A molecular marker closely linked to a drought tolerance major QTL of cowpea, primers and application thereof

Abstract

The application discloses a molecular marker closely linked with a main-effect QTL of drought resistance of cowpea and primer sequences of the molecular marker. The main-effect QTL of drought resistance of cowpea disclosed by the application is named DC3_00099, and a KASP type molecular marker 3_00099 closely linked with the main-effect QTL is located at a position of 3123287 bp of a No.4 chromosome. The marker can be used for amplifying to-be-tested materials, and whether the sample carries a key allelic variation for controlling drought resistance can be judged according to the genotype. The molecular marker and the primer provided by the application can be used for rapidly screening the main-effect QTL of drought resistance of cowpea, and compared with a traditional phenotype identification method, the application has the advantages of high detection flux, accurate and reliable result and the like, and the genotyping accuracy can reach more than 99%. The technology can significantly improve the breeding efficiency of the drought-resistant variety of cowpea and shorten the breeding period.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant molecular breeding, and relates to a molecular marker closely linked to a drought tolerance major QTL (DC3_00099) of cowpea, primers thereof and application. BACKGROUND

[0002] Cowpea (Vigna unguiculata (L.) Walp.) is an important crop of the genus Vigna in the family Fabaceae, with a genome size of about 620 Mb and a chromosome number of 2n=2x=22. As a legume crop widely cultivated in arid and semiarid regions, cowpea is one of the important edible legumes and vegetable crops.

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

[0004] With the completion of cowpea genome sequencing and the development of molecular marker technology, SNP (Single Nucleotide Polymorphism)-based molecular marker-assisted selection provides a new technical means for cowpea drought tolerance breeding. Among them, the Kompetitive Allele Specific PCR (KASP) technology has become an important tool for crop molecular breeding due to its high throughput, low cost, and high accuracy. KASP technology has achieved remarkable results in drought tolerance breeding of crops such as rice and wheat, but its application in cowpea drought tolerance breeding is still in its infancy. SUMMARY

[0005] The technical problem solved by the present application is to use KASP markers for assisted breeding of cowpea drought tolerance, to provide a molecular marker closely linked to the major QTL (DC3_00099) of cowpea drought tolerance and its KASP primers, and to establish a drought tolerance assisted breeding method based on the marker. This method identifies SNP sites related to drought tolerance through genome-wide association analysis, converts them into KASP markers for verification, and can be used to quickly identify the drought tolerance genotype of cowpea germplasm, providing molecular breeding technical support for genetic improvement of cowpea drought tolerance.

[0006] The present application identifies a major drought tolerance SNP site (DC3_00099) located at position 3123287 bp on chromosome 4 of cowpea through genome-wide association analysis (GWAS), and develops a KASP molecular marker closely linked to it. The development of this marker provides an effective technical means for cowpea drought tolerance molecular breeding, which will significantly improve breeding efficiency and shorten breeding cycle.

[0007] To achieve the above object, the present application provides the following technical solutions.

[0008] The present application provides a molecular marker closely linked to a drought tolerance major QTL of cowpea, which is 3_00099, located at 3123287 bp of chromosome 4 of the genome of a cowpea variety G98, is of KASP type, and the SNP site polymorphism is A / G.

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

[0010] The present application also provides a KASP primer for detecting the above molecular marker, which comprises a specific forward primer Primer1, a specific forward primer Primer2 and a common reverse primer Primer_Common.

[0011] 3_00099 Primer1 is shown as SEQ ID NO. 2.

[0012] 3_00099 Primer2 is shown as SEQ ID NO. 3.

[0013] 3_00099 Primer1 and 3_00099 Primer2 are two specific primers, respectively connected with different fluorescent sequences.

[0014] 3_00099 Primer_Common is shown as SEQ ID NO. 4.

[0015] Preferably, 3_00099 Primer1 is connected with a FAM group, and 3_00099 Primer2 is connected with a HEX group.

[0016] The present application also provides an application of the above KASP primer, or a reagent or kit containing the above KASP primer in detecting a drought tolerance major QTL of cowpea.

[0017] The present application also provides a method for breeding cowpea varieties with different drought tolerance by using the above KASP primer, which specifically comprises the following steps.

[0018] S1, extracting genomic DNA of a cowpea plant sample;

[0019] S2, using the genomic DNA of the cowpea plant sample as a template, and performing KASP reaction detection by using 3_00099 KASP primer;

[0020] S3, reading the fluorescence signal of the KASP reaction detection, if the Vigna unguiculata plant sample genomic DNA shows the fluorescence group signal connected by 3_00099 Primer1, it is judged that the sample carries the drought tolerance trait genotype; if the Vigna unguiculata plant sample genomic DNA shows the fluorescence group signal connected by 3_00099 Primer2, it is judged that the sample carries the drought sensitive trait genotype.

[0021] As preferred, Primer1 is connected with FAM group, and Primer2 is connected with HEX group.

[0022] As preferred, the KASP reaction detection further comprises 2X KASP Master mix reagent.

[0023] As preferred, the PCR system of the KASP reaction detection is: DNA 0.8 μl, 2x KASP Master mix 0.75 μl, Primer mix 0.05 μl. The Primer mix is the mixture of Primer1 and Primer_Common, or Primer2 and Primer_Common, each primer solution is 0.025 μl, and the concentration is 10 μM / L.

[0024] As preferred, the PCR reaction program of the KASP reaction detection is: 94℃ pre-denaturation for 15 minutes, 94℃ denaturation for 20 seconds, 61-55℃ gradient recombination for 60 seconds, each cycle recombination temperature decreases by 0.6℃, 55℃ extension for 60 seconds, 10 cycles; then 94℃ denaturation for 20 seconds, 55℃ recombination for 60 seconds, extension for 60 seconds, 26 cycles.

[0025] The name terms involved in the present application are:

[0026] Quantitative trait loci (QTL) refers to the locus of genes controlling quantitative traits in the genome.

[0027] Major QTL refers to the quantitative trait locus with greater influence or more significant effect on the quantitative traits studied.

[0028] Kompetitive Allele Specific PCR (KASP) is a kind of end-point fluorescence genotyping technology based on known SNP (single nucleotide polymorphism), which can accurately detect SNP and indel (insertion and deletion) at specific sites in DNA samples.

[0029] Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by single nucleotide variation at the genome level.

[0030] The advantages and beneficial effects of the present application are as follows:

[0031] (1) The molecular marker 3_00099 disclosed in the present application is closely linked to the main-effect QTL of drought tolerance (r²=0.95), and the typing accuracy is more than 99%, which can realize early and accurate identification of drought tolerance of cowpea.

[0032] (2) Compared with the traditional drought tolerance identification method, the present method is not limited by environmental conditions, and can be detected at the seedling stage, which significantly shortens the breeding period (from 2-3 years to 1 month) and reduces the breeding cost.

[0033] (3) The marker can effectively distinguish drought-tolerant and drought-sensitive genotypes, and provides a reliable molecular tool for breeding drought-tolerant cowpea varieties, which is particularly suitable for cowpea breeding work in coastal and drought areas. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0035] Figure 1 Figure 3 is a KASP marker genotyping diagram of the 3_00099 marker site related gene;

[0036] Figure 2 Figure 4 is a drought tolerance allelic variation difference diagram of the 3_00099 marker site. DETAILED DESCRIPTION

[0037] In order to further understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments.

[0038] Unless otherwise specified, the experimental methods used in the following examples are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0039] Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0040] The PCR amplification system used in the KASP reaction of the embodiment of the present application is: 0.8 μl of DNA, 0.75 μl of 2x KASP Master mix, and 0.05 μl of primer mix. The primer mix is a mixture of primer 1 and primer common, or primer 2 and primer common, each primer solution being 0.025 μl, and the concentration being 10 μM / L.

[0041] The PCR amplification procedure adopted by the embodiment of the present application is: 94℃ pre-denaturation for 15 minutes, 94℃ denaturation for 20 seconds, 61-55℃ gradient recombination for 60 seconds, recombination temperature decreasing by 0.6℃ for each cycle, 55℃ extension for 60 seconds, 10 cycles; then 94℃ denaturation for 20 seconds, 55℃ recombination for 60 seconds, and extension for 60 seconds, 26 cycles.

[0042] Example 1: Screening of SNP sites closely linked to the main QTL of drought resistance of cowpea

[0043] The present application collects and screens a cowpea natural population containing 344 materials for drought resistance phenotype identification. A randomized block design is used, each material is planted with 9 repeats, and under drought stress conditions (stopping watering for 40 days), leaf wilting degree, aboveground fresh weight, stem greenness and other indicators are measured for drought resistance comprehensive score, in addition, each material sets a group (three) of normal watering, and the rest conditions are consistent with the control group.

[0044] The drought resistance comprehensive score is the sum of the three drought phenotype scores of comprehensive stem greenness (0-2 points), aboveground fresh weight (0-2 points), and leaf wilting degree (0-2 points). Among them, the stem greenness: first count the color condition of the stem of cowpea under drought treatment, the typical plant with the most yellow and dry stem is 0, the typical plant with the most green and full stem is 2, the typical plant with the intermediate type is 1, and the score is 0-2 according to the fitting degree of each stem state with different typical plants; the fresh weight score is calculated according to the single plant weight percentage of the control group: greater than or equal to 60% is 2 points, in the 60%-50% (including) interval is 1.5 points, in the 50%-40% (including) interval is 1 point, in the 40%-30% (including) interval is 0.5 points, and less than 30% is 0 point; the leaf wilting degree is determined by color comparison through photography, the most green and leafy plant is 2 points, and the most withered and yellow-brown plant is 0 point, each 0.5 point is divided into five grades.

[0045] Meanwhile, the population material was resequenced by using the Illumina platform to obtain 1,856,342 high-quality SNP markers. Combined with various quality drought tolerance comprehensive scores, whole genome association analysis was carried out by mixed linear model (MLM), and a major QTL (DC3_00099) was identified at 3123287 bp of chromosome 4. The site is closely linked to a gene encoding IDD14 protein transcription factor. Based on this, KASP marker 3_00099 was developed, and the polymorphism thereof is A / G.

[0046] The genotypes of part of the materials in 344 accessions and the drought tolerance comprehensive scores thereof are shown in Table 1.

[0047] Table 1: Genotypes of part of the materials in 344 and drought tolerance comprehensive scores thereof

[0048]

[0049] The KASP marker 3_00099 screened in the embodiment is located on chromosome 4 of the cowpea variety G98 genome, and the SNP site is located at 3123287 bp, and the polymorphism is A / G.

[0050] The nucleotide sequence of the KASP marker is shown as SEQ ID NO. 1, and the SNP site is the 101st site in the sequence.

[0051] The present application designs a series of KASP primers according to the sequence. The KASP primers of the molecular marker include a specific forward primer Primer1, a specific forward primer Primer2 and a common reverse primer Primer_Common, and the sequences thereof from 5' to 3' are as follows.

[0052] 3_00099 Primer1 is shown as SEQ ID NO. 2;

[0053] 3_00099 Primer2 is shown as SEQ ID NO. 3;

[0054] 3_00099 Primer_Common is shown as SEQ ID NO. 4.

[0055] In some specific embodiments, Primer1 is connected with a FAM group, and Primer2 is connected with a HEX group.

[0056] Example 2:

[0057] In this embodiment, 58 cowpea accessions obtained from Xianghu Laboratory (Agricultural Zhejiang Provincial Laboratory) were randomly selected.

[0058] Phenotypic identification of drought tolerance was performed on 58 randomly selected cowpea germplasms. Soil culture method was used for drought treatment. Leaf wilt, aboveground fresh weight, stem greenness and other indicators were measured after 40 days of drought treatment (water deprivation) at the three-leaf stage of seedlings. In addition, a group of (three) normal watering, and the rest of the control group were set up.

[0059] Genomic DNA was extracted by CTAB method, and KASP analysis was performed using the IntelliQube genotyping platform, KASP primers of DC3_00099 designed in Example 1, and genomic DNA of the above population as amplification template. PCR reaction system: DNA 0.8 μl, 2x KASP Master Mix 0.75 μl, primer mix 0.05 μl. PCR program: 94℃ pre-denaturation for 15 min; 10 cycles of falling PCR (94℃ 20s, 61-55℃ 60s); 26 cycles of normal PCR (94℃ 20s, 55℃ 60s).

[0060] The primer mix is obtained by mixing Primer 1 and Primer Common, or Primer 2 and Primer Common, and the volume of each primer is 0.025 μl, and the concentration is 10 μM / L.

[0061] The IntelliQube machine was used for fluorescence data reading and analysis, and the analysis results are shown in Figure 1 . When the fluorescence signal is red (FAM signal), it indicates that the cowpea carries the drought-tolerant allele A (Hap I). When the fluorescence signal is blue (HEX signal), it indicates that the cowpea carries the sensitive allele G (Hap II).

[0062] As shown in Figure 1 , the KASP molecular marker of the application achieves a highly consistent and clear population typing effect in the cowpea population, and the two genotypes can be clearly distinguished and clustered, with good typing effect.

[0063] Combined with its drought tolerance performance (see Figure 2 , some data are shown in Table 2), it was found that the average drought tolerance comprehensive score of the strain carrying the A genotype was higher, while the average drought tolerance comprehensive score of the strain carrying the G genotype was relatively lower. Data analysis showed that the average drought tolerance of the strain carrying the A genotype was significantly higher than that of the G genotype (p = 0.0178), which confirmed that the marker could effectively distinguish the difference in drought tolerance of cowpea, and finally achieved the purpose of molecular marker screening.

[0064] Table 2. Genotype and drought treatment phenotype data statistics of some selected germplasms

[0065]

[0066] Note: the drought tolerance comprehensive score standard is consistent with that of Example 1, which is the sum of the three drought phenotype scores of the comprehensive chlorophyll content (0-1), the aboveground fresh weight (0-2), and the leaf wilting degree (0-2).

[0067] The above description is merely preferred examples of the present application, and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made to the present application should be included in the scope of the present application.

Claims

1. A molecular marker closely linked to a major QTL for drought tolerance in Vigna unguiculata, characterized in that, The molecular marker is 3_00099, which is a KASP type; the SNP site of the molecular marker is located at 3123287 bp on chromosome 4 of the G98 genome of the cowpea variety, and the polymorphism is A / G; the nucleotide sequence of the molecular marker is shown as SEQ ID NO.

1.

2. A KASP primer for detecting the molecular marker of claim 1, characterized in that, The KASP primer includes 3_00099 Primer1, 3_00099 Primer2 and 3_00099 Primer_Common. 3_00099 Primer1 is shown as SEQ ID NO.

2. 3_00099 Primer2 is shown as SEQ ID NO.

3. 3_00099 Primer_Common is shown as SEQ ID NO.

4.

3. A KASP primer according to claim 2, or a kit containing the KASP primer of claim 2, for use in detecting drought tolerance major QTL in cowpea.

4. A method for breeding cowpea varieties with different drought tolerance using the KASP primer of claim 2, characterized in that, The method specifically comprises the following steps: S1, extracting cowpea plant sample genomic DNA; S2, using the KASP primer of claim 2 to perform KASP reaction detection with the cowpea plant sample genomic DNA as a template; S3, reading the fluorescence signal of the KASP reaction detection, if the cowpea plant sample genomic DNA shows the FAM group signal corresponding to 3_00099 Primer1, it is judged that the sample carries the drought tolerance trait genotype; if the cowpea plant sample genomic DNA shows the HEX group signal corresponding to 3_00099 Primer2, it is judged that the sample carries the drought sensitive trait genotype.

5. The method for breeding cowpea varieties with different drought tolerance according to claim 4, characterized in that, The KASP reaction detection further includes 2X KASP Master mix reagent.

6. The method for breeding cowpea varieties with different drought tolerance according to claim 5, characterized in that, The PCR system of the KASP reaction detection is: 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 drought tolerance according to claim 4, wherein, The PCR reaction program of the KASP reaction detection is: 94℃ pre-denaturation for 15 minutes, 94℃ denaturation for 20 seconds, 61-55℃ gradient recombination for 60 seconds, recombination temperature decreasing by 0.6℃ for each cycle, 55℃ extension for 60 seconds, cycling 10 times; then 94℃ denaturation for 20 seconds, 55℃ recombination for 60 seconds, extension for 60 seconds, cycling 26 times.

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