Molecular marker closely linked with cowpea drought tolerance major QTL (quantitative trait loci) as well as primer and application of molecular marker

Abstract

The invention discloses a molecular marker closely linked with a cowpea drought tolerance major QTL (quantitative trait loci) and a primer sequence of the molecular marker. The name of the cowpea drought tolerance major QTL is DC300099, the KASP type molecular marker 300099 closely linked with the cowpea drought tolerance major QTL is located at the position of 3123287 bp on a chromosome 4, the marker can be used for amplifying a to-be-detected material, and whether a sample carries key allelic variation for controlling drought tolerance or not is judged according to the genotype of the to-be-detected material. By utilizing the molecular marker and the primer thereof provided by the invention, rapid screening of the main effect QTL of drought tolerance of cowpea can be realized, and compared with a traditional phenotype identification method, the molecular marker and the primer thereof have the advantages of high detection flux, accurate and reliable result and the like, and the typing accuracy can reach 99% or above. The technology can significantly improve the breeding efficiency of the cowpea drought-enduring variety and shorten the breeding period.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding, and relates to a molecular marker tightly linked to a major QTL (DC3_00099) of drought tolerance in cowpea, primers thereof and applications thereof. Background Art

[0002] Cowpea (Vigna unguiculata (L.) Walp.) is an important crop in the genus Vigna in the Leguminosae family. Its genome is approximately 620 Mb in size and its chromosome number is 2n=2x=22. As a legume widely cultivated in arid and semi-arid regions, it is a key edible legume and vegetable crop.

[0003] Cowpea is a model plant for drought tolerance research. Its well-developed root system, high water use efficiency, and remarkable genetic diversity in response to drought stress have been shown. Studies have shown that drought tolerance in cowpea is controlled by multiple genes, closely linked to genes encoding dehydrins and osmoregulatory proteins. However, traditional breeding relies on phenotypic screening, which is inefficient and time-consuming, making it difficult to precisely select drought-tolerant varieties.

[0004] With the completion of the cowpea genome sequencing and the development of molecular marker technology, SNP (single nucleotide polymorphism)-based molecular marker-assisted selection (MAS) has provided a new technical approach for drought-tolerant cowpea breeding. Among them, competitive allele-specific PCR (KASP) has become a key tool in crop molecular breeding due to its high throughput, low cost, and high accuracy. While KASP has achieved significant success in drought-tolerant breeding in crops such as rice and wheat, its application in cowpea drought-tolerant breeding is still in its infancy. Summary of the Invention

[0005] The technical problem addressed by this invention is the use of KASP markers for assisted breeding of cowpea for drought tolerance. The present invention provides a molecular marker tightly linked to the major QTL for drought tolerance in cowpea (DC3_00099) and its KASP primers, and establishes a method for assisted breeding of cowpea for drought tolerance based on this marker. This method identifies SNPs associated with drought tolerance through genome-wide association analysis and converts these SNPs into KASP markers for verification. This method can be used to rapidly identify drought-tolerant genotypes in cowpea germplasm, providing molecular breeding technology support for genetic improvement of drought tolerance in cowpea.

[0006] This study identified a major drought-tolerance SNP locus (DC3_00099) located at position 3123287 on chromosome 4 of cowpea through genome-wide association analysis (GWAS) and developed a closely linked KASP molecular marker. This marker provides an effective technical tool for molecular breeding of cowpea for drought tolerance, significantly improving breeding efficiency and shortening the breeding cycle.

[0007] To achieve the above object, the present application provides the following technical solutions. The present application provides a molecular marker closely linked to a drought tolerance major QTL of Vigna unguiculata, which is 3_00099, located at 3123287 bp of chromosome 4 of the Vigna unguiculata variety G98 genome, and is a KASP type, and the SNP site polymorphism is A / G.

[0008] Preferably, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.

[0009] The present application also provides a KASP primer for detecting the above-mentioned molecular marker, which comprises a specific forward primer Primer1, a specific forward primer Primer2 and a common reverse primer Primer_Common. 3_00099 Primer1 is shown as SEQ ID NO. 2. 3_00099 Primer2 is shown as SEQ ID NO. 3. 3_00099 Primer1 and 3_00099 Primer2 are two specific primers, respectively connected with different fluorescent sequences. 3_00099 Primer_Common is shown as SEQ ID NO. 4.

[0010] Preferably, 3_00099 Primer1 is connected with a FAM group, and 3_00099 Primer2 is connected with a HEX group.

[0011] The present application also provides an application of the above-mentioned KASP primer, or a reagent or kit containing the above-mentioned KASP primer in detecting a drought tolerance major QTL of Vigna unguiculata.

[0012] The present application also provides a method for breeding different drought tolerance Vigna unguiculata varieties using the above-mentioned KASP primer, which specifically comprises the following steps: S1, extracting genomic DNA of a Vigna unguiculata plant sample; S2, using the genomic DNA of the Vigna unguiculata plant sample as a template, and performing KASP reaction detection using 3_00099 KASP primers; S3. Read the fluorescent signal detected by the KASP reaction. If the genomic DNA of the cowpea plant sample shows a signal of the fluorescent group linked to 3_00099 Primer1, it is determined that the sample carries a drought-tolerant genotype; if the genomic DNA of the cowpea plant sample shows a signal of the fluorescent group linked to 3_00099 Primer2, it is determined that the sample carries a drought-sensitive genotype.

[0013] Preferably, Primer1 is connected to a FAM group, and Primer2 is connected to a HEX group.

[0014] Preferably, the KASP reaction assay further comprises 2X KASP Master mix reagent.

[0015] Preferably, the PCR system for KASP reaction detection is: 0.8 μl DNA, 0.75 μl 2x KASP Master mix, 0.05 μl Primer mix. The Primer mix is ​​a mixture of Primer1 and Primer_Common, or Primer2 and Primer_Common, with 0.025 μl of each primer solution at a concentration of 10 μM / L.

[0016] Preferably, the PCR reaction program for KASP reaction detection is pre-denaturation at 94°C for 15 minutes, denaturation at 94°C for 20 seconds, gradient annealing from 61 to 55°C for 60 seconds, the annealing temperature is reduced by 0.6°C in each cycle, extension at 55°C for 60 seconds, and cycled 10 times; then denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, extension for 60 seconds, and cycled 26 times.

[0017] The terminology of the present invention: Quantitative trait loci (QTL) refer to the loci in the genome that control quantitative traits.

[0018] A major QTL refers to a quantitative trait locus that has a greater influence or a more significant effect on the quantitative trait being studied.

[0019] Competitive allele-specific PCR (KASP) is an endpoint fluorescent genotyping technology based on known SNPs (single nucleotide polymorphisms) that can accurately detect SNPs and indels (insertions and deletions) at specific sites in DNA samples.

[0020] Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level.

[0021] The advantages and beneficial effects of the present invention are as follows: (1) The molecular marker 3_00099 disclosed in the present invention is tightly linked to the major drought tolerance QTL (r²=0.95), with a typing accuracy of over 99%, enabling early and accurate identification of cowpea drought tolerance.

[0022] (2) Compared with traditional drought tolerance identification methods, this method is not restricted by environmental conditions and can be used for testing at the seedling stage, significantly shortening the breeding cycle (from 2-3 years to 1 month) and reducing breeding costs.

[0023] (3) This marker can effectively distinguish drought-tolerant and drought-sensitive genotypes, providing a reliable molecular tool for the breeding of drought-tolerant cowpea varieties, and is particularly suitable for cowpea breeding in coastal and arid areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0025] Figure 1 This is the KASP marker typing map of the gene related to the 3_00099 marker site; Figure 2 This is a map of the drought tolerance allelic variation differences at the 3_00099 marker site. DETAILED DESCRIPTION

[0026] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples were generally carried out under conventional conditions or conditions recommended by the manufacturers.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] The PCR amplification system used in the KASP reaction of the present invention is as follows: 0.8 μl DNA, 0.75 μl 2x KASP Master mix, 0.05 μl Primer mix. The Primer mix is ​​a mixture of Primer1 and Primer_Common, or Primer2 and Primer_Common, with 0.025 μl of each primer solution at a concentration of 10 μM / L.

[0030] The PCR amplification program used in the embodiment of the present invention is pre-denaturation at 94°C for 15 minutes, denaturation at 94°C for 20 seconds, gradient annealing from 61 to 55°C for 60 seconds, the annealing temperature is reduced by 0.6°C in each cycle, extension at 55°C for 60 seconds, and 10 cycles; then denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, extension for 60 seconds, and 26 cycles.

[0031] Example 1: Screening of SNPs tightly linked to the major QTL for drought tolerance in cowpea This study collected and screened a natural population of cowpea, comprising 344 accessions, for drought tolerance phenotypic evaluation. Using a randomized block design, nine replicates of each accession were planted. Under drought stress conditions (without watering for 40 days), indicators such as leaf chlorosis, aboveground fresh weight, and stem greenness were measured to generate a comprehensive drought tolerance score. Additionally, a control group (three accessions) was maintained for each accession under normal watering conditions.

[0032] The comprehensive drought tolerance score is the sum of three drought phenotypic scores: stem greenness (0-2 points), aboveground fresh weight (0-2 points), and leaf chlorosis (0-2 points). Among them, stem greenness: first count the color conditions of cowpea stems under all drought treatments, the typical plant with the yellowest and driest stem is 0 point, the typical plant with the greenest and fullest stem is 2 points, and the intermediate typical plant is 1 point. The score of 0-2 points is calculated according to the degree of fit between the stem state of each plant and different typical plants; the fresh weight score is calculated according to the percentage of single plant mass with the control group: greater than or equal to 60% is 2 points, 60%-50% (inclusive) is 1.5 points, 50%-40% (inclusive) is 1 point, 40%-30% (inclusive) is 0.5 points, and less than 30% is zero point; the degree of leaf wilting is measured by taking pictures and comparing colors, the greenest and the leaf with intact extended morphology is scored as 2 points, the most withered and the most yellow-brown color is scored as 0 point, and each level is divided into five levels with each level of 0.5 points.

[0033] The population material was resequenced using the Illumina platform, yielding 1,856,342 high-quality SNP markers. Combined with the drought tolerance scores of various traits, genome-wide association analysis using mixed linear models (MLM) identified a major QTL (DC3_00099) at bp 3123287 on chromosome 4. This locus is tightly linked to a gene encoding the IDD14 protein transcription factor. Based on this, KASP marker 3_00099 was developed, with an A / G polymorphism.

[0034] The genotypes of some materials and their comprehensive drought resistance scores among the 344 germplasms are shown in Table 1.

[0035] Table 1 Genotypes and comprehensive drought resistance scores of some 344 accessions The KASP marker 3_00099 obtained by screening in this example is located on chromosome 4 of the genome of the cowpea variety G98. Its SNP site is located at 3123287 bp, and the polymorphism is A / G.

[0036] The nucleotide sequence of the KASP marker is shown in SEQ ID NO. 1, and the SNP site is at position 101 in the sequence.

[0037] The present invention designs a series of KASP primers based on the sequence. The molecular marker KASP primers include a specific forward primer Primer1, a specific forward primer Primer2 and a universal reverse primer Primer_Common, and the sequences from 5' to 3' are shown below.

[0038] 3_00099 Primer1 is shown in SEQ ID NO. 2; 3_00099 Primer2 is shown in SEQ ID NO. 3; 3_00099 Primer_Common is shown in SEQ ID NO.4.

[0039] In some specific embodiments, Primer1 is linked to a FAM group, and Primer2 is linked to a HEX group.

[0040] Example 2: In this example, 58 cowpea germplasms obtained from Xianghu Laboratory (Zhejiang Agricultural Laboratory) were randomly selected.

[0041] Phenotypic characterization of drought tolerance was conducted on 58 randomly selected cowpea accessions. Drought tolerance was assessed using soil culture. After 40 days of drought treatment (without water) at the three-leaf stage, leaf chlorosis, aboveground fresh weight, and stem greenness were measured to provide a comprehensive drought tolerance score. A control group (three seeds) was also established for each accession, which was kept under the same conditions but maintained under normal watering.

[0042] Genomic DNA was extracted using the CTAB method. KASP analysis was performed using the IntelliQube genotyping platform, using the KASP primers for DC3_00099 designed in Example 1, with genomic DNA from the above populations as the template. The PCR reaction system consisted of 0.8 μl of DNA, 0.75 μl of 2× KASP Master Mix, and 0.05 μl of primer mix. The PCR protocol included pre-denaturation at 94°C for 15 min, 10 cycles of touchdown PCR (94°C for 20 s, 61-55°C for 60 s), and 26 cycles of conventional PCR (94°C for 20 s, 55°C for 60 s).

[0043] The primer mix was obtained by mixing Primer1 and Primer_Common, or Primer2 and Primer_Common, with the volume of each primer being 0.025 μl and the concentration being 10 μM / L.

[0044] The fluorescence data were read and analyzed using IntelliQube machine. The analysis results were as follows: Figure 1 When the fluorescent signal is red (FAM signal), it indicates that the cowpea carries the drought-tolerant allele A (HapI). When the fluorescent signal is blue (HEX signal), it indicates that the cowpea carries the sensitive allele G (HapII).

[0045] Depend on Figure 1 As shown, the KASP molecular marker of the present invention achieved highly consistent and clearly defined population typing results in the cowpea population, and the two genotypes could be clearly distinguished and clustered, with good typing results.

[0046] Combined with its drought tolerance performance (see Figure 2 (See Table 2 for some data). It was found that strains carrying genotype A had higher average drought tolerance scores, while strains carrying genotype G had relatively lower average drought tolerance scores. Data analysis showed that strains carrying genotype A had significantly higher average drought tolerance than those carrying genotype G (p=0.0178), confirming that this marker can effectively distinguish differences in cowpea drought tolerance, ultimately achieving the goal of molecular marker screening.

[0047] Table 2 Statistics of genotypes and drought treatment phenotypes of some selected germplasm Note: The comprehensive scoring standard for drought tolerance is consistent with that in Example 1, which is the sum of the three drought phenotypic scores: chlorophyll content (0-1 points), aboveground fresh weight (0-2 points), and leaf chlorosis (0-2 points).

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations are possible within the skill of the art. Any modifications, equivalent substitutions, or improvements to the present invention are intended to fall within the scope of protection of the present invention.

Claims

1. A molecular marker tightly linked to a major QTL for drought tolerance in cowpea, characterized in that: The molecular marker is 3_00099, and the molecular marker is of KASP type; the SNP site of the molecular marker is located at 3123287 bp on chromosome 4 on the genome of cowpea variety G98, and the polymorphism is A / G.

2. The molecular marker according to claim 1, characterized in that The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1.

3. A KASP primer for detecting the molecular marker according to claim 1 or 2, characterized in that: The KASP primers include a specific forward primer Primer1, a specific forward primer Primer2 and a universal reverse primer Primer_Common; 3_00099 Primer1 is shown in SEQ ID NO. 2; 3_00099 Primer2 is shown in SEQ ID NO. 3; 3_00099 Primer1 and 3_00099 Primer2 are two specific primers, each linked to a different fluorescent sequence; 3_00099 Primer_Common is shown in SEQ ID NO.

4.

4. The KASP primer according to claim 3, characterized in that 3_00099 Primer1 is connected to the FAM group, and 3_00099 Primer2 is connected to the HEX group.

5. Use of the KASP primer according to claim 3 or 4, or a reagent or kit containing the KASP primer according to claim 3 or 4, in detecting a major QTL for drought tolerance in cowpea.

6. A method for breeding cowpea varieties with different drought tolerance using the KASP primers according to claim 3 or 4, characterized in that: The method specifically comprises the following steps: S1. Extract genomic DNA from cowpea plant samples; S2, using cowpea plant sample genomic DNA as template and 3_00099 KASP primer for KASP reaction detection; S3. Read the fluorescent signal detected by the KASP reaction. If the genomic DNA of the cowpea plant sample shows a signal of the fluorescent group linked to 3_00099 Primer1, it is determined that the sample carries a drought-tolerant genotype. If the genomic DNA of the cowpea plant sample shows a signal of the fluorescent group linked to 3_00099 Primer2, it is determined that the sample carries a drought-sensitive genotype.

7. The method for breeding cowpea varieties with different drought tolerance according to claim 6, characterized in that: Primer1 is connected to the FAM group and Primer2 is connected to the HEX group.

8. The method for breeding cowpea varieties with different drought tolerance according to claim 7, characterized in that: The KASP reaction assay also includes 2X KASP Master mix reagent.

9. The method for breeding cowpea varieties with different drought tolerance according to claim 8, characterized in that: The PCR system for KASP reaction detection is: DNA 0.8μl, 2x KASP Master mix 0.75μl, Primer mix 0.05μl.

10. The method for breeding cowpea varieties with different drought tolerance according to claim 6, characterized in that: The PCR reaction program for KASP reaction detection was pre-denaturation at 94°C for 15 minutes, denaturation at 94°C for 20 seconds, gradient annealing from 61 to 55°C for 60 seconds, with the annealing temperature decreasing by 0.6°C in each cycle, extension at 55°C for 60 seconds, and 10 cycles; then denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, and extension for 60 seconds, and 26 cycles.

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