim 9 is used to screen out soybean recurrent selection population. MS1ms1 Heterozygous genotype, homozygous genotype soybean recurrent selection population is obtained for breeding.

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