A snp molecular marker related to low fertility of mung bean bud dehiscence, primer set, kit and application

Abstract

This invention discloses a SNP molecular marker, primer set, kit, and application associated with the low fertility trait of flower bud splitting in mung beans, belonging to the field of plant molecular marker screening and molecular genetic breeding technology. The molecular marker is located at position 43652302 on chromosome 4 of the mung bean genome, on the EVM0022977 gene; the SNP molecular marker is the nucleotide sequence shown in SEQ ID NO.1, where the 120th base is either T or A. The SNP molecular marker and primer set provided by this invention are specific, accurate, reliable, and easy to operate, enabling rapid identification of mung bean flower bud splitting low fertility mutants, meeting the needs of assisted mung bean molecular breeding and shortening the breeding cycle.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular marker screening and molecular genetic breeding technology, and in particular to an SNP molecular marker, primer set, kit, and application associated with the low fertility trait of mung bean flower bud splitting. Background Technology

[0002] Mung bean is an important edible legume crop in my country. As a typical cleistogamous plant, its natural cross-pollination rate is only 1.68%, which limits the utilization of heterosis to some extent. Therefore, creating new germplasm suitable for hybridization and identifying key genes regulating floral organ development are of great significance for elucidating the molecular mechanisms of floral organ development and improving breeding efficiency. Previously, our team obtained a mutant with premature flower bud dehiscence and reduced fertility by EMS mutagenesis of mung bean 'Weilv 11' (WL11), named the Flower Bud Dehiscence Low-fertility Mutant (WL11). Dehiscent Bud and Low Fertility , dblf Its phenotype during the vegetative growth phase is similar to that of WL11, but after entering the reproductive growth phase, dblf The mutant exhibited premature bud splitting, pollen adhesion, and short pod formation, with a significantly lower number of pods per plant compared to WL11. dblf The trait of flower bud splitting can be stably inherited, so it is expected to be applied to the mung bean hybrid seed production system.

[0003] Phenotypic identification of mung bean bud-cracking low fertility mutants requires field investigation during the flowering period, supplemented by pollen staining tests, which is time-consuming and labor-intensive. Therefore, developing molecular markers related to the bud-cracking low fertility trait has the advantages of accuracy, economy, and speed. Functional molecular markers are molecular markers developed based on polymorphic sequences within functional genes closely related to phenotypic traits. Their advantages include accurate and reliable identification of target genes, accurate reflection of genetic variation in functional alleles, and more reliable genetic effect values. SNPs are polymorphisms arising from single nucleotide variants, which are difficult to distinguish using conventional PCR and gel electrophoresis techniques based on differences in length. Summary of the Invention

[0004] The purpose of this invention is to provide an SNP molecular marker, primer set, kit, and application for the association of low fertility trait in mung bean flower bud splitting, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is an SNP molecular marker associated with the low fertility trait of mung bean flower bud splitting. The molecular marker is located at position 43652302 on chromosome 4 of the mung bean genome, on the EVM0022977 gene. The SNP molecular marker is the nucleotide sequence shown in SEQ ID NO.1, where the 120th base is either T or A.

[0006] The second technical solution of the present invention is a primer set for specific detection of the molecular marker, including primers as shown in SEQ ID NO.2-4.

[0007] The third technical solution of the present invention is a kit for specifically detecting the molecular marker, comprising the primer set.

[0008] The fourth technical solution of the present invention is the application of the molecular marker, the primer set, or the kit in identifying or assisting in the identification of the low fertility trait of mung bean flower bud splitting.

[0009] The fifth technical solution of the present invention is a method for identifying the low fertility trait of mung bean flower bud dehiscence using the molecular marker, the primer set, or the kit, comprising the following steps: Using the genomic DNA of the mung bean sample to be tested as a template, PCR amplification is performed using the primer set or the kit, and the genotype of the corresponding single nucleotide polymorphism site of the SNP molecular marker in the amplification product is detected. The phenotype of the flower organs is determined based on the genotype.

[0010] The sixth technical solution of the present invention is the application of the molecular marker, the primer set, or the kit in breeding for the low fertility trait of mung bean flower bud splitting.

[0011] Based on the above technical solution, the present invention has the following technical effects: The SNP molecular markers and primer sets provided by this invention are specific, accurate, reliable and easy to operate, and can quickly identify low-fertility mutants of mung bean flower bud cracking, thus meeting the needs of assisting mung bean molecular breeding and shortening the breeding cycle. Attached Figure Description

[0012] Figure 1 Fine mapping of genes in mung bean flower bud splitting low fertility mutant.

[0013] Figure 2 190 copies dblf × Sulv1 F2 group (A) and 149 copies dblf VrSNP-32 molecular marker typing map of the × V2709 F3 population (B). Detailed Implementation

[0014] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0015] This invention provides an SNP molecular marker associated with the low fertility trait of mung bean flower bud splitting. The molecular marker is located at position 43652302 on chromosome 4 of the mung bean genome, on the EVM0022977 gene. The SNP molecular marker is the nucleotide sequence shown in SEQ ID NO.1, where the 120th base is either T or A.

[0016] In some specific implementations, the molecular marker is present in the genotype TT, AA, or TA.

[0017] Embodiments of the present invention also provide a primer set for specific detection of the molecular marker, including primers as shown in SEQ ID NO.2-4.

[0018] This invention also provides a kit for the specific detection of the molecular marker, including the primer set.

[0019] This invention also provides the application of the molecular marker, the primer set, or the kit in identifying or assisting in the identification of low fertility traits in mung bean flower bud dehiscence.

[0020] This invention also provides a method for identifying the low fertility trait of mung bean flower bud dehiscence using the molecular marker, the primer set, or the kit, comprising the following steps: Using the genomic DNA of the mung bean sample to be tested as a template, PCR amplification is performed using the primer set or the kit, and the genotype of the corresponding single nucleotide polymorphism site of the SNP molecular marker in the amplification product is detected. The phenotype of the flower organs is determined based on the genotype.

[0021] In some specific implementation schemes, the method for determining floral organ phenotype based on genotype is as follows: If the genotype of the SNP molecular marker is TT or TA, then the mung bean plant has a normal phenotype. If the genotype of the SNP molecular marker is AA, then the mung bean plant exhibits a phenotype of low fertility due to flower bud splitting.

[0022] In some specific implementations, the PCR amplification reaction system is as follows: 1 µL template DNA 50 ng / µL, 5 µL Hi Geno 2x Probe Mix, 0.14 µL of 10 μM primer 1, 0.14 µL of 10 μM primer 2 and 0.14 µL of 10 μM primer 3, and 3.58 µL of ddH2O; The reaction program was as follows: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 20 s, annealing and extension at 61-55℃ with a decrease of 0.6℃ per cycle for 45 s, for 10 cycles; denaturation at 95℃ for 20 s, annealing and extension at 55℃ for 45 s, for 30-34 cycles.

[0023] The embodiments of the present invention also provide the application of the molecular marker, the primer set or the kit in the breeding of mung bean flower bud cracking and low fertility traits.

[0024] Example 1 Design SNP molecular markers associated with mung bean flower bud cracking and low fertility traits Using dblf × Sulv1 (Sulu No. 1) F2 population (190 individuals) for the mung bean flower bud cracking and low fertility mutant dblf For preliminary mapping: From the dblf × Sulv1 F2 population, extreme phenotype individuals were screened, 20 F2 individuals with flower bud cracking and low fertility phenotypes and 20 F2 individuals with normal phenotypes were selected to construct Mutant-pool and Normal-pool. Sulv1 and dblf were used as two parental pools. Then, BSA pooled sequencing was used for analysis. After filtering the original sequencing data to obtain high-quality sequencing data (cleandata), association analysis was performed. For the reliable SNP sites, two calculation methods were used to analyze the SNP sites and Indel sites obtained by sequencing. The intersection of the ED value calculation method and the SNP-index analysis results was taken to obtain the SNP association region, which is located at the end of chromosome 4, with a size of approximately 2.0 Mb, and the reference genome is Sulu No. 1 (Submitted GenBank assembly: GCA_050395885.1; BioSample ID: SAMN46312187).

[0025] Initially, 12 SNP molecular markers were designed based on some genes with non-synonymous mutations within the fine mapping interval, including VrSNP-1 (43056490), VrSNP-15 (43503239), VrSNP-16 (43527712), VrSNP-18 (43531564), VrSNP-22 (43556385), VrSNP-25 (43565410), VrSNP-28 (43575731), VrSNP-31 (43610633), VrSNP-32 (43652302), VrSNP-50 (43818290), VrSNP-54 (43857655), VrSNP-56 (43945469); according to the single nucleotide differences, the KASP primer sets containing 12 SNP differences were designed using Primer5.0 software respectively, and each primer set contains 2 specific primers and 1 universal primer.

[0026] This invention utilizes the AQP™ genotyping system, also known as an allele-specific quantitative PCR genotyping system, and a real-time PCR instrument to acquire fluorescence signals.

[0027] Based on the genotyping results, the nine molecular markers VrSNP-1, VrSNP-15, VrSNP-16, VrSNP-18, VrSNP-22, VrSNP-25, VrSNP-31, VrSNP-32, and VrSNP-56 can effectively genotype the organisms. Further combining SNP molecular marker mapping with population exchange individual plant mapping, [the following data is missing from the original text]. dblf The site was located at 43531564-43945469 bp within the LG04 region. Twelve genes within this region exhibited nonsynonymous mutations. Among them, the EVM0022977 gene (LOC106760156) had a T-to-A mutation at position 321, resulting in a premature stop codon (Stopgain SNV). This caused premature termination of translation of the encoded protein. Combined with homologous gene functional analysis, this gene was identified as... dblf The key candidate gene for the trait, and the VrSNP-32 molecular marker falls on the mutant base of this gene, is a core functional molecular marker, based on the nucleotide difference at position 43652302 on chromosome 4 of mung bean. The sequence of the VrSNP-32 molecular marker, a total of 240 bp, is shown in SEQ ID NO.1, with VrSNP-32 at position 120, and the polymorphism is T / A. The genotyping results are as follows. Figure 2 As shown in A, its counterpart dblf The detection rate of floral organ mutation phenotype in the F2 population of × Sulv1 reached 100%, so it can be used to identify the low fertility trait of mung bean flower bud splitting.

[0028] VrSNP-32: SEQ ID NO.1: GGCAATTTGCAAGCGGGAGGAGGAGGAGGACCGGTTCATCATCATCATCACCATCATCACCAGCAGCAGCAGCAGCAGCATGAACATCATCATCATCATGACGGTCACGGAACTTG T[A] TCAAACGCCAAGGAAGTAGTGGAGAAAGAGCACATGTTTGACAAAGTGGTGACACCGAGCGATGTGGGGAAGCTGAACAGGCTGGTGATACCGAAGCAGCACGCGGAGAAGTACTTCCCC.

[0029] Note: The underlined position is the VrSNP-32 site.

[0030] The primer sequences for amplifying the VrSNP-32 molecular marker are as follows: Primer 1: VrSNP32-F-FAM (SEQ ID NO. 2): 5' GAAGGTGACCAAGTTCATGCTATCATGACGGTCACGGAACTTGt 3'.

[0031] Primer 2: VrSNP32-F-HEX (SEQ ID NO. 3): 5' GAAGGTCGGAGTCAACGGATTATCATGACGGTCACGGAACTTGa 3'.

[0032] Primer 3: VrSNP32-R (SEQ ID NO.4): 5' GGTATCACCAGCCTGTTCAGCTT 3'.

[0033] Table 1 dblf × Correspondence between genotypes and floral organ phenotypes of the F2 segregating population of Sulv1

[0034] Note: "--" indicates that no signal was detected by PCR.

[0035] Example 2 Application of SNP markers associated with low fertility due to flower bud dehiscence in mung bean for screening mung bean mutants use dblf Genomic DNA was extracted from 149 individual mung bean plants (see Table 2) from the F3 segregating population of × V2709, and the concentration of each DNA was determined using a spectrophotometer. Using the DNA of the mung bean variety to be tested as a template, PCR amplification was performed using the above primer set, i.e., AQPTM reaction detection.

[0036] Table 2 dblf Correspondence between genotypes and floral organ phenotypes of the F3 segregating population of × V2709

[0037] Note: "--" indicates that no signal was detected by PCR.

[0038] PCR reaction system (10µl): 1 µL template DNA (50 ng / µL), 5 µL Hi Geno 2x Probe Mix, 0.14µl each of primer 1 (10 μM), primer 2 (10 μM) and primer 3 (10 µM), and 3.58 µL ddH2O.

[0039] PCR reaction program: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 20 s, annealing and extension at 61-55℃ with a decrease of 0.6℃ per cycle for 45 s, 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing and extension for 45 s, 30-34 cycles.

[0040] After PCR amplification cycles, fluorescence values ​​were read using an Omega F SNP genotyping instrument at 35°C. The KASP genotyping system uses FAM and HEX fluorophores to distinguish between two isogenetic loci. ROX was used to correct for signal differences between wells due to reaction volume errors. The results were analyzed using Klustercaller software. In this software, FAM and HEX data were plotted on the x and y axes, respectively. Samples were clustered based on relative fluorescence values, and genotypes were further determined based on these clusters.

[0041] If only the base T is detected at the SNP locus VrSNP32 (43652302), the mung bean sample to be tested is determined to be a homozygous genotype with a normal floral organ phenotype; if only the base A is detected, the mung bean sample to be tested is determined to be a homozygous genotype with a low fertility trait of flower bud dehiscence; if both the bases T and A are detected, the mung bean sample to be tested is determined to be a heterozygous genotype with a normal floral organ phenotype.

[0042] A field survey of the flowering organ characteristics of various mung bean lines was conducted. The specific steps are as follows: Sowing will take place in mid-June 2025 at the plant experimental field located at the Jiangsu Academy of Agricultural Sciences (Nanjing, Jiangsu, 118.88°E, 32.038°N). dblf The F3 segregating population of × V2709, totaling 149 individual plants, was investigated in the field after entering the reproductive growth stage. If the flower buds showed premature splitting, it was determined to be a low-fertility trait of mung bean flower bud splitting; otherwise, it was considered a normal flower phenotype. The phenotypes of each line were collected and recorded, and compared with the corresponding genotypes for analysis.

[0043] The correspondence between T / A genotypes and floral organ phenotypes is shown in Table 1. Genotyping results are as follows: Figure 2As shown in Figure B, among 149 individual plants, 42 AA genotypes were detected, of which 40 exhibited the stunted fertility trait of flower bud dehiscence, with an accuracy rate of 95.24%. 31 TT genotypes and 72 TA genotypes were detected, of which 102 exhibited the normal floral organ phenotype, accounting for 99.03%. The remaining 4 samples did not show any phenotypes. Therefore, the overall accuracy rate of the molecular marker VrSNP-32 was 97.93%.

[0044] As shown in Table 1, the SNP markers disclosed in this invention for detecting the low fertility trait associated with flower bud splitting in mung beans are specific, accurate, reliable, and easy to operate. They can quickly identify flower bud splitting low fertility mutants, assist in molecular breeding, and shorten the breeding cycle.

[0045] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A SNP molecular marker associated with low fertility due to dehiscence of mung bean flower buds, characterized in that, The molecular marker is located at position 43652302 on chromosome 4 of the mung bean genome, on the EVM0022977 gene; The SNP molecular marker is the nucleotide sequence shown in SEQ ID NO.1, where the 120th base is either T or A.

2. The SNP molecular marker according to claim 1, characterized in that, The molecular markers are present in genotypes TT, AA, or TA.

3. A primer set for specific detection of the molecular marker described in claim 1 or 2, characterized in that, Includes primers as shown in SEQ ID NO. 2-4.

4. A kit for specific detection of the molecular marker of claim 1 or 2, characterized in that, Includes the primer set described in claim 3.

5. The application of the molecular marker of claim 1 or 2, the primer set of claim 3, or the kit of claim 4 in identifying or assisting in the identification of the low fertility trait of mung bean flower bud splitting.

6. A method for identifying the low fertility trait of mung bean flower bud dehiscence using the molecular marker of claim 1 or 2, the primer set of claim 3, or the kit of claim 4, characterized in that, Includes the following steps: Using the genomic DNA of the mung bean sample to be tested as a template, PCR amplification is performed using the primer set described in claim 3 or the kit described in claim 4. The genotype of the corresponding single nucleotide polymorphism site of the SNP molecular marker in the amplification product is detected, and the phenotype of the flower organs is determined based on the genotype.

7. The method according to claim 6, characterized in that, The method for determining floral organ phenotype based on genotype is as follows: If the genotype of the SNP molecular marker is TT or TA, then the mung bean plant has a normal phenotype. If the genotype of the SNP molecular marker is AA, then the mung bean plant exhibits a phenotype of low fertility due to flower bud splitting.

8. The method according to claim 6, characterized in that, The PCR amplification reaction system consisted of: 1 µL template DNA 50 ng / µL, 5 µL Hi Geno 2x Probe Mix, 0.14 µL of 10 μM primer 1, 0.14 µL of 10 μM primer 2 and 0.14 µL of 10 μM primer 3, and 3.58 µL of ddH2O. The reaction program was as follows: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 20 s, annealing and extension at 61-55℃ with a decrease of 0.6℃ per cycle for 45 s, for 10 cycles; denaturation at 95℃ for 20 s, annealing and extension at 55℃ for 45 s, for 30-34 cycles.

9. The application of the molecular marker of claim 1 or 2, the primer set of claim 3, or the kit of claim 4 in the breeding of mung bean flower bud splitting fertility trait.

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