A molecular marker closely linked to a heat tolerance major-effect qtl of vigna unguiculata, primers and application thereof

Abstract

The application discloses a heat-resistant major QTL closely linked molecular marker of cowpea, primers thereof and application. The heat-resistant major QTL of the cowpea is named HS-Pn-1.1, and the KASP molecular marker 3_00175 closely linked therewith is located at the position of 1157933 bp of the No.1 chromosome. The sample whether carrying a key allelic variation of heat resistance can be accurately determined according to the genotype by carrying out PCR amplification on the to-be-tested material through the marker. The molecular marker and the primers provided by the application can realize efficient and accurate screening of the heat-resistant major QTL of the cowpea. Compared with a traditional phenotype identification method, the method has the advantages of high detection flux, stable and reliable results and the like, the typing accuracy can reach more than 99%, and the technology can significantly improve the cowpea heat-resistant variety breeding efficiency and effectively shorten the breeding period.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, and relates to a molecular marker tightly linked to a major QTL (HS-Pn-1.1) for heat resistance in cowpea, its primers, and its applications. Background Technology

[0002] cowpea( Vigna unguiculata Cowpeas (L. Walp.) are a legume vegetable crop widely cultivated in tropical and subtropical regions. Their genome size is approximately 620 Mb, and their chromosome number is 2n=2x=22. Cowpeas play an irreplaceable role in ensuring the daily vegetable supply for residents, stabilizing vegetable production capacity, and supporting the processing of specialty foods.

[0003] High-temperature stress is one of the key environmental factors restricting the growth, development, and yield of cowpea. In southern regions, during summer open-field cultivation and protected off-season cultivation, persistent high temperatures often lead to inhibited flower bud differentiation, reduced flower quantity, and a significant decrease in pod setting rate, thus severely impacting the economic benefits of cultivation. Net photosynthetic rate (Pn) can directly characterize the carbon assimilation capacity of cowpea leaves and reflect the degree of physiological damage caused by high-temperature stress, and is therefore widely established as a core physiological indicator for evaluating the heat tolerance of cowpea.

[0004] Studies have shown significant genetic differences in the tolerance of cowpea to high-temperature stress among varieties. Heat tolerance, as a complex quantitative trait regulated by multiple genes and highly responsive to environmental changes, is difficult to traitly improve through traditional breeding methods that rely on phenotypic identification. This process is not only time-consuming but also susceptible to environmental interference, leading to poor selection accuracy and low efficiency. The completion of cowpea genome sequencing has provided fundamental data for its genetic improvement of stress resistance. Molecular marker technology based on SNPs (single nucleotide polymorphisms) is also gradually being applied in breeding practices, providing a new technical pathway for targeted improvement of cowpea heat tolerance. Among these methods, Kompetitive Allele Specific PCR (KASP) has become a commonly used detection method in crop molecular breeding due to its high throughput, low cost, and accurate genotyping. Currently, KASP technology has been successfully applied in heat tolerance breeding of crops such as soybean and maize, but its application in high-temperature stress breeding of cowpea is still in its early stages. Summary of the Invention

[0005] This invention aims to address key technical bottlenecks in heat-assisted breeding of cowpea, providing a highly stable and practical KASP molecular marker and its specific primers that are tightly linked to the major heat-resistant QTL (HS-Pn-1.1), and establishing a molecular-assisted selection breeding technology system based on this marker. This system uses genome-wide association analysis (GWAS) to identify SNP loci significantly associated with cowpea heat resistance, further developing them into KASP markers and performing genetic validation across multiple populations. The obtained markers enable efficient identification of heat-resistant alleles in cowpea germplasm resources, providing reliable molecular breeding tools for genetic improvement of cowpea heat resistance.

[0006] This invention identifies a major-effect QTL (HS-Pn-1.1) of heat tolerance located at 1157933 bp on chromosome 1 of cowpea and associated with net photosynthetic rate using GWAS, and develops a KASP molecular marker closely linked to it.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In one aspect, this invention provides a KASP molecular marker 3_00175 that is closely linked to the major QTL of heat tolerance in cowpea. This marker is located at 1157933 bp on chromosome 1 of the reference genome of cowpea variety G98, and its SNP site polymorphism is A / G.

[0009] Preferably, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1.

[0010] In another aspect, the present invention provides a KASP primer combination for detecting the above-mentioned molecular markers, comprising two specific forward primers (Primer1 and Primer2) and one universal reverse primer (Primer_Common).

[0011] 3_00175 Primer1 is shown as SEQ ID NO.2;

[0012] 3_00175 Primer2 is shown in SEQ ID NO.3;

[0013] 3_00175 Primer1 and 3_00175 Primer2 are two specific primers, each linked to a different fluorescent sequence;

[0014] 3_00175 Primer_Common is shown in SEQ ID NO.4.

[0015] Preferably, 3_00175 Primer1 is linked to the FAM group, and 3_00175 Primer2 is linked to the HEX group.

[0016] In another aspect, the present invention provides the application of the above-mentioned KASP primers, detection reagents or kits containing the KASP primers in the detection of major QTLs for heat resistance in cowpeas.

[0017] In another aspect, this invention provides a molecularly assisted selection breeding method for heat resistance of cowpea based on the above-mentioned KASP primers, specifically including the following steps:

[0018] S1. Extract genomic DNA from the leaf tissue of cowpea plants;

[0019] S2. Using cowpea leaf tissue genomic DNA as a template, KASP genotyping reaction was performed using the 3_00175 KASP primer system;

[0020] S3. Read the fluorescence signal of the KASP reaction: If the sample shows a specific fluorescence signal of 3_00175 Primer1 (FAM marker), it is determined that the cowpea individual carries the heat-resistant trait allele; if it shows a specific fluorescence signal of 3_00175 Primer2 (HEX marker), it is determined that it carries the heat-sensitive trait allele.

[0021] Preferably, Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0022] As a preferred option, the KASP reaction assay also includes 2X KASP Master mix reagent.

[0023] As a preferred option, the PCR system for KASP reaction detection is: DNA 0.8 μl, 2x KASP Master mix 0.75 μl, Primer mix 0.05 μl.

[0024] As a preferred method, the PCR reaction program for KASP reaction detection is as follows: pre-denaturation at 94℃ for 15 minutes, denaturation at 94℃ for 20 seconds, gradient annealing at 61~55℃ for 60 seconds, with the annealing temperature decreasing by 0.6℃ per cycle, extension at 55℃ for 60 seconds, for 10 cycles; then denaturation at 94℃ for 20 seconds, annealing at 55℃ for 60 seconds, extension for 60 seconds, for 26 cycles.

[0025] The terminology involved in this invention includes:

[0026] Quantitative trait loci (QTLs) are genomic regions that control quantitative traits and have detectable genetic effects.

[0027] A major effect QTL is a QTL that has a large genetic effect on the target quantitative trait (e.g., phenotypic variation R² ≥ 10%) or significant statistical support.

[0028] Kompetitive Allele-Specific PCR (KASP) is a fluorescent genotyping technique based on known SNP sites, suitable for accurate bicelestem typing of target SNPs and small insertions / deletions (Indels) in DNA samples.

[0029] Single nucleotide polymorphism (SNP) refers to a variation in DNA sequence caused by a single nucleotide substitution at the same physical site in the genome.

[0030] The advantages and beneficial effects of this invention are as follows:

[0031] (1) The molecular marker 3_00175 disclosed in this invention is closely linked to the major QTL of heat resistance (R²=0.5819), and the genotyping accuracy rate is over 99%, which can realize the rapid and accurate identification of heat resistance of cowpea.

[0032] (2) Compared with traditional heat resistance phenotypic identification methods, this molecular marker-assisted selection method is not affected by field environmental fluctuations and can be stably carried out during the cowpea seedling stage, shortening the heat resistance identification cycle from the conventional 2-3 years to about one month, significantly shortening the breeding time and reducing the breeding cost.

[0033] (3) This marker can effectively distinguish between heat-resistant and heat-sensitive genotypes, providing a reliable molecular tool for the breeding of heat-resistant cowpea varieties. It is particularly suitable for cowpea breeding in areas with frequent high temperatures and in facility cultivation environments. Attached Figure Description

[0034] To clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings involved in each embodiment are briefly described below.

[0035] Figure 1 KASP marker genotyping diagram of genes associated with the 3_00175 marker site;

[0036] Figure 2 Plot showing the differences in allelic variation in heat resistance at the 3_00175 marker site. Detailed Implementation

[0037] To facilitate a thorough understanding of the present invention, the technical solutions therein are described clearly and completely below with reference to specific embodiments. It should be understood that the following embodiments are only some implementations of the present invention, and not all embodiments.

[0038] Unless otherwise specified, the experimental methods described in the following examples are performed in accordance with the manufacturer's instructions.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] The PCR amplification system used in the KASP reaction of this invention is as follows: 0.8 μl DNA, 0.75 μl 2x KASP Master mix, and 0.05 μl primer mixture. The primer mixture is obtained by mixing Primer1 with Primer_Common, or Primer2 with Primer_Common, with each primer having a volume of 0.025 μl and a concentration of 10 μM / L.

[0041] The PCR amplification program used in this embodiment of the invention is as follows: pre-denaturation at 94℃ for 15 minutes, denaturation at 94℃ for 20 seconds, gradient annealing at 61~55℃ for 60 seconds, with the annealing temperature decreasing by 0.6℃ in each cycle, extension at 55℃ for 60 seconds, for 10 cycles; then denaturation at 94℃ for 20 seconds, annealing at 55℃ for 60 seconds, extension for 60 seconds, for 26 cycles.

[0042] Example 1: Screening of SNP sites closely linked to the major QTL of heat resistance in cowpea

[0043] This invention identified the heat resistance of 344 cowpea core germplasm materials from the Zhejiang Academy of Agricultural Sciences. The experiment was conducted in August 2025 in a greenhouse at the Toupeng Base of the Xiaoshan District Agricultural Science and Technology Research Institute in Hangzhou. Seedlings were raised in plug trays under conventional conditions until the trifoliate compound leaves were fully expanded. Subsequently, the greenhouse ventilation net was closed for three consecutive days of high-temperature treatment, utilizing the greenhouse's heat retention to stabilize the daytime maximum temperature at 55-60℃ and the nighttime temperature at 38-42℃. A randomized block design was used, with three biological replicates per material, each replicate containing four plants, for a total of 12 individual plants. After the high-temperature stress period, the net photosynthetic rate (µmol·m³) of each individual plant was measured. -2 ·s -1 The average value was used as the representative heat resistance phenotype value of the material. Whole-genome resequencing of the population was performed using the Illumina platform, yielding 1,856,342 high-quality SNP markers after rigorous quality control. Genome-wide association analysis using a mixed linear model (MLM) identified a major-effect QTL (HS-Pn-1.1) at 1157933 bp on chromosome 1. The minor allele frequency (MAF) was 0.3, and this locus was tightly linked to a gene encoding an E3 ubiquitin ligase. Based on this, a specific KASP marker, 3_00175, with an A / G dimorphic allele, was developed.

[0044] Table 1 shows the statistical results of genotypes and net photosynthetic rates of some materials in the 344 germplasm accessions after high temperature stress.

[0045] Table 1. Statistics on genotypes and net photosynthetic rates after high-temperature stress for some materials from 344 germplasm accessions.

[0046]

[0047] In this embodiment, the KASP marker 3_00175 was successfully developed. It is located on chromosome 1 of the reference genome of cowpea variety G98, and its SNP site is located at 1157933 bp, corresponding to an A / G bimorphic SNP site.

[0048] The nucleotide sequence of the KASP-tagged sequence is shown in SEQ ID NO.1, where the SNP site is located at the 101st base of the sequence.

[0049] Based on this sequence, the present invention designed a series of KASP primers, the sequences from 5' to 3' of which are shown below.

[0050] The KASP primers for this molecular marker include a specific forward primer Primer1, a specific forward primer Primer2, and a universal reverse primer Primer_Common, the sequences of which are shown below.

[0051] 3_00175 Primer1 is shown as SEQ ID NO.2;

[0052] 3_00175 Primer2 is shown in SEQ ID NO.3;

[0053] 3_00175 Primer_Common is shown in SEQ ID NO.4.

[0054] In some specific implementations, Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0055] Example 2:

[0056] In this embodiment, 50 cowpea germplasm samples were randomly selected from the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences.

[0057] The heat tolerance phenotype of 50 randomly selected cowpea germplasms was identified. Net photosynthetic rate was measured after the seedlings were subjected to a high-temperature treatment of 55 / 35℃ for 3 days when the trifoliate compound leaves of the seedlings were fully expanded.

[0058] Genomic DNA was extracted from 50 cowpea germplasm accessions using the CTAB method. KASP analysis was performed using the IntelliQube genotyping platform and the KASP primers 3_00175 designed in Example 1, with the genomic DNA of the above population as a template. The PCR reaction system and PCR procedure are detailed in the specific implementation instructions.

[0059] Analysis results as follows Figure 1 As shown in the diagram, a red fluorescence signal (FAM signal) indicates that the cowpea germplasm carries the heat-resistant allele A (HapI). A blue fluorescence signal (HEX signal) indicates that the germplasm carries the heat-sensitive allele G (HapII).

[0060] Depend on Figure 1 As shown, the KASP marker 3_00175 exhibited highly consistent and well-defined genotyping results in 50 cowpea germplasms. The two genotypes were clearly separated and clustered stably in the fluorescence signal space, with no overlap, indicating that the KASP marker has high genotyping accuracy and good population applicability.

[0061] Considering its heat resistance (net photosynthetic rate, Pn, µmol·m) -2 ·s -1 ) performance (see Figure 2 (Some data are shown in Table 2). It was found that germplasm carrying the G genotype had a lower net photosynthetic rate, while germplasm carrying the A genotype had a relatively higher net photosynthetic rate. Data analysis showed that the net photosynthetic rate of germplasm carrying the A genotype was significantly higher than that of the G genotype (p=0.0004). This result confirms a significant genetic association between the KASP marker 3_00175 and the heat tolerance phenotype in cowpea, and can be effectively used for genotype-assisted screening of heat-tolerant germplasm, achieving the purpose of molecular marker screening.

[0062] Table 2. Statistical data on genotypes of selected germplasm and net photosynthetic rates after high-temperature stress.

[0063]

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made to the present invention should be included within the scope of protection of the present invention.

Claims

1. A molecular marker tightly linked to a major QTL for heat resistance in cowpea, characterized in that, The molecular marker is 3_00175, and the molecular marker is of the KASP type; the SNP site of the molecular marker is located at 1157933 bp on chromosome 1 of the cowpea variety G98 genome, and the polymorphism is A / G; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1.

2. A KASP primer for detecting the molecular marker as described in claim 1, characterized in that, The KASP primers include 3_00175 Primer1, 3_00175 Primer2, and 3_00175 Primer_Common; 3_00175 Primer1 is shown as SEQ ID NO.2; 3_00175 Primer2 is shown in SEQ ID NO.3; 3_00175 Primer_Common is shown in SEQ ID NO.4; 3_00175 Primer1 is linked to the FAM group, and 3_00175 Primer2 is linked to the HEX group.

3. The application of the KASP primer of claim 2 or a kit containing the KASP primer of claim 2 in detecting the major QTL of heat resistance in cowpea.

4. A method for breeding cowpea varieties with different heat resistance using the KASP primers described in claim 2, characterized in that, The method specifically includes the following steps: S1. Extract genomic DNA from cowpea plant samples; S2. Using cowpea plant sample genomic DNA as a template, KASP reaction detection was performed using the KASP primers described in claim 2; S3. Read the fluorescence signal detected by the KASP reaction. If the genomic DNA of the cowpea plant sample shows the signal of the FAM group of the fluorescent group attached to 3_00175 Primer1, then the sample is determined to carry the heat-resistant genotype. If the genomic DNA of the cowpea plant sample shows the signal of the HEX group of the fluorescent group attached to 3_00175 Primer2, then the sample is determined to carry the heat-sensitive genotype.

5. The method for breeding cowpea varieties with different heat resistance according to claim 4, characterized in that, The KASP reaction assay also includes the 2X KASP Master mix reagent.

6. The method for breeding cowpea varieties with different heat resistance according to claim 5, characterized in that, The PCR system for KASP reaction detection was as follows: DNA 0.8 μl, 2x KASP Master mix 0.75 μl, Primer mix 0.05 μl.

7. The method for breeding cowpea varieties with different heat resistance according to claim 4, characterized in that, The PCR reaction program for KASP reaction detection is as follows: pre-denaturation at 94℃ for 15 minutes, denaturation at 94℃ for 20 seconds, gradient annealing at 61~55℃ for 60 seconds, with the annealing temperature decreasing by 0.6℃ per cycle, extension at 55℃ for 60 seconds, for 10 cycles; then denaturation at 94℃ for 20 seconds, annealing at 55℃ for 60 seconds, extension for 60 seconds, for 26 cycles.

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