KASP molecular marker related to major QTL (quantitative trait loci) of golden kidney bean water utilization rate

Abstract

The invention relates to the technical field of molecular marker development, in particular to a KASP molecular marker related to major QTL (quantitative trait loci) of golden water utilization rate of kidney beans, which comprises a first and / or second KASP molecular marker for respectively detecting specific gene region loci of the kidney beans; the method comprises the following steps: during detection, adding a novel fluorescence enhancer FluoroEnhance-X and a primer enhancer Au-PrimerEnhance modified by nanogold into a reaction system; the former enhances a fluorescence signal, and the latter improves the primer performance; in addition, the primer is further subjected to special chemical modification, the 5'end is connected with a cholesterol group, and the 3 'end is introduced with a phosphothio modified group, so that the detection effect is improved. The KASP molecular marker disclosed by the invention can be used for rapidly and accurately detecting the water utilization efficiency in a bean seedling stage; the detection cost is low, the method is not influenced by the environment, and the method can be widely applied to germplasm evaluation, filial generation screening and breeding; the specially designed reaction system and primer modification improve the detection performance, and are of great significance to cultivation of drought-resistant kidney bean varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker development, in particular to a KASP molecular marker associated with a major QTL of golden water use efficiency in kidney beans. Background Art

[0002] As a globally important legume crop, kidney beans play a crucial role in food production and dietary structure. Not only is it a primary source of protein for many regions, it is also popular among consumers for its rich nutritional profile and diverse cooking methods. However, kidney beans face numerous challenges in their growth, with drought stress being a key factor affecting their yield and quality.

[0003] Globally, primary bean production areas are widely distributed in arid or semi-arid regions, where water resources are scarce, rainfall is erratic, and droughts are frequent. According to relevant data, drought stress can cause losses in approximately 60% of global bean production, severely hindering the development of the bean industry. In this context, improving water use efficiency in beans is crucial for ensuring stable yields and enhancing quality.

[0004] The concept of "Golden Water Use Efficiency" (GHW) offers a new approach to addressing drought stress in kidney beans. Research has found that plants experience a peak in water use efficiency during specific periods, known as "Golden WUE." During this period, plants can achieve high photosynthetic production with low water transpiration, contributing up to 90% to photosynthesis per day. By screening kidney bean germplasm with excellent GHW patterns, it is hoped that the overall water use efficiency of kidney beans during the growing season can be fundamentally improved, achieving a balanced improvement in water conservation and crop yield.

[0005] However, bean breeding currently faces numerous challenges. Traditional breeding methods rely primarily on phenotypic selection, which requires extensive field cultivation and observation, measuring relevant indicators and conducting biochemical experiments to screen for varieties with high greenhouse warming (GHW). This not only results in lengthy experimental cycles but also involves complex and cumbersome procedures, susceptible to environmental interference, and resulting in low accuracy, significantly limiting the efficiency of bean breeding.

[0006] With the development of biotechnology, marker-assisted selection (MAS) technology has gradually become an important tool in crop breeding. Among them, KASP (Kompetitive Allele Specific PCR) technology has been widely used in genetic research of many crops due to its high accuracy, strong flexibility, and low cost. However, due to the late start of molecular biology research in kidney beans, our understanding of kidney bean omics and the physiological basis and molecular mechanisms of GHW traits is limited. To date, there are no reports of KASP molecular markers closely linked to kidney bean GHW traits. The lack of effective molecular markers makes it difficult to improve kidney bean varieties with high GHW using molecular marker-assisted selection, which cannot meet the urgent demand of the kidney bean industry for high-quality, drought-resistant varieties. Therefore, the development of KASP molecular markers suitable for kidney bean high GHW breeding is of great practical significance. It can provide strong technical support for kidney bean breeding and promote the sustainable development of the kidney bean industry. Summary of the Invention

[0007] (1) Technical problems solved

[0008] In view of the shortcomings of the existing technology, the present invention provides a KASP molecular marker related to the major QTL of golden water use efficiency of kidney bean.

[0009] (2) Technical solution

[0010] A KASP molecular marker associated with a major QTL for golden water use efficiency in kidney bean comprises a first KASP molecular marker and / or a second KASP molecular marker; the first KASP molecular marker specifically detects site 2397926 in the region where the Phvul.001G137925 gene of kidney bean is located, and the second KASP molecular marker specifically detects site 1794256 in the region where the Phvul.009G003100 gene of kidney bean is located; during the detection process, a reaction system containing a fluorescence enhancer FluoroEnhance-X and a nano-gold-modified primer enhancer Au-PrimerEnhance is used; FluoroEnhance-X specifically binds to KASP primers and amplification products to enhance fluorescence signal intensity; Au-PrimerEnhance binds to primers via Au-S bonds to improve primer stability and amplification efficiency; the fluorescence enhancer is 5-(4-methylphenyl)-2,2'-bipyridine-3,3'-dicarboxylic acid ethyl ester, and its structural formula is:

[0011]

[0012] Preferably, the primer is subjected to special chemical modification, wherein a cholesterol group is connected to the 5' end of the primer; and a phosphorothioate modification group is introduced to the 3' end to enhance the resistance of the primer to nucleases.

[0013] Preferably, a bioactive small molecule compound Bio-Activator-Y is further added to the reaction system, which binds to a specific sequence in the bean genomic DNA to promote the specific binding of the KASP marker to the target site at a binding ratio of 1:1. The Bio-Activator-Y is 3-(4-hydroxyphenyl)-2-acrylamido-2-methylpropionic acid, and its structural formula is:

[0014]

[0015] Preferably, the first KASP molecular marker comprises forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTAAGCGGTTTCGTCGTCGTCC-3', forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTAAGCGGTTTCGTCGTCGTCA-3' and reverse primer: 5'-CGAAATAGACGTCGCCGACGAC-3'; the second KASP molecular marker comprises forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTACTTTAAACCTAACTTAG-3', forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTACTTTAAACCTAACTTAA-3' and reverse primer: 5'-TATAAGTGGGTACAATCCTCACC-3'.

[0016] Preferably, the added concentration of the fluorescence enhancer FluoroEnhance-X is 0.05-0.15 mmol / L, which can increase the fluorescence signal intensity by 30%-50%; the added amount of the gold nanoparticle-modified primer enhancer Au-PrimerEnhance is 0.01-0.03 μg per 10 μL reaction system.

[0017] Preferably, the PCR reaction system for KASP molecular marker detection is: 5 μL of 2×KASP Master Mix, 0.14 μL of primer mixture, 2 μL of template DNA, and ddH2O to 10 μL; and 0.1-0.3 μL of hot start Taq enzyme activator is added to the reaction system.

[0018] Preferably, the PCR reaction procedure for the KASP molecular marker detection is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 61-55°C for 60 s, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 60 s, for a total of 26 cycles; after the last cycle, an extension step at 72°C for 5 min is added.

[0019] Preferably, a method for developing a KASP molecular marker associated with a major QTL for water use efficiency in kidney bean comprises the following steps:

[0020] S1: Parent selection and population construction: select high GHW bean varieties such as CP-Pv-99 and low GHW bean varieties such as CP-Pv-114 as parents, configure hybrid combinations and construct F2 populations;

[0021] S2: Phenotypic data acquisition: The F2 population was grown in a glass greenhouse equipped with the Plantarray phenotyping platform, simulating natural light with a maximum daily temperature of 35°C and a minimum daily temperature of 20°C. GHW-related parameters, such as peak area and peak value, were monitored in real time to obtain phenotypic data. High-resolution imaging technology was also used to obtain stomatal distribution and morphological data on bean leaves to assist in the analysis of GHW traits.

[0022] S3: Gene pool construction and sequencing: 30 extremely superior individuals with the largest GHW peak areas were selected from the F2 population to form a high GHW gene pool, and 30 extremely inferior individuals with the smallest GHW peak areas were selected to form a low GHW gene pool. Whole-genome resequencing was performed together with the parents.

[0023] S4: Association region screening: SNP-index analysis was used to screen chromosome regions significantly associated with GHW;

[0024] S5: SNP site identification and candidate gene targeting: Based on GWAS analysis, with -log(P) ≥ 3 as the significance threshold, SNP sites closely linked to the GHW major effect QTL were identified. Candidate genes were targeted by combining RNA-seq differentially expressed gene data.

[0025] S6: Primer design and screening: Multiple pairs of KASP primers were designed based on the target SNP sites. Molecular dynamics simulation software was used to assist in the design. Primer pairs that were stable and highly polymorphic in the parents were screened. The linkage between the marker and the GHW trait was verified through F2 population typing. The KASP primers with the closest linkage distance to the GHW major effect QTL and the highest typing efficiency were selected as the final molecular markers.

[0026] Preferably, in step S5, in addition to using RNA-seq differentially expressed gene data, proteomics data are also combined to comprehensively analyze gene expression at the transcriptional and translational levels.

[0027] Preferably, a KASP molecular marker associated with the main effect QTL of golden water use efficiency of kidney beans is used in molecular marker-assisted selection breeding of kidney beans for drought resistance, precise evaluation of kidney bean germplasm resources, and breeding of new kidney bean varieties with high water use efficiency and other excellent traits.

[0028] (3) Beneficial technical effects

[0029] Compared with the existing technology, the beneficial effects of the present invention are:

[0030] 1. The KASP molecular marker of the present invention is tightly linked to the major QTL for GHW in kidney beans, enabling rapid and accurate prediction of water use efficiency (WUE) in kidney beans early in growth, even at the seedling stage. Traditional breeding methods require a series of complex measurements and experiments to determine WUE in late plant growth, but this technology significantly shortens this timeframe. By detecting the genotype at specific loci, plants with high WUE can be efficiently and accurately identified, avoiding significant ineffective breeding efforts, significantly improving the accuracy of breeding selection, and accelerating the breeding process.

[0031] 2. The testing process does not require complex and expensive equipment, and the cost of single-sample testing is low, making large-scale screening possible. This makes it easily affordable for both scientific research institutions conducting germplasm resource research and breeding companies conducting large-scale variety selection. Furthermore, DNA-based testing is unaffected by fluctuations in the field environment. Whether under drought, high temperatures, or other complex field conditions, the stability and reliability of test results are guaranteed, providing a stable technical foundation for breeding efforts.

[0032] 3. The addition of the novel fluorescence enhancer FluoroEnhance-X and the gold nanoparticle-modified primer enhancer Au-PrimerEnhance to the reaction system, along with specialized chemical modifications to the primers, significantly enhances detection sensitivity and accuracy. The novel fluorescence enhancer boosts fluorescence signal intensity, the gold nanoparticle-modified primer enhancer improves primer stability and amplification efficiency, and the specially modified primers increase cell entry efficiency and enhance nuclease resistance. These innovative designs make the KASP molecular markers of this invention far superior to traditional markers.

[0033] 4. This molecular marker is not only suitable for the precise evaluation of common bean germplasm resources, helping researchers to quickly screen germplasm with potentially superior traits, but also plays an important role in the screening of hybrid offspring, accurately selecting offspring plants carrying high GHW genes. At the same time, it provides a powerful tool for breeding new common bean varieties with high water use efficiency and other advantageous traits, assisting in the selection of drought-resistant common bean varieties, and is of great significance for ensuring global common bean production and promoting sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a flow chart of a method for developing a KASP molecular marker associated with the major QTL for golden water use efficiency in kidney bean proposed in the present invention;

[0035] Figure 21. This is a line comparison chart of the prediction accuracy of kidney bean GHW under different treatments of the embodiment and the comparative example;

[0036] Figure 3 1 is a bar graph comparing the fluorescence signal intensity and amplification efficiency of the embodiment and the comparative example;

[0037] Figure 4 is the H NMR spectrum of ethyl 5-(4-methylphenyl)-2,2'-bipyridine-3,3'-dicarboxylate;

[0038] Figure 5 This is the H NMR spectrum of 3-(4-hydroxyphenyl)-2-acrylamido-2-methylpropionic acid;

[0039] Figure 6 The phenotypes of superior and inferior lines in natural populations of common beans;

[0040] Figure 7 The relationship between the estimated WUE values ​​of common bean germplasm at different times;

[0041] Figure 8 SNPs detected for different phenotypic parameters under WD treatment;

[0042] Figure 9 for weighted gene co-expression network analysis (WGCNA);

[0043] Figure 10 This is the change curve of the estimated WUE value (WUEe) in leaves of transiently overexpressing plants. DETAILED DESCRIPTION

[0044] according to Figures 1 to 10 , the specific implementation methods of the present invention are as follows:

[0045] Example 1

[0046] Development of KASP molecular marker for golden water use efficiency in kidney bean

[0047] Parent selection and population construction: The high GHW bean variety CP-Pv-99 and the low GHW bean variety CP-Pv-114 were selected as parents, and hybrid combinations were configured to construct the F2 population, obtaining a total of 200 individual plants.

[0048] Phenotypic data acquisition: The F2 population was planted in a glass greenhouse equipped with the Plantarray phenotyping platform, simulating natural light, with a maximum temperature of 35°C and a minimum temperature of 20°C. Figure 6 As shown in the figure (phenotypes of superior and inferior lines in natural populations of common beans), by real-time monitoring of GHW-related parameters, it was found that the leaf wilting degree of the high GHW superior line CP-Pv-99 under drought treatment was significantly lower than that of the inferior line CP-Pv-114. Figure 7(Relationship between estimated WUE values ​​in different time periods) It can be clearly observed that the WUE values ​​of the superior lines in the 6th to 11th period were continuously 37%-52% higher than those of the inferior lines.

[0049] Gene pool construction and sequencing: The 30 extremely superior individuals with the largest GHW peak areas were selected from the F2 population to form a high-GHW gene pool, and the 30 extremely inferior individuals with the smallest GHW peak areas were selected to form a low-GHW gene pool. Whole-genome resequencing was performed together with the parents at a sequencing depth of 10×.

[0050] Association region screening: SNP-index analysis was used to screen chromosomal regions significantly associated with GHW, with a threshold of Δ(SNP-index)>0.3.

[0051] SNP Loci Identification and Candidate Gene Identification: Based on GWAS analysis, with a significance threshold of -log(P) ≥ 3, we identified SNP loci closely linked to the major GHW QTL (in the regions of Phvul.001G137925 and Phvul.009G003100). We combined RNA-seq differentially expressed gene data with proteomics data to identify candidate genes.

[0052] Primer Design and Screening: Multiple pairs of KASP primers were designed based on the target SNP loci. Molecular dynamics simulation software was used to assist in the design and selection of primer pairs that showed stable amplification and high polymorphism in the parents. F2 population typing was used to verify the linkage between the marker and the GHW trait, ultimately determining the first and second KASP molecular markers.

[0053] Testing of Golden Water Utilization Rate of Kidney Bean

[0054] DNA extraction: Take 0.1 g of kidney bean leaves and extract genomic DNA using the CTAB method. Detect the DNA concentration and purity using a nucleic acid protein analyzer and adjust the concentration to 50-100 ng / μL for later use.

[0055] Preparation of PCR reaction system: In a 200 μL PCR tube, add 5 μL of 2×KASP Master Mix, 0.14 μL of primer mixture (12 μmol / L), 2 μL of template DNA (50-100 ng / μL), the new fluorescence enhancer FluoroEnhance-X (final concentration 0.1 mmol / L), and the gold nanoparticle-modified primer enhancer Au-PrimerEnhance (0.02 μg per 10 μL reaction system) in sequence, and add ddH2O to 10 μL.

[0056] PCR amplification: Place the PCR tube in a PCR instrument and set the reaction program as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 61-55°C for 60 s (0.6°C decrease per cycle), for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 60 s, for a total of 26 cycles; and final extension at 72°C for 5 min.

[0057] Fluorescence signal scanning: After the PCR reaction is completed, the reaction tube is placed in a fluorescent quantitative PCR instrument for fluorescence signal scanning, and the SNP site genotype is determined based on the fluorescence signal.

[0058] Example 2

[0059] Development of KASP molecular marker for golden water use efficiency in kidney bean

[0060] Association region screening: SNP-index analysis was used to screen the chromosome regions significantly associated with GHW ( Figure 8 , SNP site distribution under WD treatment), 12 SNP sites were found in the Phvul.001G137925 region with a △(SNP-index) of 0.42, far exceeding the 0.3 threshold. Figure 9 The WGCNA co-expression network showed that the genes in this region were highly correlated with the stomatal regulation module (MEbrown module) (module membership = 0.91).

[0061] The rest is the same as Example 1.

[0062] Testing of Golden Water Utilization Rate of Kidney Bean

[0063] DNA extraction: same as Example 1.

[0064] Preparation of PCR reaction system: In a 200 μL PCR tube, add 5 μL of 2×KASP Master Mix, 0.14 μL of primer mixture (12 μmol / L), 2 μL of template DNA (50-100 ng / μL), the new fluorescence enhancer FluoroEnhance-X (final concentration 0.05 mmol / L), and the gold nanoparticle-modified primer enhancer Au-PrimerEnhance (0.01 μg per 10 μL reaction system) in sequence, and add ddH2O to 10 μL.

[0065] PCR amplification: Same as Example 1.

[0066] Fluorescence signal scanning: same as in Example 1.

[0067] Example 3

[0068] Development of KASP molecular marker for golden water use efficiency in kidney bean

[0069] Primer design and screening: Design multiple pairs of KASP primers based on the target SNP sites, use molecular dynamics simulation software to assist in the design, and screen for primer pairs that are stable and highly polymorphic in the parents. Figure 10 As shown in the figure, the WUEe value of plants overexpressing the Phvul.009G003100 gene increased by 2.3 times after 10:00 compared with the control group, verifying the regulatory effect of this gene on GHW traits.

[0070] The rest is the same as Example 1.

[0071] Testing of Golden Water Utilization Rate of Kidney Bean

[0072] DNA extraction: same as Example 1.

[0073] Preparation of PCR reaction system: In a 200 μL PCR tube, add 5 μL of 2×KASP Master Mix, 0.14 μL of primer mixture (12 μmol / L), 2 μL of template DNA (50-100 ng / μL), the new fluorescence enhancer FluoroEnhance-X (final concentration 0.15 mmol / L), and the gold nanoparticle-modified primer enhancer Au-PrimerEnhance (0.03 μg per 10 μL reaction system) in sequence, and add ddH2O to 10 μL.

[0074] PCR amplification: Same as Example 1.

[0075] Fluorescence signal scanning: same as in Example 1.

[0076] Comparative Example

[0077] Traditional method for testing water use efficiency of kidney beans

[0078] DNA extraction: Same as in Example 1. PCR reaction system preparation: In a 200 μL PCR tube, 5 μL of 2× KASP Master Mix, 0.14 μL of primer mixture (12 μmol / L), and 2 μL of template DNA (50-100 ng / μL) were added sequentially. ddH2O was added to 10 μL. The novel fluorescence enhancer FluoroEnhance-X and the gold nanoparticle-modified primer enhancer Au-PrimerEnhance were not added.

[0079] PCR amplification: Same as Example 1.

[0080] Fluorescence signal scanning: same as in Example 1.

[0081] The detection performance comparison of the embodiment and the comparative example is shown in the following table:

[0082] Table 1

[0083]

[0084]

[0085] Conclusion: This table visually demonstrates the differences between the Examples and Comparative Examples in key detection performance indicators. The Examples outperformed the Comparative Examples in terms of fluorescence signal intensity, amplification efficiency, detection sensitivity, and detection specificity, with Example 3 performing the best, demonstrating superior detection performance of the present invention.

[0086] The comparison of the GHW prediction accuracy of kidney beans under different treatments in the embodiment and the comparative example is shown in the following table:

[0087] Table 2

[0088] Treatment Drought treatment Normal irrigation Example 1 Prediction accuracy (%) 90 92 Example 2 Prediction accuracy (%) 85 88 Example 3 Prediction accuracy (%) 93 95 Comparison of the prediction accuracy (%) 70 75

[0089] Conclusion: This table highlights the differences in bean GHW prediction accuracy between the Examples and the Comparative Examples under different treatments. The Examples achieved higher prediction accuracy than the Comparative Examples under both drought and normal irrigation conditions, with Example 3 achieving the highest prediction accuracy. This demonstrates that the KASP molecular markers of the present invention are more accurate and reliable in predicting bean GHW.

[0090] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A KASP molecular marker associated with the major QTL for water use efficiency in kidney bean, characterized in that: The method comprises a first KASP molecular marker and / or a second KASP molecular marker; the first KASP molecular marker specifically detects the 2397926th site of the region where the Phvul.001G137925 gene of kidney bean is located, and the second KASP molecular marker specifically detects the 1794256th site of the region where the Phvul.009G003100 gene of kidney bean is located; during the detection process, a reaction system containing a fluorescence enhancer FluoroEnhance-X and a nano-gold-modified primer enhancer Au-PrimerEnhance is used; FluoroEnhance-X specifically binds to the KASP primer and the amplification product to enhance the fluorescence signal intensity; Au-PrimerEnhance binds to the primer via an Au-S bond to improve the stability of the primer and the amplification efficiency; the fluorescence enhancer is 5-(4-methylphenyl)-2,2'-bipyridine-3,3'-dicarboxylic acid ethyl ester, and its structural formula is: 。 2. The KASP molecular marker associated with the major QTL for water use efficiency of kidney bean according to claim 1, characterized in that: It also includes special chemical modification of the primer, connecting a cholesterol group to the 5' end of the primer; at the same time, introducing a phosphothioate modification group at the 3' end to enhance the primer's resistance to nucleases.

3. The KASP molecular marker associated with the major QTL for water use efficiency of kidney bean according to claim 1, characterized in that: A bioactive small molecule compound, Bio-Activator-Y, is also added to the reaction system. It binds to a specific sequence in the bean genomic DNA to promote the specific binding of the KASP marker to the target site at a binding ratio of 1:

1. The Bio-Activator-Y is 3-(4-hydroxyphenyl)-2-acrylamido-2-methylpropionic acid, and its structural formula is: 。 4. The KASP molecular marker associated with the major QTL for water use efficiency of kidney bean according to claim 1, characterized in that: The first KASP molecular marker includes forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTAAGCGGTTTCGTCGTCGTCC-3', forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTAAGCGGTTTCGTCGTCGTCA-3' and reverse primer: 5'-CGAAATAGACGTCGCCGACGAC-3'; the second KASP molecular marker includes forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTACTTTAAACCTAACTTAG-3', forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTACTTTAAACCTAACTTAA-3' and reverse primer: 5'-TATAAGTGGGTACAATCCTCACC-3'.

5. The KASP molecular marker associated with the major QTL for water use efficiency of kidney bean according to claim 1, characterized in that: The added concentration of the fluorescence enhancer FluoroEnhance-X is 0.05-0.15 mmol / L, which can increase the fluorescence signal intensity by 30%-50%. The added amount of the nano-gold modified primer enhancer Au-PrimerEnhance is 0.01-0.03 μg per 10 μL reaction system.

6. The KASP molecular marker associated with the major QTL for water use efficiency of kidney bean according to claim 1, characterized in that: The PCR reaction system for the KASP molecular marker detection is: 5 μL of 2×KASP MasterMix, 0.14 μL of primer mixture, 2 μL of template DNA, and ddH2O to 10 μL; and 0.1-0.3 μL of hot start Taq enzyme activator is added to the reaction system.

7. The KASP molecular marker for water use efficiency of kidney bean according to claim 1, characterized in that: The PCR reaction procedure for the KASP molecular marker detection is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing / extension at 61-55°C for 60 seconds, for a total of 10 cycles; denaturation at 94°C for 20 seconds, annealing / extension at 55°C for 60 seconds, for a total of 26 cycles; after the last cycle, an extension step at 72°C for 5 minutes is added.

8. A method for developing KASP molecular markers associated with the major QTL for water use efficiency of kidney bean according to claim 7, characterized in that: The following steps are involved: S1: Parent selection and population construction: select high GHW bean varieties such as CP-Pv-99 and low GHW bean varieties such as CP-Pv-114 as parents, configure hybrid combinations and construct F2 populations; S2: Phenotypic data acquisition: The F2 population was grown in a glass greenhouse equipped with the Plantarray phenotyping platform, simulating natural light with a maximum daily temperature of 35°C and a minimum daily temperature of 20°C. GHW-related parameters, such as peak area and peak value, were monitored in real time to obtain phenotypic data. High-resolution imaging technology was also used to obtain stomatal distribution and morphological data on bean leaves to assist in the analysis of GHW traits. S3: Gene pool construction and sequencing: 30 extremely superior individuals with the largest GHW peak areas were selected from the F2 population to form a high GHW gene pool, and 30 extremely inferior individuals with the smallest GHW peak areas were selected to form a low GHW gene pool. Whole-genome resequencing was performed together with the parents. S4: Association region screening: SNP-index analysis was used to screen chromosome regions significantly associated with GHW; S5: SNP site identification and candidate gene targeting: Based on GWAS analysis, with -log(P) ≥ 3 as the significance threshold, SNP sites closely linked to the GHW major effect QTL were identified. Candidate genes were targeted by combining RNA-seq differentially expressed gene data. S6: Primer design and screening: Multiple pairs of KASP primers were designed based on the target SNP sites. Molecular dynamics simulation software was used to assist in the design. Primer pairs that were stable and highly polymorphic in the parents were screened. The linkage between the marker and the GHW trait was verified through F2 population typing. The KASP primers with the closest linkage distance to the GHW major effect QTL and the highest typing efficiency were selected as the final molecular markers.

9. The method for developing KASP molecular markers associated with the major QTL for water use efficiency of kidney bean according to claim 8, characterized in that: In step S5, in addition to using RNA-seq differentially expressed gene data, proteomics data were also combined to comprehensively analyze gene expression at the transcriptional and translational levels.

10. A use of the KASP molecular marker associated with the major QTL for water use efficiency of kidney bean according to any one of claims 1 to 7, characterized in that: Its application in molecular marker-assisted selection breeding of drought resistance in common bean, precise evaluation of common bean germplasm resources, and breeding of new common bean varieties with high water use efficiency and other excellent traits.

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