Molecular marker primer related to flowering period of mung bean and its application and identification method

Abstract

The application discloses a molecular marker primer related to flowering period of mung bean, and an application and identification method thereof. A major QTL qDF3 of the flowering period of the mung bean is fine-mapped to an interval of 69.5 kb between markers R3-36 and R3-19 on a chromosome 3 of a mung bean genome, and a physical position is 8604.6 kb-8674.1 kb. A gene coding PHYA in the positioning interval is an optimal candidate gene of the qDF3. Meanwhile, a functional marker R3-HM is developed according to nucleotide sequence differences of the candidate gene in parents, is located at 8626 kb on the chromosome 3, and is used for phenotype identification of the flowering period of mung bean resources. Phenotype and genotype show extremely significant correlation, and a correct rate of the genotype for predicting the flowering period phenotype can reach 93.0 %. The application associates the phenotypic traits with the stable molecular marker, avoids the restrictive influence of environment and artificial factors on the phenotypic identification, and makes the result more stable and accurate.

Description

Technical Field

[0001] This invention relates to a molecular marker primer and its application and method in the field of molecular biology, and particularly to a molecular marker primer related to the flowering period of mung beans, the application of the primer, and a method for identifying the flowering period of mung beans. Background Technology

[0002] As a legume, mung beans have nitrogen-fixing capabilities, which can enrich and improve soil. Therefore, mung beans are often rotated with rice and wheat, offering advantages such as reduced nitrogen fertilizer application, less damage from diseases and pests, increased crop yield, and enhanced economic benefits. Mung beans are characterized by their preference for warm temperatures, tolerance to poor soil, shade tolerance, short growing season, long suitable sowing period, and high economic benefits, playing a vital role in high-yield, high-quality, and high-efficiency agricultural production. Mung beans are rich in nutrients, with a protein content as high as 19.5%–33.1%, higher than that of cereal crops such as rice, wheat, and corn. They are a high-protein, medium-starch, and low-fat food. Furthermore, mung beans are rich in various minerals, vitamins, and active substances, possessing detoxifying, antibacterial, anti-allergic, lipid-lowering, blood pressure-lowering, anti-tumor, and cancer-preventing effects, making them a dual-purpose crop for both food and medicine. Mung beans can be processed into various forms of food such as powder, soup, porridge, and noodles, and are widely popular. Their straw can also be processed into animal feed.

[0003] The flowering period of mung beans is closely related to traits such as varietal adaptability and yield. Identification of mung bean flowering period often relies on direct observation during field planting, a method easily limited by the planting season. Furthermore, the flowering period trait is easily affected by external environmental factors such as water, fertilizer, temperature, and light, thus impacting the accuracy of the results. This also poses certain difficulties for mung bean introduction. Secondly, there are few QTL studies related to mung bean flowering period, and reports on molecular markers related to genes regulating flowering period are scarce; there are no reports on marker-based identification of flowering period. Therefore, a simple method for predicting and identifying the flowering period trait of mung beans is needed. Summary of the Invention

[0004] To address the limitations of the growing season on mung bean flowering time identification and reduce the impact of external environmental and human factors on this identification, and to address the current situation of weak basic research on mung bean flowering time regulatory genes and the lack of relevant molecular markers for marker-assisted breeding, this invention provides a molecular marker primer related to mung bean flowering time, the application of the primer, and a method for identifying mung bean flowering time. This invention develops a functional marker based on high-resolution melting analysis (HRM) technology and applies this marker to the prediction and identification of flowering time in mung bean germplasm resources. This marker can be used for marker-assisted selection, enabling precise screening of mung bean flowering time in early generations of breeding, reducing breeding workload and improving selection efficiency.

[0005] This invention is achieved using the following technical solution: a molecular marker primer related to the flowering period of mung beans, wherein the molecular marker of the primer is the functional marker R3-HM of the major QTL qDF3 candidate gene affecting the flowering period of mung beans. The functional marker R3-HM is located at a physical position of 8626 kb on chromosome 3 of the mung bean genome. The forward primer sequence of the functional marker R3-HM is shown in Seq NO.3 of the sequence listing, and the reverse primer sequence is shown in Seq NO.4 of the sequence listing. The major QTL qDF3 is located in the 69.5 kb interval between markers R3-36 and R3-19 on chromosome 3 of the mung bean genome, with a physical position of 8604.6 kb to 8674.1 kb.

[0006] As a further improvement to the above scheme, a genetic linkage map was constructed using the F6 population obtained by crossing Sulv 16-10 and Weilv 11 to preliminarily locate the major QTL qDF3 on chromosome 3 between markers R3-15 and R3-19.

[0007] Furthermore, by utilizing four Fs that exchange between R3-15 and R3-19... 4:5 Fine-tuning of qDF3 was performed using family lineages and two BC4F2 groups.

[0008] As a further improvement to the above scheme, the gene encoding phytochrome A within the localization region is the optimal candidate gene for qDF3. The CDS coding sequence of this gene is shown in Seq ID NO.1, and the amino acid sequence is shown in Seq ID NO.2.

[0009] This invention also provides the application of molecular marker primers related to the flowering period of mung beans in the identification of the flowering period of mung bean resources. The molecular markers are used to predict or identify the flowering period of mung bean germplasm resources, and the genotype and phenotype of mung bean resources show good consistency: if the amplification product of marker R3-HM on mung bean resources is consistent with Weilv 11, then the mung bean resources have an earlier flowering period phenotype; if the amplification product of marker R3-HM on mung bean resources is consistent with Sulv 16-10, then the mung bean resources have a later flowering period phenotype.

[0010] This invention also provides the application of molecular marker primers related to the flowering period of mung beans in screening for the flowering period of mung beans in the early generations of breeding, wherein the materials are genotyped in the early generations of breeding to screen for materials with the desired flowering period phenotype.

[0011] This invention also provides a method for identifying the flowering period of mung beans, which includes the following steps:

[0012] A polymorphic molecular marker was developed within the mapping interval of the major QTL qDF3 affecting the flowering period of mung bean: the functional marker R3-HM, located at a physical position of 8626 kb on chromosome 3 of the mung bean genome. The forward primer for the functional marker R3-HM is shown in Seq NO.3, and the reverse primer is shown in Seq NO.4. The major QTL qDF3 was located within a 69.5 kb interval on chromosome 3 of the mung bean genome between markers R3-36 and R3-19, with a physical position of 8604.6 kb to 8674.1 kb.

[0013] The flowering period of mung bean resources was identified using functional markers.

[0014] As a further improvement to the above scheme, a major QTL qDF3 regulating the flowering period of mung beans was identified using the F6 population obtained by crossing the late-flowering variety Sulv 16-10 and the early-flowering variety Weilv 11.

[0015] Further improvements were made using the F1 generation obtained from the cross between Sulv 16-10 and Weilv 11. 4:5 The family lineage and BC4F2 group were used to finely locate the primary effect QTLqDF3.

[0016] As a further improvement to the above scheme, if the amplification product of the R3-HM marker for mung bean resources is consistent with that of Weilv 11 (86bp), then the mung bean resources have an earlier flowering phenotype; if the amplification product of the R3-HM marker for mung bean resources is consistent with that of Sulv 16-10 (88bp), then the mung bean resources have a later flowering phenotype.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) This invention identified a major QTL qDF3 that regulates the flowering period of mung beans, completed the fine mapping of this major QTL qDF3 and the cloning of candidate genes, and developed functional markers for this gene. This invention associates the flowering period trait of mung beans with stable molecular markers, and the analysis process can be carried out indoors at any time. This not only avoids the disadvantages of traditional identification methods that require time-consuming and laborious field planting, but also solves the problem of the influence of the flowering period identification being limited by the mung bean growing season. In addition, it reduces the interference of external environmental and human factors on the results, making the results more stable, accurate, and highly reproducible.

[0019] (2) The amplification product of the gene functional marker R3-HM developed in this invention can be analyzed using high-resolution melting analysis (HRM). Compared to conventional PCR, which requires electrophoresis or sequencing after the PCR reaction, HRM technology has many advantages, including high throughput, high sensitivity, good specificity, good reproducibility, ease of operation, and low cost. Therefore, this marker can be used for high-throughput and more convenient genotyping of resources when used for marker-assisted selection. Precise screening during the flowering period of mung beans in early generations of breeding reduces the workload of breeding and improves selection efficiency.

[0020] (3) The molecular marker developed in this invention can accurately predict the flowering period of mung bean resources. If the amplification product of marker R3-HM on mung bean resources is consistent with Weilv 11 (86bp), then the mung bean resources have an earlier flowering phenotype; if the amplification product of marker R3-HM on mung bean resources is consistent with Sulv 16-10 (88bp), then the mung bean resources have a later flowering phenotype. Validation using 86 mung bean resources with different flowering period phenotypes showed that the accuracy rate of marker identification was as high as 93.0%. Therefore, this marker can be used to accurately predict or identify the flowering period of mung bean resources. Attached Figure Description

[0021] Figure 1 This is a histogram showing the frequency distribution of flowering periods in the F6 population of this invention.

[0022] Figure 2 This is the peak value diagram of the main effect QTL qDF3 LOD during the flowering period obtained by the present invention using F6 population analysis.

[0023] Figure 3 For the present invention to utilize F 4:5 A schematic diagram of the fine mapping of qDF3 in families and BC4F2 populations; the black box represents the Sulv16-10 genotype, the gray box represents the heterozygous genotype, and the white box represents the Weilv11 genotype.

[0024] Figure 4 This is a schematic diagram illustrating how the deletion of two bases in Weilv11 in qDF3 of the present invention causes premature termination of translation.

[0025] Figure 5 This is a melting curve obtained by amplifying mung bean resources using the R3-HM label in this invention.

[0026] Figure 6 This is a schematic diagram illustrating the flowering period analysis of two groups of mung bean genotypes. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] Example 1

[0029] This embodiment provides a major-effect QTL related to the flowering period of mung beans. This QTL is located on chromosome 3 between markers R3-15 and R3-19, with a LOD value of 74.2, an additive effect value of 12.2, and a contribution rate of 83.47%. The allele from Weilv 11 can shorten the flowering period. The determination of the major-effect QTL will be described below.

[0030] First, a segregation population was constructed during the flowering period of mung beans.

[0031] Early-flowering, purple-stemmed mung bean variety Weilv 11 was selected as the male parent, and late-flowering, green-stemmed mung bean variety Sulv 16-10 was selected as the female parent to prepare a hybrid combination. During hybridization, the stamens of the female parent were removed after 4 PM the day before hybridization, and pollination was performed the following morning by applying pollen from the male parent to the stigma of the female parent. After the hybrid pods matured, they were harvested and planted in the field. The F1 generation, with purple young stems, was the true hybrid. Seeds from the true F1 plants were harvested to obtain the F2 generation. The F2 generation was propagated using the single-seed method until the F5 generation. After harvesting individual F5 plants, they were planted in rows to obtain the F6 generation, which served as the QTL mapping population.

[0032] Secondly, the flowering phenotype was investigated in the F6 segregating population.

[0033] From the F6 generation population, 180 phenotypically stable and consistent plant rows were selected for constructing a genetic linkage map. The flowering period of each plant row was investigated, and the results are as follows: Figure 1 As shown, the flowering periods of the parents, Sulv 16-10 and Weilv 11, differed significantly, reaching a highly significant level. The flowering period showed continuous variation in the F6 population, but did not conform to a normal distribution; the phenotypic data were shifted towards both ends. These results indicate that the flowering period trait of mung beans is a quantitative trait regulated by multiple genes, and there may be major-effect QTLs with significant genetic effects, which can be analyzed using QTL mapping.

[0034] Next, a genetic linkage map was constructed and QTL analysis was performed.

[0035] Fresh leaves from 180 mung bean plant rows were selected, and genomic DNA was extracted from the mung bean leaves using a plant genomic DNA extraction kit (catalog number: DP305-03), following the kit's instructions. A self-developed molecular marker with good polymorphism was used to perform PCR on the DNA from the 180 plant rows to obtain genotype data for constructing a genetic linkage map. The PCR reaction system consisted of 10 μL of 10 μmol / L genomic DNA, 0.5 μL of 10 μmol / L forward primer, 0.5 μL of 10 μmol / L reverse primer, 5 μL of 2×T5 PCR Mix (Qingke Biotechnology), and 3 μL of ddH2O. The PCR amplification parameters were set as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 10 s, annealing (55–60℃) for 10 s, 72℃ extension for 10 s, 35 cycles; and a final extension at 72℃ for 10 min, followed by storage at 8℃. After PCR, electrophoresis was performed using 8% polyacrylamide. Following electrophoresis, silver nitrate staining was used to analyze band patterns. If the band pattern of a single-plant DNA amplified with markers matched that of Sulv 16-10, it was recorded as 2; if it matched that of Weilv 11, it was recorded as 0; heterozygotes were recorded as 1. The Map function of QTL IciMapping V4.10 software was used to construct a genetic linkage map. The LOD (likelihood of odds) value was set to 4.0 to complete linkage group division. The Order command was used to sort the markers, the Rippling command to adjust the marker order, and the Outputting command to output the results. Finally, the Map command was used to construct the map. The BIP function of QTL IciMapping V4.10 software was used for QTL localization, employing the complete interval addition / dominance (ICIM-ADD) method. The Pin was set to 0.001, the step size to 1 cM, and the LOD threshold to 2.5 to complete QTL localization and effect size estimation. QTL analysis revealed a major QTL regulating the flowering period of mung beans between markers R3-15 and R3-19 on chromosome 3. Figure 2 The QTL, located at 8504.9 kb–8674.1 kb, is named qDF3. This QTL has a LOD value of 74.2, an additive effect value of 12.2, and a phenotypic variation explained of 81.47%. Late flowering is dominant over early flowering, and the allele from Weilv 11 can shorten the flowering period.

[0036] Example 2

[0037] This embodiment utilizes F 4:5Fine mapping of the major QTL qDF3 from Example 1 was performed on segregating families and the BC4F2 population, narrowing the mapping interval of qDF3 to a 69.5 kb region between chromosome 3 markers R3-36 and R3-19, with a physical location of 8604.6 kb to 8674.1 kb. This mapping interval contained six candidate genes, among which the gene encoding phytochrome A (PHYA) was the optimal candidate gene for qDF3. Based on the nucleotide sequence differences of this gene between parents, the functional marker R3-HM, a molecular marker primer associated with the flowering period of mung beans, was developed. This example, based on Example 1, completed the fine mapping and candidate gene analysis of the major QTL qDF3 for flowering period and developed the functional marker R3-HM.

[0038] To locate qDF3 within a smaller genomic region, we utilized four F-terms that exchange between R3-15 and R3-19. 4:5 Fine localization of qDF3 was performed using family pedigrees (numbered 400, 101, 125, and 11), with the following results: Figure 3 As shown in Figure A, fine mapping narrowed qDF3 down to a 69.5 kb interval between R3-36 and R3-19, with a physical location of 8604.6 kb to 8674.1 kb, containing six candidate genes. Considering the potential impact of genetic background on F... 4:5 The phenotype of the family line has an influence, so we constructed two high-generation backcross populations with Weilv 11 as the genetic background. The specific method is as follows: using Weilv 11 as the backcross parent, we selected... Figure 3 Plants with genotypes 101-W and 125-S in group A were backcrossed. Genotype segregation generations were selected using markers R3-15, R3-30, and R3-19 to obtain two BC4F1 populations with relatively consistent genetic backgrounds. The genotypes are shown in Table 1.

[0039] Table 1. Genotypes of BC4F1 families

[0040]

[0041] Note: BB indicates the homozygous Weilv 11 genotype; A / B indicates the heterozygous type.

[0042] Fine gene mapping was performed on the BC4F2 population generated from these two BC4F1 populations, and the results are as follows: Figure 3As shown in B. The localization results are consistent with previous results. Analysis of candidate genes within the localization interval revealed a gene highly homologous to phytochrome A (PHYA) in soybean, presumably related to flowering period regulation in mung beans, with gene number Vradi03g07170 (symbol in https: / / www.ebi.ac.uk / ). Sequencing of the gene revealed its CDS nucleotide sequence, as shown in Seq ID NO.1, with a full length of 3393 bp. The amino acid sequence of the protein encoded by this gene, as shown in Seq ID NO.2, contains 1130 amino acids. In Weilv 11, the first exon of this gene contains a deletion of two bases (TA). Figure 4 A), which leads to translation termination in exon 1 ( Figure 4 B) This deficiency affects protein function. To address these two base deletions, a molecular marker, R3-HM, based on high-resolution melting analysis (HRM) technology was designed. Its forward primer is shown in Seq NO.3 (5'-3'), and its reverse primer is shown in Seq NO.4 (5'-3'), located at 8626 kb on chromosome 3. Compared to conventional PCR, which requires electrophoresis or sequencing after the PCR reaction, HRM technology offers numerous advantages, including high throughput, high sensitivity, good specificity, good reproducibility, ease of operation, and low cost. Therefore, this functional marker can be used for high-throughput and convenient genotyping of mung bean resources.

[0043] Example 3

[0044] This embodiment provides the application of a molecular marker primer related to the flowering period of mung beans in the identification of the flowering period of mung bean resources. The molecular marker primer is the functional marker R3-HM of the major QTL candidate gene qDF3 for flowering period of mung beans in Example 2. If the mung bean resource has the allele genotype Sulv 16-10 at R3-HM, that is, the amplified product of the marker is 88bp, then the Tm value of the PCR reaction is consistent with that of Sulv 16-10, and the melting curve in HRM program analysis tends to be similar to that of Sulv 16-10, then the resource has a later flowering phenotype. If the mung bean has the same genotype as Weilv 11 at R3-HM, that is, the amplified product of the marker is 86bp, then the Tm value of the PCR reaction is consistent with that of Weilv 11, and the melting curve in HRM program analysis tends to be similar to that of Weilv 11, then the resource has an earlier flowering period.

[0045] To verify the effectiveness of the functional marker R3-HM in identifying the flowering time of mung beans, we selected 86 mung bean resources with different flowering times for genotypic and phenotypic consistency verification. Resource information is shown in Table 2. Resource information and identification results are also shown in Table 2. Figure 5PCR amplification and high-resolution melting curve analysis were performed on a Roche Light Cycler 96 real-time PCR instrument. Reagents were obtained from Roche (Roche, USA) Light Cycler 96. 480 High Resolution Melting Master Kit (REF: 04909631001; Version 05). The reaction system consisted of 20 μL containing 1.0 μL of genomic DNA (50 ng / μL), 0.5 μL each of forward and reverse primers (10 μmol / L), 10 μL of 2×ResoLight Mix, 1.6 μL of 2.0 mmol / L MgCl2, and 6.4 μL of dH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 10 min, followed by 45 cycles of denaturation at 95℃ for 10 s, annealing at 61℃ for 15 s, and extension at 72℃ for 15 s. The HRM detection program for the amplified products was as follows: 98℃ for 1 min, 40℃ for 1 min, 62℃ for 1 s. During the temperature rise from 62℃ to 95℃, the signal was continuously acquired at a frequency of 20 times / ℃ to collect fluorescence information. Finally, the temperature was lowered to 40℃ for high-resolution melting curve analysis to obtain genotype data for each resource. Table 2 shows that the genotypes and phenotypes of the 86 mung bean accessions at the qDF3 locus were consistent with the overall phenotype, indicating that the qDF3 locus plays a dominant role in regulating the flowering period of mung beans. Correlation analysis showed that the correlation coefficient between the genotype at the R3-HM marker and the number of days to maturity in mung beans was 0.65, and the correlation was significant at the 0.01 level. Figure 5 It can be seen that the melting curves of the tested resources clearly cluster into two types: Weilv 11 and Sulv 16-10. This indicates that the marker R3-HM can effectively distinguish between the two genotypes of mung bean resources, thus classifying the tested resources into two categories based on genotype. Of the 86 mung bean resources, 33 were homozygous for the Weilv 11 genotype at R3-HM, with only one resource exhibiting a later flowering period; the remaining 32 resources all showed an earlier flowering phenotype (flowering period ≤ 40 days), with a genotypic consistency rate of 97.0%. Of the 53 resources, 48 ​​were homozygous for the Sulv 16-10 genotype at R3-HM, exhibiting a late flowering (flowering period ≥ 40 days), with a genotypic consistency rate of 90.6%. Grouping and statistically analyzing the flowering period of the two types of mung beans revealed that the average flowering period of the 33 homozygous Weilv 11 mung bean resources was 34.3 days, while the average flowering period of the 53 homozygous Sulv 16-10 mung bean resources was 49.6 days, and the difference in flowering period between the two groups was highly significant. Figure 6 These results indicate that the R3-HM marker can effectively identify the flowering phenotype of mung beans, with an overall accuracy rate of 93.0%. In actual production, varieties / strains exhibiting the Weilv 11 type at R3-HM should be prioritized to obtain the early flowering trait.

[0046] Table 2. Flowering time and genotype of 86 mung bean resources

[0047]

[0048]

[0049]

[0050] In summary, the major QTL qDF3 for flowering time was precisely mapped to a 69.5 kb region on chromosome 3 between markers R3-36 and R3-19, with a physical location of 8604.6 kb to 8674.1 kb. The gene encoding phytochrome A (PHYA) within this region is the optimal candidate gene for qDF3. Based on the nucleotide sequence differences of the candidate gene between the parents, the functional marker R3-HM was developed, located at 8626 kb on chromosome 3. The amplification product of this marker can be analyzed using high-resolution melting curve analysis. Using the marker to identify the flowering time of mung bean resources, a highly significant correlation was observed between genotype and phenotype, with a genotype-to-phenotype prediction accuracy of 93.0%, indicating that this marker can effectively identify the flowering time phenotype of mung bean resources. This invention associates the flowering time trait of mung bean with a stable molecular marker, making the results more stable and accurate. The R3-HM marker, when used for marker-assisted selection, can be used for high-throughput and more convenient genotyping of resources. Precise screening of mung bean flowering period in early generations of breeding reduces the workload of breeding and improves selection efficiency.

[0051] Example 4

[0052] This embodiment discloses a method for identifying the flowering period of mung beans. This is a specific application of the molecular marker primers related to the flowering period of mung beans in Example 3. The method for identifying the flowering period of mung beans includes the following steps:

[0053] (1) A polymorphic molecular marker was developed within the mapping interval of the major QTL qDF3 that affects the flowering period of mung bean: the functional marker R3-HM, which is located at a physical position of 8626 kb on chromosome 3 of the mung bean genome. The forward primer of the functional marker R3-HM is shown in Seq NO.3 of the sequence listing, and the reverse primer is shown in Seq NO.4 of the sequence listing. The major QTL qDF3 was located in the 69.5 kb interval between markers R3-36 and R3-19 on chromosome 3 of the mung bean genome, with a physical position of 8604.6 kb to 8674.1 kb.

[0054] (2) Use functional markers to identify the flowering period of mung bean resources.

[0055] In specific implementation, this invention can be carried out using the following steps and corresponding verification:

[0056] (1) Construction of segregating population during flowering period of mung bean: early flowering and late flowering mung bean varieties were selected for hybridization to construct RIL(F6) segregating population;

[0057] (2) Investigation of flowering phenotype in the F6 segregating population;

[0058] (3) Construction of genetic function map and identification of major QTLs at flowering time;

[0059] (4) Develop polymorphic molecular markers within the QTL localization region;

[0060] (5) Fine mapping of the major QTL qDF3 during flowering: using F1 and F2 gene sequences that segregate phenotype and genotype. 4:5 Fine-grained QTL localization using family pedigrees and the BC4F2 population;

[0061] (6) Develop functional marker R3-HM for major QTLqDF candidate genes during flowering period;

[0062] (7) Identify the flowering period of mung bean resources using functional R3-HM markers.

[0063] These seven detailed steps have already been described in detail in Examples 1-3, so they will not be repeated in Example 4.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a detection primer for a molecular marker related to the flowering period of mung beans in the identification of the flowering period of mung bean resources, characterized in that, The primers include forward primers and reverse primers. The forward primer is shown in SEQ ID NO: 3, and the reverse primer is shown in SEQ ID NO:

4. The primers are used to amplify the genomic DNA of the mung bean resource to be tested. If the amplification product is consistent with Weilv 11 and has a length of 86 bp, then the mung bean resource has an earlier flowering phenotype. If the amplification product is consistent with Sulv 16-10 and has a length of 88 bp, then the mung bean resource has a later flowering phenotype.

2. The application of a detection primer for a molecular marker associated with the flowering period of mung beans in screening for the flowering period of mung beans in early generations of breeding, characterized in that... Genotyping of materials was performed in early generations of breeding to screen for materials with the desired flowering period phenotype. The primers included forward primers and reverse primers. The forward primer is shown in SEQ ID NO: 3, and the reverse primer is shown in SEQ ID NO:

4. The genomic DNA of the mung bean resource to be tested was amplified using the primers. If the amplification product was consistent with Weilv 11 and had a length of 86 bp, the mung bean resource had an earlier flowering period phenotype. If the amplification product was consistent with Sulv 16-10 and had a length of 88 bp, the mung bean resource had a later flowering period phenotype.

3. A method for identifying the flowering period of mung beans, characterized in that, It includes the following steps: The genomic DNA of the mung bean resource to be tested was amplified using primers. The flowering period of the mung bean was determined based on the size of the amplification product. The primers included forward primers and reverse primers. The forward primer is shown in SEQ ID NO: 3, and the reverse primer is shown in SEQ ID NO:

4. If the amplification product is consistent with Weilv 11 and has a length of 86 bp, the mung bean resource has an earlier flowering period phenotype. If the amplification product is consistent with Sulv 16-10 and has a length of 88 bp, the mung bean resource has a later flowering period phenotype.

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