A molecular marker, method and application for identifying green-core soybeans

Abstract

This invention relates to the field of biotechnology and discloses a molecular marker, method, and application for identifying green-core soybeans. The molecular markers for identifying green-core soybeans are D1-dCAPS and D2-Indel. The method comprises: 1. Amplifying the D1-dCAPS molecular marker using a semi-nested PCR method with the DNA of the soybean material as a template; 2. Amplifying the D2-Indel molecular marker using a triplet PCR method with the DNA of the soybean material as a template; 3. Simultaneously satisfying the identification in step 1 as D1-dCAPS. Δ Soybean materials identified as having the D2-In genotype in step two are considered green-core soybeans. The method of this invention can distinguish different molecular marker banding patterns using agarose gel electrophoresis, which offers advantages over non-denaturing acrylamide gel electrophoresis in terms of safety, speed, and ease of operation. The molecular marker and identification method described in this invention can be used for the identification and screening of green-core soybean phenotypes, and can rapidly, accurately, and effectively breed green-core soybean varieties, accelerating the crop breeding process and greatly improving breeding efficiency and success rate.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a molecular marker, method, and application for identifying green-core soybeans. Background Technology

[0002] In recent years, with the adjustment of people's dietary structure and the development of the soybean deep processing industry, the demand for soybeans has developed towards diversification and multi-purpose applications, highlighting the specialization and unique characteristics of soybeans. Green-core soybeans are a special type of green-kernel soybean, named for the green color of their seeds and leaves. They include green-skinned green-core soybeans and black-skinned green-core soybeans. Green-skinned green-core soybeans, also known as double-green soybeans, are neutral in nature and sweet in taste. They are rich in unsaturated fatty acids and soybean phospholipids, which help maintain blood vessel elasticity, improve brain function, and prevent fatty liver formation. Black-skinned green-core soybeans, also known as green-kernel black soybeans or green-core black soybeans, are rich in anthocyanins, B vitamins, and minerals, and have certain antioxidant, blood sugar regulating, and digestive-promoting effects. Green-core soybeans are both visually appealing and nutritionally superior, and are commonly used in the production of pickles, tofu, soy milk, tofu pudding, pickled sprouts, roasted nuts, and baking ingredients. They have a large demand in both international trade and the domestic market.

[0003] Molecular marker-assisted soybean breeding helps in the selection of superior varieties. Developing molecular markers that co-segregate or are closely linked to target traits can improve the accuracy and efficiency of soybean variety selection, and shorten the breeding cycle. The key to molecular marker-assisted breeding lies in developing efficient, stable, and easily detectable molecular markers. Such markers have universality in soybean breeding and are of great significance for the selection of new soybean varieties.

[0004] During the seed maturation process of yellow-fleshed soybeans, the chlorophyll in the cotyledons is gradually degraded, resulting in yellow cotyledons in the final mature seed. In contrast, the chlorophyll degradation process in the cotyledons of green-fleshed soybeans is impaired, preventing chlorophyll degradation and resulting in green cotyledons in the final mature seed. Previous studies have demonstrated that the D1 and D2 genes (D1 gene number SoyZH13_01G197200 (Zhonghuang 13 genome version) or Glyma.01G214600 (Williams82 genome version); D2 gene number SoyZH13_11G026500 (Zhonghuang 13 genome version) or Glyma.11G027400 (Williams82 genome version)) encode two SGR proteins in soybeans, which are key genes controlling chlorophyll degradation. Summary of the Invention

[0005] This invention provides a molecular marker, method, and application for identifying green-core soybeans.

[0006] The present invention identifies the molecular markers for green-core soybean as D1-dCAPS and D2-Indel, wherein the nucleotide sequence of D1-dCAPS is shown in SEQ ID NO.1 and the nucleotide sequence of D2-Indel is shown in SEQ ID NO.2.

[0007] Furthermore, the Y at position 58 of the D1-dCAPS nucleotide sequence indicates polymorphism, where Y is either A or Δ, and Δ indicates that a base is missing at that position.

[0008] Furthermore, the insertion or deletion of 1075 bp from position 557 to position 1631 in the D2-Indel sequence is polymorphic.

[0009] The present invention utilizes the above-mentioned molecular markers to identify green-core soybeans, and is carried out according to the following steps:

[0010] I. Using the DNA of the soybean material to be tested as a template, the D1-dCAPS molecular marker was amplified by a semi-nested PCR method. After the first and second rounds of PCR amplification, the D1-dCAPS amplification product was 214 bp or 213 bp in length. After PmeI digestion, the 214 bp band could not be digested, remaining as a 214 bp main band. Therefore, the material to be tested was labeled as D1-dCAPS. A Genotype; the product of the 213 bp band can be digested with PmeI enzyme, and the digestion product contains two bands of 159 bp and 54 bp, which are labeled as D1-dCAPS soybean material. Δ genotype;

[0011] 2. Using the DNA of the soybean material to be tested as a template, the D2-Indel molecular marker was amplified by triple PCR. If the PCR product contains a 754 bp band, the soybean material to be tested is labeled as D2-In. If the PCR product contains a 571 bp band, the soybean material to be tested is labeled as D2-Del.

[0012] III. Simultaneously satisfying the criteria in step one for identification as D1-dCAPS Δ The soybean material to be tested that has the D2-In genotype identified in step two is the green-core soybean.

[0013] Furthermore, in the semi-nested PCR method of step one: the nucleotide sequence of the upstream primer for the first round of PCR is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; the nucleotide sequence of the upstream primer for the second round of PCR is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0014] Furthermore, the first round of PCR system consisted of: 10 μL of 2×Mix, 1.5 μL of DNA template, 1 μL each of upstream and downstream primers, and 6.5 μL of ddH2O in a 20 μL reaction system; the PCR amplification program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s; a total of 20 cycles; and a final extension at 72℃ for 5 min.

[0015] The second round of PCR consisted of the following: 10 μL of 2×Mix, 1.5 μL of DNA template, 1 μL each of upstream and downstream primers, and 6.5 μL of ddH2O in a 20 μL reaction system; PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s; 33 cycles in total; 72℃ extension for 5 min.

[0016] Furthermore, the enzyme digestion reaction system in step one is as follows: 10 μL of the second round PCR product, 1.2 μL of rCutSmart, 0.3 μL of PmeI enzyme and 0.5 μL of ddH2O; the enzyme digestion reaction conditions are: 37°C, and the digestion is carried out for 2 h.

[0017] Furthermore, in step two, the triple PCR method is as follows: the nucleotide sequence of the upstream primer is shown in SEQ ID NO.6, the nucleotide sequence of one downstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of another downstream primer is shown in SEQ ID NO.8. The PCR system is as follows: in a 20 μL reaction system, there are 10 μL of 2X Mix, 1 μL each of the upstream and downstream primers, 1.5 μL of DNA template, and 6.5 μL of ddH2O. The PCR reaction conditions are: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 33 cycles, followed by a final extension at 72℃ for 10 min.

[0018] The application of the molecular markers described in this invention in the identification of green-core soybeans.

[0019] Furthermore, the application of molecular markers in identifying the cotyledon color of soybean seeds and in molecular marker-assisted breeding of green-core soybeans.

[0020] The beneficial effects of the technical solution described in this invention are as follows: using the primer pairs of this invention, different molecular marker band patterns can be distinguished by agarose gel electrophoresis, which has the advantages of being safer, faster, and simpler to operate than non-denaturing acrylamide gel electrophoresis. Furthermore, the molecular marker and identification method described in this invention can be used for the identification and screening of green-core soybean phenotypes, and can rapidly, accurately, and effectively breed green-core soybean varieties, accelerating the crop breeding process and greatly improving breeding efficiency and success rate. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of sequence variations in the d1 / d2 mutant;

[0022] Figure 2 The observation of cotyledon color in Dongnong 50 and d1 / d2 mutants after seed maturation and rounding;

[0023] Figure 3 Electrophoresis images of the D1-dCAPS molecular marker primers of the present invention before and after enzyme digestion in the yellow-core soybean variety Dongnong 50, the green-core soybean variety Jiqing 3 and their F1 generation.

[0024] Figure 4 This is an electrophoresis diagram of the amplification products of the D2-Indel molecular marker primers of the present invention in the yellow-core soybean variety Dongnong 50, the green-core soybean variety Jiqing 3 and their F1 generation;

[0025] Figure 5 This is an electrophoresis diagram showing the validation results of the D1-dCAPS molecular marker primers of the present invention in detecting 22 soybean germplasm resources with known cotyledon color phenotypes.

[0026] Figure 6 This is an electrophoresis diagram showing the validation results of the D2-Indel molecular marker primers of the present invention in detecting 22 soybean germplasm resources with known cotyledon color phenotypes. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0029] Example 1: Development and validation of the molecular markers described in this invention.

[0030] I. Development of the Molecular Markers Described in this Invention

[0031] 1. Double mutation of the D1 and D2 genes can produce green-core soybeans.

[0032] Cas9 target sgRNAs were designed and synthesized targeting the D1 and D2 gene sequences. The target sequence of the sgRNA is 5'-GCCATATTTGAAGCCTCAAA-3'. A CRISPR-Cas9 knockout vector (containing a herbicide resistance selection marker) specifically targeting the D1 and D2 genes was constructed. The constructed vector was transformed into Agrobacterium tumefaciens EHA105. Using the yellow-fleshed soybean variety Dongnong 50 as the recipient material, soybean transformation experiments were conducted using the soybean cotyledon node genetic transformation method. T0 generation plants were obtained, and herbicide-resistant plants were detected using BAR transgenic test strips. After obtaining resistant T0 generation plants, primers were designed to amplify the DNA fragment containing the target site. The upstream primer sequence for D1 was 5'-AATTAGGGCAACCATACCACG-3', and the downstream primer sequence was 5'-GGTCGGCAGTATGATTCACATG-3'; the upstream primer sequence for D2 was 5'-ATTGGGGAAACCAAACCACG-3', and the downstream primer sequence was 5'-GTCGGCAGTATGATTCACATG-3'. PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 35 cycles, followed by a final extension at 72℃ for 10 min. After separation of the PCR products by agarose gel electrophoresis, a target strip of approximately 1000 bp was excised and sent to a sequencing company for sequencing using the amplification primers. Based on the D1 and D2 gene sequences, a d1d2 double mutant with a 1 bp insertion in the D1 gene and a 1 bp deficiency in the D2 gene was identified in T0 generation resistant plants. Figure 1 As shown ( Figure 1 (This is a schematic diagram of sequence variations in the d1 / d2 mutant).

[0033] The near-isogenic line d1d1 / d2d2, based on existing literature, exhibits a phenotype of green leaves and a green seed coat at the D1 and D2 loci. The d1 / d2 double mutant created in this invention also exhibits a phenotype of green leaves and a light green seed coat. However, the main focus of this invention is the green cotyledon color in the seeds of the d1 / d2 double mutant. This invention observes the cotyledon color after the seeds are rounded; the cotyledons of the yellow-core soybean variety Dongnong 50 are yellow, while the cotyledons of the d1d2 double mutant are green. Figure 2 As shown ( Figure 2 (This refers to the observation of cotyledon color in Dongnong 50 and the d1 / d2 mutant after the seeds have matured and rounded). These results indicate that double mutation of the D1 and D2 genes produces green-core soybean seeds.

[0034] 2. Development of D1-dCAPS molecular markers

[0035] Based on the variant analysis of the D1 gene (SoyZH13_01G197200 (Zhonghuang 13 genome version) or Glyma.01G214600 (Williams82 genome version)) published by the National Center for Biotechnology Information (https: / / ngdc.cncb.ac.cn / soyomics / index), the main variant type of the D1 gene was found to be soy31895591. Among the 5691 soybean resource data resequencing, 5780 soybean resources carried TA. 56991967 89 soybean resources carry T 56991967 The mutation is a frameshift mutation caused by the deletion of one amino acid. Primers were designed and PCR amplified to develop the D1-dCAPS marker. The nucleotide sequence of the D1-dCAPS molecular marker is shown in SEQ ID NO.1. The Y at position 58 of this sequence indicates a polymorphism, which is either A or Δ (Δ indicates that one base is deleted at this site).

[0036] 3. Development of D2-Indel molecular markers

[0037] Using chromosome walking, a large insertion variant was discovered in the D2 gene (SoyZH13_11G026500 (Zhonghuang 13 genome version) or Glyma.11G027400 (Williams82 genome version)), which leads to premature termination of protein translation. The inventors designed primers and performed PCR amplification targeting this Indel variant, thus developing the D2-Indel marker. The nucleotide sequence of the D2-Indel molecular marker is shown in SEQ ID NO.2; an insertion or deletion of 1075 bp from position 557 to position 1631 in this sequence is polymorphic.

[0038] II. Validation of Molecular Markers

[0039] The polymorphisms of D1-dCAPS and D2-Indel molecular markers were verified using yellow-core soybean variety Dongnong 50 and green-core soybean variety Jiqing 3, as well as F1 generation plants bred from them as parents.

[0040] 1. Validation of D1-dCAPS molecular marker

[0041] The D1-dCAPS molecular marker was amplified using a semi-nested PCR method. The nucleotide sequence of the upstream primer for the first round of PCR is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4. The nucleotide sequence of the upstream primer for the second round of PCR is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0042] First round of PCR: High-quality genomic DNA isolated from the leaves of soybean varieties Dongnong 50 and Jiqing 3, as well as their F1 hybrids, was used as a template. PCR amplification was performed using SEQ ID NO.3 and SEQ ID NO.4 as primers. The PCR system consisted of 10 μL of 2×Mix, 1.5 μL of DNA template, 1 μL each of upstream and downstream primers, and 6.5 μL of ddH2O in a 20 μL reaction mixture. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s; 20 cycles in total; 72℃ extension for 5 min.

[0043] Second round PCR: Using a 50-fold dilution of the first round PCR product as a template, and SEQ ID NO.4 and SEQ ID NO.5 as primers, PCR amplification was performed. The PCR system consisted of: 10 μL of 2×Mix, 1.5 μL of DNA template, 1 μL each of upstream and downstream primers, and 6.5 μL of ddH2O in a 20 μL reaction volume. The PCR amplification program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s; for a total of 33 cycles; 72℃ extension for 5 min.

[0044] Enzyme digestion reaction: 10 μL of second-round PCR product, 1.2 μL of rCutSmart, 0.3 μL of PmeI enzyme, and 0.5 μL of ddH2O. The digestion conditions were: 37°C for 2 h.

[0045] Agarose gel electrophoresis: Take 4 g of agarose powder, add 100 ml of 1×TAE buffer, and heat in a microwave oven until fully melted. Perform agarose gel electrophoresis on the products of the second round of PCR and the enzyme digestion products. Set the electrophoresis apparatus to 110V and the electrophoresis time to approximately half an hour.

[0046] Based on the two rounds of PCR amplification, the D1-dCAPS amplification product length is 214 bp or 213 bp, as shown in SEQ ID NO. 1. The Y at position 58 of this sequence indicates polymorphism, which is either A or Δ (Δ indicates a deletion of one base at this site). According to the recognition sequence 5'-GTTT⇅AAAC-3' of the restriction endonuclease PmeI, if the SNP site is A, it cannot be digested and is labeled as D1-dCAPS. A If the SNP site is Δ, it can be cleaved into two bands of 159 bp and 54 bp, labeled as D1-dCAPS. Δ .

[0047] Based on the detection results of Dongnong 50, Jiqing 3, and F1, before PmeI digestion, the main band sizes of the three materials were 214 bp or 213 bp, differing by only one base and thus indistinguishable by gel electrophoresis. After PmeI digestion, Dongnong 50 could not be digested and retained its 214 bp main band; Jiqing 3 could be digested into two main bands of 54 bp and 159 bp; F1 had three main bands of 214 bp, 159 bp, and 54 bp. Figure 3 As shown ( Figure 3 This is an electrophoresis diagram of the amplification products of the D1-dCAPS molecular marker primers of the present invention in the yellow-core soybean variety Dongnong 50, the green-core soybean variety Jiqing 3, and their F1 generations, before and after enzyme digestion. The above results demonstrate that D1-dCAPS has stable polymorphism.

[0048] 2. Validation of D2-Indel molecular markers

[0049] The D2-Indel molecular marker was amplified using a triple PCR method. The nucleotide sequence of the upstream primer for amplifying the molecular marker is shown in SEQ ID NO.6, the nucleotide sequence of one downstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the other downstream primer is shown in SEQ ID NO.8.

[0050] PCR: High-quality genomic DNA was isolated from the leaves of soybean varieties Dongnong 50 and Jiqing 3, as well as their F1 hybrids, using the CTAB method. PCR amplification was performed using SEQ ID NO. 6-8 as primers. The PCR system consisted of: 10 μl of 2X Mix, 1 μl each of upstream and downstream primers, 1.5 μl of DNA template, and 6.5 μl of ddH2O in a 20 μL reaction volume. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 33 cycles, followed by a final extension at 72℃ for 10 min.

[0051] Agarose gel electrophoresis: Take 3 g of agarose powder, add 100 ml of 1×TAE buffer, and heat in a microwave oven until fully melted. Perform agarose gel electrophoresis on the PCR products. Set the electrophoresis apparatus to 110 V and the electrophoresis time to approximately half an hour.

[0052] The nucleotide sequence of the molecular marker D2-Indel is shown in SEQ ID NO.2. An insertion or deletion of 1075 bp from position 557 to 1631 in this sequence is polymorphic. If the polymorphic region is present, PCR can be performed to obtain an amplified fragment with a length of 754 bp using primers SEQ ID NO.6 and SEQ ID NO.7, labeled as the D2-In genotype. If the polymorphic region is absent, PCR can be performed to obtain an amplified fragment with a length of 571 bp using primers SEQ ID NO.6 and SEQ ID NO.8, labeled as the D2-Del genotype.

[0053] Based on the detection results of Dongnong 50, Jiqing 3, and F1, Dongnong 50 can amplify a 571 bp main band; Jiqing 3 can amplify a 754 bp main band; and F1 contains two main bands, with sizes of 754 bp and 571 bp respectively. Figure 4 As shown ( Figure 4 This is an electrophoresis diagram of the amplification products of the D2-Indel molecular marker primers of the present invention in the yellow-core soybean variety Dongnong 50, the green-core soybean variety Jiqing 3, and their F1 generation. The above results demonstrate that D2-Indel has stable polymorphism.

[0054] Example 2: Identification of green-core soybeans using the molecular markers described in this invention.

[0055] 1. Test materials

[0056] This invention utilizes 5 green-core soybean germplasm resources and 12 yellow-core soybean germplasm resources, with specific soybean varieties shown in Table 1 (Table 1 shows the phenotypic and genotypic statistics of cotyledon color for 17 soybean germplasm resources) to validate the D1-dCAPS and D2-Indel molecular markers. All germplasm materials used were obtained from the inventors' team.

[0057] 2. Specific experimental methods,

[0058] A method for identifying green-core soybeans includes the following steps:

[0059] (1) Identification of D1-dCAPS molecular marker

[0060] The D1-dCAPS molecular marker was amplified using a semi-nested PCR method. The nucleotide sequence of the upstream primer for the first round of PCR is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4. The nucleotide sequence of the upstream primer for the second round of PCR is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0061] First round of PCR: High-quality genomic DNA was isolated from the leaves of soybean varieties Dongnong 50 and Jiqing 3 and their F1 hybrids using the CTAB method. PCR amplification was performed using SEQ ID NO.3 and SEQ ID NO.4 as primers.

[0062] Second round of PCR: Using a 50-fold dilution of the first round PCR product as a template, and SEQ ID NO.4 and SEQ ID NO.5 as primers, PCR amplification was performed.

[0063] Enzyme digestion reaction: 10 μL of second-round PCR product, 1.2 μL of rCutSmart, 0.3 μL of PmeI enzyme, and 0.5 μL of ddH2O. The digestion conditions were: 37°C for 2 h.

[0064] (2) Identification of D2-Indel molecular markers

[0065] The D2-Indel molecular marker was amplified using a triple PCR method. The nucleotide sequence of the upstream primer for amplifying the molecular marker is shown in SEQ ID NO.6, the nucleotide sequence of one downstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the other downstream primer is shown in SEQ ID NO.8. High-quality genomic DNA isolated from the leaves of soybean varieties Dongnong 50 and Jiqing 3, as well as their F1 hybrids, was used as a template for PCR amplification.

[0066] (3) The above PCR system is as follows: in a 20 μL reaction system, 10 μL of 2XMix, 1 μL each of upstream and downstream primers, 1.5 μL of DNA template, and ddH2O to make up to 20 μL. The PCR reaction conditions are: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 35 s, the first round of semi-nested PCR is performed for 20 cycles, the other PCRs are performed for 33 cycles, and then the extension is performed at 72℃ for 10 min.

[0067] (4) Agarose gel electrophoresis: Take 4g of agarose powder, add 100ml of 1×TAE buffer, and heat in a microwave oven until fully melted. Perform agarose gel electrophoresis on the PCR products. Set the electrophoresis apparatus to 110V and the electrophoresis time to about half an hour.

[0068] (5) Genotype markers

[0069] Following semi-nested PCR amplification, the D1-dCAPS molecular marker amplification product has a length of 214 bp or 213 bp, as shown in SEQ ID NO.1. After PmeI enzyme digestion, if the SNP site is A, it cannot be digested, retaining a 214 bp master band, and is labeled as D1-dCAPS. A If the SNP site is Δ, it can be cleaved into two major bands of 159 bp and 54 bp, labeled as D1-dCAPS. Δ .

[0070] Based on triple PCR amplification, if the D2-Indel molecular marker amplifies to a 754 bp major band, it is labeled as the D2-In genotype; if the D2-Indel molecular marker amplifies to a 557 bp major band, it is labeled as the D2-Del genotype.

[0071] 3. Results Analysis

[0072] After identification using the D1-dCAPS molecular marker, soybean germplasm resources 1-5 were all identified as D1-dCAPS. Δ All soybean germplasm resources 6-17 are D1-dCAPS. A ,like Figure 5 As shown ( Figure 5 This is an electrophoresis diagram showing the validation results of the D1-dCAPS molecular marker primers of this invention in detecting 22 soybean germplasm resources with known cotyledon color phenotypes;).

[0073] After identification using the D2-Indel molecular marker, soybean germplasm resources 1-5 were all D2-In, and soybean germplasm resources 6-17 were all D2-Del. Figure 6 As shown ( Figure 6 This is an electrophoresis diagram showing the validation results of the D2-Indel molecular marker primers of this invention in detecting 22 soybean germplasm resources with known cotyledon color phenotypes.

[0074] Furthermore, it was discovered that green-core soybean germplasm resources 1-5 are D1-dCAPS. Δ And the D2-In genotype, while the yellow-core soybean germplasm resource 6-17 is D1-dCAPS. A And the D2-Del genotype.

[0075] The above results demonstrate that D1-dCAPS and D2-Indel molecular markers can be used to identify green-core soybeans. When the test material is D1-dCAPS... Δ The D1-dCAPS and D2-Indel molecular markers were used to test green-core soybean varieties, along with the D2-In genotype. These markers can be applied to marker-assisted breeding of green-core soybean varieties.

[0076] The phenotypes and genotypes of cotyledon color in 17 soybean germplasm resources were statistically analyzed, and the results are shown in Table 1:

[0077]

Claims

1. A molecular marker for identifying green-core soybeans, characterized in that... The molecular markers for identifying green-core soybeans are D1-dCAPS and D2-Indel. The nucleotide sequence of D1-dCAPS is shown in SEQ ID NO.1; the nucleotide sequence of D2-Indel is shown in SEQ ID NO.

2. The Y at position 58 of the D1-dCAPS nucleotide sequence indicates polymorphism, where Y is A or Δ, and Δ indicates the deletion of one base at that position. The 1075 bp from position 557 to position 1631 of the D2-Indel sequence represents insertion or deletion polymorphism.

2. The method for identifying green-core soybeans using the molecular markers described in claim 1, characterized in that... The method for identifying green-core soybeans is as follows: I. Using the DNA of the soybean material to be tested as a template, the D1-dCAPS molecular marker was amplified by semi-nested PCR. After the first and second rounds of PCR amplification, the length of the D1-dCAPS amplification product was 214 bp or 213 bp. After PmeI digestion, the 214 bp band could not be digested and remained as the main 214 bp band. Therefore, the material to be tested was labeled as D1-dCAPS. A Genotype; the product of the 213 bp band can be digested with PmeI enzyme, and the digestion product contains two bands of 159 bp and 54 bp, which are labeled as D1-dCAPS soybean material. Δ Genotype; The semi-nested PCR method: The nucleotide sequence of the upstream primer for the first round of PCR is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the upstream primer for the second round of PCR is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; Using the DNA of the soybean material to be tested as a template, the D2-Indel molecular marker was amplified by triple PCR. If the PCR product contained a 754 bp band, the soybean material to be tested was labeled as D2-In; if the PCR product contained a 571 bp band, the soybean material to be tested was labeled as D2-Del. The triple PCR method consisted of the following: the nucleotide sequence of the upstream primer was shown in SEQ ID NO.6; the nucleotide sequence of one downstream primer was shown in SEQ ID NO.7; and the nucleotide sequence of the other downstream primer was shown in SEQ ID NO.

8. The PCR system consisted of 10 μl of 2X Mix, 1 μl each of the upstream and downstream primers, 1.5 μl of DNA template, and 6.5 μl of ddH2O in a 20 μL reaction system. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 33 cycles, followed by a final extension at 72℃ for 10 min. III. Simultaneously satisfying the criteria in step one for identification as D1-dCAPS Δ The soybean material to be tested that has the D2-In genotype identified in step two is the green-core soybean.

3. The method for identifying green-core soybeans using molecular markers according to claim 2, characterized in that... The first round of PCR consisted of: 10 μL of 2×Mix, 1.5 μL of DNA template, 1 μL each of upstream and downstream primers, and 6.5 μL of ddH2O in a 20 μL reaction system; the PCR amplification program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s; a total of 20 cycles; and a final extension at 72℃ for 5 min. The second round of PCR consisted of the following: 10 μL of 2×Mix, 1.5 μL of DNA template, 1 μL each of upstream and downstream primers, and 6.5 μL of ddH2O in a 20 μL reaction system; PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s; 33 cycles in total; 72℃ extension for 5 min.

4. The method for identifying green-core soybeans using molecular markers according to claim 3, characterized in that... The enzyme digestion reaction system in step one was as follows: 10 μL of the second round PCR product, 1.2 μL of rCutSmart, 0.3 μL of PmeI enzyme and 0.5 μL of ddH2O; the enzyme digestion reaction conditions were: 37°C, and the digestion was carried out for 2 h.

5. Specific detection of the application of the primers for the molecular markers described in claim 1 in the identification of green-core soybeans.

6. The application according to claim 5, characterized in that... The application used was to identify the cotyledon color of soybean seeds and to identify molecular marker-assisted breeding of green-core soybeans.

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