A molecular marker associated with soybean petiole opening and application thereof

Abstract

The present application relates to the field of crop genetic breeding technology, in particular to a molecular marker related to soybean petiole open and application thereof. The molecular marker is A>C mutation at 33746105 of gene number Glyma.03g124400. The present application obtains a flower organ petiole open mutant (OFC) through mutagenesis, determines the related functional site by using map-based cloning, and develops an identification marker, which can detect the related traits conveniently and quickly, can screen excellent germplasm and provide utilization, and provides materials, theory and technical support for utilization of soybean hybrid vigor and improvement of cross-pollination rate.

Description

Technical Field

[0001] This invention relates to the field of crop genetics and breeding technology, and in particular to a molecular marker related to the opening of soybean keel lobes and its application. Background Technology

[0002] With a rapidly growing population, Jonathan and Tilman et al. predict that current crop yields must double by 2050 to meet the food consumption demands of the world's growing population. To meet this demand, crop yields need to increase at a rate of at least 2.4% annually. Cultivated soybean [Glycine max (L.) Merr.], belonging to the Leguminosae family, Papilionate subfamily, Glycine genus, Soja subgenus, is an important annual herbaceous plant, a major source of plant protein and oil in people's lives, and also an important feed crop, accounting for approximately 56% of global edible oil production. Ray et al. mentioned that the current annual yield increase rate of soybeans is about 1.3%, far below the expected requirements. Therefore, finding an efficient breeding strategy to select high-yielding and high-quality soybean varieties is the most urgent problem that soybean breeders need to solve. In soybean breeding methods, the methods have evolved from the initial natural variation selection and hybridization breeding to mutation breeding, introduction of new varieties, heterosis breeding, high light efficiency breeding, pollen tube pathway breeding, marker-assisted selection breeding, and transgenic breeding, among others. Molecular breeding, in particular, has transformed traditional phenotypic selection-based methods into methods that directly target genotype selection, thus improving selection efficiency. Currently, heterosis is widely used in various crops and plays a crucial role in increasing crop yields.

[0003] Soybeans are self-pollinating crops with a cross-pollination rate ranging from 0.03% to 3.62%. The unique butterfly-shaped petal structure of soybeans creates mechanical pollination barriers, hindering cross-pollination and making hybrid seed production difficult. This limits the potential for increasing soybean yield through heterosis. Therefore, understanding the structure of soybean floral organs, creating materials with open petals, and improving the cross-pollination rate are of great value in increasing soybean yield.

[0004] Molecular marker breeding, as an important auxiliary tool in current breeding work, hinges on the development of efficient molecular markers. Molecular markers that are tightly linked to and stable with target traits, and are easy and quick to detect, can directly select for genotypes, greatly improving the accuracy and efficiency of selection in breeding and significantly shortening the breeding cycle. Therefore, the development of efficient molecular markers is of great significance for the breeding of new varieties for variety improvement. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a molecular marker related to the opening of soybean keel petals and its application. The molecular marker provided by this invention can distinguish between soybeans with open keel petals and soybeans with closed keel petals, providing technical support for creating materials with open petals and improving the cross-pollination rate of soybeans.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a molecular marker associated with the opening of soybean keel flaps, wherein the molecular marker is an A>C mutation at position 33746105 of gene number Glyma.03g124400.

[0008] The present invention also provides a protein associated with the opening of soybean keel flaps, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] The present invention also provides a gene encoding the above-mentioned protein, the CDS sequence of which is shown in SEQ ID NO.2.

[0010] The present invention also provides a primer pair for amplifying the above-mentioned molecular marker, the primer pair comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer being shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer being shown in SEQ ID NO.4.

[0011] The present invention also provides the use of the above-mentioned molecular markers, proteins, genes, or primer pairs in one or more of the following aspects, including:

[0012] 1) Prepare, identify, or screen products made from soybeans with open keel-shaped petals;

[0013] 2) Identify or screen soybeans with open keel lobes;

[0014] 3) Assist in the cultivation of soybean varieties with open keel petals.

[0015] The present invention also provides a kit for identifying or screening soybeans with open keel lobes, the kit comprising the primer pairs described above.

[0016] This invention also provides a method for identifying or screening soybeans with open keel lobes, comprising the following steps:

[0017] PCR amplification was performed using the genomic DNA of the soybean to be identified as a template to obtain amplification products; the primers used in the PCR amplification included the primer pairs mentioned above.

[0018] The traits of soybean keel flaps are determined based on the genotype of the site corresponding to the molecular marker described in claim 1 in the amplification product, as follows:

[0019] If the genotype at the corresponding locus is AA or AC, then the soybean to be identified is a closed keel lobe.

[0020] If the genotype at the corresponding locus is CC, then the soybean to be identified is an open keel flap.

[0021] Preferably, the method for determining the genotype includes sequencing or electrophoresis;

[0022] When the determination method is electrophoresis, it includes: digesting the amplification product with AluⅠ enzyme and then performing electrophoretic analysis on the digested product;

[0023] If there is only one 282bp electrophoresis band, then the genotype of the site corresponding to the molecular marker is CC;

[0024] If there is only one 257bp electrophoretic band, or only two 257bp and 25bp electrophoretic bands respectively, then the genotype of the site corresponding to the molecular marker is AA;

[0025] If there are two electrophoretic bands of 282bp and 257bp respectively, or three electrophoretic bands of 282bp, 257bp and 25bp respectively, then the genotype of the site corresponding to the molecular marker is AC.

[0026] Preferably, the PCR amplification reaction process includes: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

[0027] The present invention also provides a major QTL for soybean keel flap opening, wherein the major QTL is located on soybean chromosome 3, physically between SSR1043 and SSR1045, with an interval size of 58Kb.

[0028] Beneficial effects:

[0029] This invention provides a molecular marker associated with the opening of the keel petals in soybean. The marker is defined as an A>C mutation at gene position 33746105 of Glyma.03g124400. This invention obtains a mutant with an open keel petal in the floral organ through mutagenesis, uses map-based cloning to identify relevant functional sites, and develops identification markers. This allows for convenient and rapid detection of related traits, screening for superior germplasm, and providing material, theoretical, and technical support for utilizing soybean heterosis and improving outcrossing rates. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0031] Figure 1 A comparative diagram of the floral organ phenotypes of wild-type WT and ofc provided by this invention;

[0032] Figure 2 The candidate gene structure diagram provided by this invention;

[0033] Figure 3 The analysis of BSA sequencing results provided by this invention preliminarily determined the candidate region map;

[0034] Figure 4 This invention provides a fine mapping of the open gene of the keel flap.

[0035] Figure 5-1 and Figure 5-2 The expression characteristics of genes within the finely mapped candidate regions provided by this invention in floral organs, leaves, and other tissues between parents; * indicates P < 0.01, ** indicates P < 0.05;

[0036] Figure 6 This is a diagram showing the amino acid sequence differences between the wild-type and mutant candidate genes provided by the present invention.

[0037] Figure 7 This invention provides verification electrophoresis images of the molecular marker OFC17 from 54 known genotype materials of domestic and foreign germplasm resources;

[0038] Figure 8 The image shows the identification results of the dCAPS marker OFC17 in 83 keel flap open (ofc) and keel flap closed (LY) populations provided by this invention. Detailed Implementation

[0039] This invention provides a molecular marker associated with the opening of soybean keel flaps, wherein the molecular marker is an A>C mutation at position 33746105 of gene number Glyma.03g124400.

[0040] This invention utilizes the cloven-petaled variety Zhongpin 661 through mutagenesis treatment, combined with phenotypic identification through field observation of flower organs during full bloom, to obtain open-petaled material ofc (i.e., open-petaled mutant, also known as OFC). Hybrid seeds are obtained by crossing LY (closed-petaled mutant) with ofc. The hybrid seeds are then self-pollinated in the F2 segregating population to construct closed-petaled and open-petaled individuals, respectively. BSA-seq sequencing is performed, and linkage analysis is conducted in conjunction with the segregating population to confirm the relevant regions. A gene Gmofc associated with open-petaled buds is identified. This gene is then compared with open-petaled buds... This invention relates to the Gmofc gene of soybean flower organs and keel petal closure materials. Molecular markers associated with soybean keel petal opening were screened at exon 33746105 on chromosome 3 (Gmofc gene number Glyma.03g124400 in Genbank database version V2.1). Based on these markers, the dCAPS marker OFC17 was designed. This marker can be used to identify OFC for constructing hybrid populations and to determine the keel petal opening habits of soybean flower organs both domestically and internationally. It also allows for the screening of superior germplasm and its utilization, providing material, theoretical, and technical support for improving the outcrossing rate of soybean heterosis.

[0041] This invention also provides a protein associated with the opening of soybean keel lobes, the amino acid sequence of which is shown in SEQ ID NO.1, and is as follows:

[0042] MREILHVQGGQCGNQIGSKFWEVVCDEHGIDPTGKYVGNSDLQLERVNVYYNEASCGRFVPRAVLMDLEPGTMDSVRTGPYGQIFRPDNFVFGQSGAGNNWAKGHYTEGAELIDSVLDVVRKEAENCDCLQGFQVCHSLGGGTGSGMGTLLISKIREEYPDRMMLTFSVFPSPKVSDTVVEPYNATLSVHQ LVENADECMVLDNEALYDICFRTLKLTTPSFGDLNHLISATMSGVTCCLRFPGQLNSDLRKLAVNLIPFPRLHFFMVGFAPLTSRGSQQYRALTVPELTQQMWDAKNMMCAADPRHGRYLTASAMFRGKMSTKEVDEQMINVQNKNSSYFVEWIPNNVKSSVCDIAPRGLSMASTFIGNSTSIQEMFRRVSE P FTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATAEDDGEYEDEEDDDVEADHM.

[0043] The protein described in this invention is the protein expressed by the Gmofc gene in the above-mentioned keel petal opening material ofc, that is, the protein expressed by keel petal opening soybean, which can regulate the opening of the keel petal. The amino acid (proline) in the above-mentioned amino acid sequence is due to the A>C mutation occurring at the above-mentioned molecular marker in wild-type soybean plants, which causes the amino acid encoding the 384th position to change from glutamine to proline.

[0044] The present invention also provides a gene encoding the above-mentioned protein, the CDS sequence of which is shown in SEQ ID NO.2, as follows:

[0045]

[0046] The present invention also provides a primer pair for amplifying the above-mentioned molecular marker, the primer pair comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer being as shown in SEQ ID NO.3, specifically: CATGATGTGTGCTGCAGATCCA; the nucleotide sequence of the downstream primer being as shown in SEQ ID NO.4, specifically: TTCCTCCTAAACATGGCCGTGAGC.

[0047] The primer pairs provided by this invention can specifically amplify fragments containing the above-mentioned molecular markers. After the amplified fragments are digested with AluI enzyme, the genotype of the material to be identified at the above-mentioned molecular marker sites can be determined by electrophoresis, providing technical support for screening soybeans with open keel lobes.

[0048] The present invention also provides the application of the above-mentioned molecular markers or proteins or genes or primer pairs in one or more of the following aspects: 1) preparing products for identifying or screening open keel lobe soybeans; 2) identifying or screening open keel lobe soybeans; 3) assisting in the breeding of open keel lobe soybean varieties.

[0049] The present invention also provides a kit for identifying or screening soybeans with open keel lobes, the kit comprising the primer pairs described above.

[0050] This invention also provides a method for identifying or screening soybeans with open keel lobes, comprising the following steps:

[0051] Using the genomic DNA of the soybean to be identified as a template, PCR amplification was performed to obtain the amplification product; the primers used in the PCR amplification included the primer pairs mentioned above or the primer pairs in the kit mentioned above.

[0052] The traits of soybean keel flaps are determined based on the genotypes at the corresponding sites of the molecular markers mentioned above in the amplification products, as follows:

[0053] If the genotype at the corresponding locus is AA or AC, then the soybean to be identified is a closed keel lobe.

[0054] If the genotype at the corresponding locus is CC, then the soybean to be identified is an open keel flap.

[0055] In this invention, the preferred PCR amplification reaction procedure includes: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min.

[0056] In this invention, the PCR amplification reaction system is preferably 20 μL, and preferably includes 1-2 μL of template, 0.6 μL each of upstream and downstream primers, 2 μL of 10×EasyTaq Buffer, 2 μL of dNTPs, 0.2 μL of EasyTaq polymerase, and the balance ddH2O.

[0057] In this invention, the method for determining the genotype preferably includes sequencing or electrophoresis, more preferably electrophoresis; the electrophoresis method preferably includes non-denaturing polyacrylamide gel electrophoresis.

[0058] When the determination method is electrophoresis, it preferably includes: digesting the amplification product with AluⅠ enzyme and then performing electrophoretic analysis on the digested product;

[0059] If there is only one 282bp electrophoresis band, then the genotype of the site corresponding to the molecular marker is CC;

[0060] If there is only one 257bp electrophoretic band, or only two 257bp and 25bp electrophoretic bands respectively, then the genotype of the site corresponding to the molecular marker is AA;

[0061] If there are two electrophoretic bands of 282bp and 257bp respectively, or three electrophoretic bands of 282bp, 257bp and 25bp respectively, then the genotype of the site corresponding to the molecular marker is AC.

[0062] In this invention, during electrophoretic analysis, it is preferable to use 282bp and 257bp electrophoretic bands as controls to determine the size of the electrophoretic bands of the enzyme digestion products.

[0063] This invention utilizes AluI enzyme to digest the amplification products, allowing for rapid and accurate determination of the genotype of the sample to be identified based on the digestion results. Since the genotype bands are not significantly different in size, conventional agarose gel electrophoresis cannot effectively distinguish them. Therefore, this invention uses non-denaturing polyacrylamide gel electrophoresis to effectively differentiate the electrophoretic bands after enzyme digestion. Furthermore, theoretically, wild-type amplification products should produce two electrophoretic bands of 257bp and 25bp after enzyme digestion. However, due to limitations in current electrophoresis technology, only 257bp and 282bp bands can currently be observed simultaneously to distinguish different genotypes. Therefore, during the identification process, observing the presence of 257bp and 282bp bands is sufficient to differentiate the genotype of the sample, without the need for further observation of the 25bp electrophoretic band.

[0064] This invention also provides a major QTL for soybean keel flap opening, which is located on soybean chromosome 3, physically between SSR1043 (BARCSOYSSR_03_1043) and SSR1045 (BARCSOYSSR_03_1045), with a range of 58 kb. The major QTL provided by this invention can achieve co-segregation of soybeans with both closed and open keel flap traits.

[0065] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a molecular marker related to the opening of soybean keel flaps and its application, should not be construed as limiting the scope of protection of the present invention.

[0066] In this example, the wild-type soybean germplasm Zhongpin 661 (WT) keel petal closure and keel petal opening mutant ofc were provided by the Institute of Crop Science, Chinese Academy of Agricultural Sciences. The F1 hybrid population constructed by the keel petal closure mutant LY and the keel petal opening mutant ofc was identified by Professor Sun Rujian of Hulunbuir Agricultural and Animal Husbandry Research Institute through generational breeding.

[0067] Example 1

[0068] Construction and fine mapping of Gmofc gene population in soybean

[0069] 1. Genetic Population Construction: In 2018, at the Beijing Changping Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, a hybrid combination was constructed using the open-valve-petal mutant ofc as the male parent and the closed-valve-petal mutant LY as the female parent to obtain hybrid seeds. In 2019, F1 generation plants were propagated and seeds were harvested at the Zhalantun Hulunbuir Agricultural and Animal Husbandry Research Institute in Inner Mongolia. In 2020, the F2 generation population was planted at the Beijing Changping Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. Furthermore, in 2020, the F3 generation population was planted at the Hainan Nanbin Farm Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and phenotypic identification was conducted to verify the F2 generation genotype.

[0070] 2. Population genetic analysis: In 2020, the differences in keel petal opening habits were statistically analyzed in the F2 population, and genetic analysis was performed (Table 1) to verify the genetic law of keel petal opening. The results showed that the keel petal opening phenotype is controlled by a recessive single gene. Through comparison of floral organs, ofc was identified as the keel petal opening material, and the wild type as the closed material. Figure 1 )

[0071] Table 1. Genetic analysis of the keel flap opening gene Gmofc

[0072]

[0073] 3. Initial BSA localization of soybean keel petal opening: Thirty individuals with closed keel petals and 30 individuals with open keel petals were selected from the F2 population of the LY×ofc hybrid to construct keel petal closure and keel petal opening pools, respectively. The parental materials were also used. Deep resequencing of the DNA pools from the four materials was performed on the HiSeq 2500 platform. Using the Williams 82 genome sequence as a reference, single nucleotide polymorphism (SNP) sites were identified between the two pools. Using the SNP-index association algorithm, one candidate region related to the trait was obtained, with a total length of 18.33 Mb; using the ED association algorithm, one candidate region related to the trait was obtained, with a total length of 18.90 Mb. Taking the intersection of the two methods yielded another candidate region related to the trait, with a total length of 18.33 Mb. The associated region contained a total of 938 genes, including 10 non-synonymous mutant genes. Figure 3 ).

[0074] Fine localization of soybean keel petal opening: Genomic DNA was extracted from individual plants in the F2 population of LY×ofc. Candidate gene locations were preliminarily determined using annotation information of candidate genes in the initial localization interval, combined with phenotype (keel petal opening or closing trait). Polymorphic markers were developed using parental differences in InDel to identify the genotypes of the F2 population of 338 plants. Combined with the F3 phenotype, the candidate interval was narrowed down to between SSR1043 (BARCSOYSSR_03_1043) and SSR1045 (BARCSOYSSR_03_1045), with a interval size of 58 kb. Figure 4 ).

[0075] Using the soybean genome annotation website (https: / / soybase.org / ), the annotated genes in the reference genome within the 58Kb region were analyzed. The results are shown in Table 2. The results show that there are 3 annotated genes in the located region, including 1 known protein-coding gene and 2 genes with unknown functions. No previously reported soybean keel flap opening-related genes were found in the region, indicating that the Gmofc (i.e., Gene 1 in Table 2) keel flap opening gene located in this invention is a novel gene.

[0076] Table 2 Gene annotation information within the localization interval

[0077]

[0078] Using quantitative real-time PCR, the gene expression differences within candidate regions were compared among sepal, petal, stem, bud, and flower bud tissues of the wild-type keel-petal-closed Zhongpin 661 population, the parental keel-petal-closed LY mutant, and the keel-petal-open mutant ofc. Figure 5-1 and Figure 5-2The results showed that, except for Gene1, which had the highest expression levels in all tissues across both parents with highly significant differences, Gene2 and Gene3 had lower expression levels in all tissues across both parents with no significant differences. Based on gene annotation and sequencing results, Gmofc was identified as a candidate gene.

[0079] Example 2

[0080] Isolation and structural analysis of the Gmofc gene in soybean

[0081] 1. Isolation of the Gmofc gene

[0082] Total RNA was extracted from the wild-type soybean variety ZP661. The total RNA was reverse transcribed to obtain cDNA, which was then used as a template for PCR amplification using both the forward primer (SEQ ID NO. 5: ATGAGGGAGATCCTTCACGTTCAG) and the reverse primer (SEQ ID NO. 6: TGTTGAAGCAGACCACATGTGA). The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 120 s, 35 cycles; 72℃ extension for 8 min. The PCR amplification reaction system consisted of: 5 μL template (100 ng / μL), 1 μL each of the forward and reverse primers, and 10×Easy Taq Buffer (Mg-free). 2+ 25 μL of dNTPs (2.5 mM), 10 μL of Easy Taq polymerase (All-Gold, AP111), and 7.4 μL of ddH2O were added. A 1350 bp cDNA fragment of the Gmofc gene was obtained, as shown in SEQ ID NO.7.

[0083]

[0084] The amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.8, consisting of 449 amino acids. Figure 6 The WT in the text is as follows:

[0085] MREILHVQGGQCGNQIGSKFWEVVCDEHGIDPTGKYVGNSDLQLERVNVYYNEASCGRFVPRAVLMDLEPGTMDSVRTGPYGQIFRPDNFVFGQSGAGNNWAKGHYTEGAEL IDSVLDVVRKEAENCDCLQGFQVCHSLGGGTGSGMGTLLISKIREEYPDRMMLTFSVFPSPKVSDTVVEPYNATLSVHQLVENADECMVLDNEALYDICFRTLKLTTPSFGDL NHLISATMSGVTCCLRFPGQLNSDLRKLAVNLIPFPRLHFFMVGFAPLTSRGSQQYRALTVPELTQQMWDAKNMMCAADPRHGRYLTASAMFRGKMSTKEVDEQMINVQNKN SSYFVEWIPNNVKSSVCDIAPRGLSMASTFIGNSTSIQEMFRRVSEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATAEDDGEYEDEEDDDVEADHM.

[0086] 2. Gmofc gene structure analysis

[0087] DNA was extracted from young leaves of the wild-type soybean variety zp661. Using this genomic DNA as a template, the Gmofc genomic fragment was obtained by amplification using two overlapping primer pairs (the products amplified by the two primer pairs were sequenced separately and then the sequencing results were combined). The primer pairs were primer pair 1 OFC1244-1F: GACTCAAATCGAATTGCACCCA (SEQ ID NO. 9) and OFC1244-1R: CTCCCTCATTTTCCCCTGCA (SEQ ID NO. 10), and primer pair 2 OFC1244-2F: CCAAGGGCATATCCGTCACA (SEQ ID NO. 11) and OFC1244-2R: GGTGGCTCCTTTACCAGTTCA (SEQ ID NO. 12). The reaction procedure and process of primer pair 1 and primer pair 2 were the same as in step 1.

[0088] The Gmofc genome fragment is 2939 bp in length and contains 2 exons and 2 introns. Figure 2 Specifically, as shown in SEQ ID NO.13:

[0089]

[0090] Example 3

[0091] Development and utilization of functional markers for Gmofc gene in soybean

[0092] By utilizing the sequence differences between wild-type soybean zp661 and the mutant ofc, primers were designed based on the conserved sequences at both ends of the SNP site, and a dCAPS marker was successfully developed for population identification. The dCAPS marker information is shown in Table 3.

[0093] Table 3dCAPS Marking Information

[0094]

[0095] The genotypes of 1238 F2 recessive individuals of the open keel flap mutant ofc and the closed keel flap mutant LY from Example 1 were identified. The specific identification method is as follows:

[0096] The genomic DNA of the soybean to be identified was used as a template for PCR amplification to obtain the amplification product;

[0097] The amplification products were digested with AluⅠ enzyme, and the digested products were analyzed by electrophoresis.

[0098] If there is only one 282bp electrophoresis band, the genotype of the site corresponding to the molecular marker is CC, which is characterized by open keel flap;

[0099] If there is only one 257bp electrophoretic band, the genotype of the site corresponding to the molecular marker is AA, which is characterized by keel flap closure.

[0100] If there are two electrophoretic bands of 282bp and 257bp respectively, then the genotype of the site corresponding to the molecular marker is AC, which is characterized by keel flap closure.

[0101] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

[0102] The PCR amplification reaction system was as follows: 2 μL template (100 ng / μL), 0.6 μL each of upstream and downstream primers (as shown in Table 3), and 10×EasyTaq Buffer (without Mg). 2+ 2 μL of dNTPs (2.5 mM), 2 μL of Easy Taq polymerase (all-gold, AP111), and 12.6 μL of ddH2O.

[0103] The method for non-denaturing polyacrylamide gel electrophoresis is as follows:

[0104] Add 4 μL of 10× Loading Buffer to the enzyme digestion product, mix well, and then take 1.5 μL of sample for detection using non-denaturing polyacrylamide gel electrophoresis (Non-9PAGE). Plate preparation: Clean the glass plates and concave plates and place them upright on a horizontal table. Wipe them with 70% ethanol to ensure no gel residue remains. Place the two plates together, with the concave plate under the plate, leaving the cavity for gel filling. Secure the sides with clips to prevent leakage. Gel pouring: Prepare 30 mL of the pre-mixed gel, add 1% (v / v) ammonium persulfate (APS) and 1‰ (v / v) TMED, mix horizontally, and gently pour the gel between the two plates. Insert a clean comb, with the teeth extending approximately 1.0 cm into the gel. Allow to solidify at room temperature for 30 min–1 h, observing frequently to prevent gel shrinkage and deformation. Plate Preparation: Remove the clamps from the solidified gel and rinse thoroughly (to remove residual gel). Gently pull out the comb, ensuring the wells are intact and free of residual gel. Place the two plates upright, facing each other, and secure them to the electrophoresis tanks with clamps, ensuring no leakage of buffer solution. Add 0.5×TBE buffer to each electrophoresis tank, ensuring the lower tank covers the card slots and the upper tank covers the wells. Spotting: Clean the wells with a syringe, removing air bubbles and gel fragments. Using a micropipette, inject 1.0–1.5 mL of non-denaturing PCR product (with a small amount of bromophenol blue indicator added) into each well. Inject D2000 DNA markers into both ends of each well as band markers. Electrophoresis: Run at 250V for 30–40 minutes, observing the bromophenol blue indicator bands continuously. The running time depends on the band size. Silver staining: After electrophoresis, remove the clamps from the electrophoresis tank, detach the gel plate, and pry open the flat and concave plates. Remove the gel and gently place it into a prepared 1% AgNO3 aqueous solution for silver staining. Gently shake to ensure complete contact of the gel with the silver staining solution. After 3-5 minutes, remove the gel and rinse with deionized water for 30 seconds. Development: Place the gel in developing solution (1L deionized water, with 1.5g NaOH and 5-10mL formaldehyde mixed well). Observe continuously until the bands on the gel are clear. Then, transfer the gel to deionized water to wash away the developing solution. Sealing: Clean the work surface, lay a layer of plastic wrap, sprinkle with a little water, place the developed gel flat on the plastic wrap, sprinkle with a little more water, and seal with plastic wrap. Use a plastic sheet to filter out excess water and air from the plastic wrap. Place the gel horizontally in a fume hood for a period of time to remove formaldehyde.

[0105] The genotypes of all 1238 open keel petal plants were found to be identical. Figure 8 (Develop identification photographs for 83 individual dCAPS markers).

[0106] Genotyping of domestic and international planting resources was conducted using the developed dCAPS markers (identification method as above). 39 keel-valve closure genotypes were found to be identical to the wild-type zp661, validating the efficiency of dCAPS marker development in this study and concluding that dCAPS marker development was successful. Figure 7 (Photographs for the development and identification of 45 varieties using the dCAPS marker).

[0107] Table 4. Opening characteristics of keel petals in 45 known domestic and international soybean varieties.

[0108]

[0109]

[0110] Note that items 3 to 47 in Table 4 were provided by the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and their phenotypic identification indicates they are keel-petal closure materials.

[0111] In summary, the molecular markers provided by this invention can distinguish between soybeans with open keel petals and those with closed keel petals, providing technical support for creating materials with open petals and improving the cross-pollination rate of soybeans.

[0112] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a primer pair in one or more of the following aspects, said primer pair consisting of an upstream primer and a downstream primer, the nucleotide sequence of said upstream primer being shown in SEQ ID NO.3, and the nucleotide sequence of said downstream primer being shown in SEQ ID NO.4, characterized in that, include: 1) Prepare soybean products for identification or screening of open keel lobes; 2) Identify or screen soybeans with open keel lobes; The method for identifying or screening open-lobed soybeans includes: using the genomic DNA of the soybean to be identified as a template, performing PCR amplification using the primer pair to obtain the amplification product; The keel lobe trait of soybean was determined based on the genotype at the corresponding site of the molecular marker in the amplification product. The molecular marker was defined as an A>C mutation at position 1151 bp of the gene's CDS sequence, as shown in SEQ ID NO.2, and is detailed below: If the genotype at the corresponding locus is AA or AC, then the soybean to be identified is a closed keel lobe. If the genotype at the corresponding locus is CC, then the soybean to be identified is an open keel flap.

2. A method for identifying or screening soybeans with open keel lobes, characterized in that, Includes the following steps: PCR amplification was performed using the genomic DNA of the soybean to be identified as a template to obtain the amplification product; The primer pair used in the PCR amplification consists of an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

4. The keel lobe trait of soybean was determined based on the genotype at the corresponding site of the molecular marker in the amplification product. The molecular marker was defined as an A>C mutation at position 1151 bp of the gene's CDS sequence, as shown in SEQ ID NO.2, and is detailed below: If the genotype at the corresponding locus is AA or AC, then the soybean to be identified is a closed keel lobe. If the genotype at the corresponding locus is CC, then the soybean to be identified is an open keel flap.

3. The method according to claim 2, characterized in that, The methods for determining the genotype include sequencing or electrophoresis; When the determination method is electrophoresis, it includes: using Alu I. The amplification product was digested with enzyme, and the digested product was analyzed by electrophoresis; If there is only one 282bp electrophoresis band, then the genotype of the site corresponding to the molecular marker is CC; If there is only one 259bp electrophoretic band, or only two 259bp and 23bp electrophoretic bands respectively, then the genotype of the site corresponding to the molecular marker is AA; If there are two electrophoretic bands of 282bp and 259bp respectively, or three electrophoretic bands of 282bp, 259bp and 23bp respectively, then the genotype of the site corresponding to the molecular marker is AC.

4. The method according to claim 2, characterized in that, The PCR amplification reaction program includes: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

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