Cowpea SNP (Single Nucleotide Polymorphism) molecular marker combination, SNP chip and application thereof

Abstract

The invention discloses a cowpea SNP molecular marker combination, and belongs to the technical field of molecular breeding. The cowpea SNP molecular marker combination is composed of 53131 SNP molecular markers, the physical positions of the 53131 SNP molecular markers are determined by comparison based on a cowpea reference genome IT97K-499-35, and the physical positions of the SNP molecular markers are shown in the specification table 1. Compared with the prior art, the SNP molecular marker combination is uniformly distributed on 11 chromosomes of cowpea, and the polymorphism and the detection rate are high. The invention further provides an SNP liquid phase chip for detecting the cowpea SNP molecular marker combination, theoretical and data support is provided for work such as cowpea new variety identification, gene positioning, molecular marker-assisted selection breeding, whole genome selection and population genetic diversity analysis, and the SNP liquid phase chip has important theoretical value and application significance.

Description

Technical Field

[0001] The invention relates to a cowpea SNP molecular marker combination, a SNP chip and application thereof, and belongs to the technical field of molecular breeding. Background Art

[0002] Cowpea (Vigna unguiculata L.Walp) is an important legume vegetable crop in my country. It is rich in lysine and low in sulfur amino acids. It has high nutritional value and can be complemented with cereal crops to improve the protein quality in the national diet. China is considered to be the secondary origin center of cowpea and has rich germplasm resources. At present, my country has collected and preserved more than 5,000 cowpea germplasm resources from home and abroad. Many domestic research institutions have analyzed the genetic diversity of different germplasms through agronomic trait surveys and molecular marker identification, providing a theoretical basis for germplasm innovation. However, compared with major crops, cowpea is a minor crop. Although the whole genome sequence has been published, most studies are still using traditional markers such as SSR and RAPD with limited flux and accuracy, which cannot meet the actual needs of modern biological breeding for precision and efficiency. The production seeds are messy, the resource base is insufficient, the genetic research is lagging behind, and the genetic diversity and kinship information of resources are limited, which greatly hinders the genetic improvement of cowpea in my country and the development of the cowpea industry.

[0003] The rapid development of high-throughput sequencing technology and the continuous reduction of sequencing costs have made it possible to obtain effective genetic information of germplasm resources accurately and at high throughput. As a key support for genomics, genetics and molecular breeding, genotyping platforms have been maturely applied in multinational seed companies with high-throughput, automated, large-scale and shared facilities. Targeted sequencing genotyping detection technology based on liquid phase probe capture has the characteristics of high throughput, low cost, wide adaptability and flexible application. It has been developed and utilized in more than 20 major crops such as rice, corn, and wheat, and is widely used in germplasm resource evaluation, molecular biological breeding, variety rights protection, seed quality monitoring and other fields. However, there is no cowpea liquid phase detection chip product available yet. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the above-mentioned prior art and provide a cowpea SNP molecular marker combination and a SNP chip. The SNP marker combination is evenly distributed on the 11 chromosomes of cowpea, and has high polymorphism and detection rate. The liquid probe chip based on the SNP marker has the characteristics of high labeling density, strong automation, and high detection throughput, and can be used for the identification of new cowpea varieties, gene positioning, molecular marker-assisted selection breeding, whole genome selection, population genetic diversity analysis, etc.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A cowpea SNP molecular marker combination, the molecular marker combination consists of 53131 SNP molecular markers, the physical positions of the 53131 SNP sites are determined based on the cowpea reference genome IT97K-499-35 comparison, the specific SNP molecular marker situation is shown in Table 1, and the position and variation information of the SNP site are expressed in the form of chromosome_physical position: reference genotype / variant allele type in Table 1.

[0007] Table 1 SNP molecular marker information

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[0082] The present invention is based on the whole genome resequencing data of 463 cowpea germplasm resources collected, and after filtering and analysis, 53131 SNP molecular markers are screened out, which are evenly distributed on the 11 chromosomes of cowpea, with high polymorphism and detection rate.

[0083] The present invention also provides a SNP chip for detecting the above cowpea SNP molecular marker combination, and the chip is provided with probes for detecting the above SNP molecular marker combination.

[0084] Furthermore, the SNP chip is a liquid phase chip.

[0085] The present invention also provides the application of the SNP chip in the identification of new cowpea varieties, gene positioning, molecular marker-assisted selection breeding, whole genome selection, and population genetic diversity analysis.

[0086] Beneficial effects of the present invention:

[0087] The present invention discloses a cowpea SNP molecular marker combination, a SNP chip and an application thereof. The SNP molecular marker combination consists of 53131 SNP molecular markers. The physical positions of the SNP molecular markers on the cowpea reference genome IT97K-499-35 are shown in Table 1 of the specification. The SNP molecular marker combination is obtained by filtering, analyzing and screening 463 copies of whole genome resequencing data of cowpea resource materials after alignment to the cowpea reference genome IT97K-499-35. The SNP molecular marker combination is evenly distributed on 11 chromosomes of cowpea, and has high polymorphism and detection rate. The present invention also provides a SNP liquid phase chip for detecting the cowpea SNP molecular marker combination, which provides theoretical and data support for the identification of new cowpea varieties, gene positioning, molecular marker-assisted selection breeding, whole genome selection, population genetic diversity analysis and other work, and has important theoretical value and application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is the PCA scatter plot of 463 cowpea resource samples based on 237,329 SNP loci;

[0089] Figure 2 The cluster analysis results of 463 cowpea resource samples based on 237,329 SNP loci;

[0090] Figure 3This is the density distribution map of 53131 SNP markers on the genome;

[0091] Figure 4 This is the PCA scatter plot of 463 cowpea resource samples based on 53,131 SNP loci;

[0092] Figure 5 The cluster analysis results of 463 cowpea resource samples based on 53,131 SNP loci;

[0093] Figure 6 PCA scatter plot of 263 cowpea test samples based on 53,131 SNP loci;

[0094] Figure 7 Cluster analysis results based on 53,131 SNP loci for 263 cowpea test samples. DETAILED DESCRIPTION

[0095] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0096] Example 1

[0097] A method for screening SNP molecular marker combinations and designing SNP chips based on 463 cowpea resource whole genome resequencing data specifically includes the following process:

[0098] (1) The genomic DNA of 150 cowpea germplasm leaf tissues was extracted by magnetic bead method. After the DNA quality inspection was qualified, the whole genome resequencing library was constructed using DNA library preparation reagent kit. Fragments in the range of 260-360 bp were selected, enriched and amplified, and the sample concentration was detected by Qubit fluorescence quantification instrument. The length of sample fragments was detected by Agilent2100 system. After the library inspection was qualified, sequencing was performed using BGI DNBSEQ-T7. The average sequencing depth was 10×, and the sequencing amount of each sample was about 6.5 Gb.

[0099] (2) The clean data obtained by sequencing in step (1) was integrated with the clean data of 313 published cowpea resource resequencing data collected, and the cowpea IT97K-499-35 genome was used as the reference genome. The BWA software was used to align the clean data of all 463 cowpeas to the reference genome, and the GATK software was used to detect and filter the variant sites to obtain 3,081,228 high-quality SNP sites.

[0100] (3) The SNP sites selected in step (2) were further filtered using vcftools software to select dimorphic SNP sites with heterozygosity rate <0.2, site deletion rate <0.2, minimum allele frequency >0.01, and sequencing coverage depth >5×, and a total of 237,329 high-quality SNP sites were obtained.

[0101] The population structure of 463 cowpea resources was analyzed using 237,329 SNP loci. Figure 1 and Figure 2 . Figure 1 This is a PCA (principal component analysis) scatter plot obtained through Plink software analysis. Figure 2 The results of calculating the genetic distance matrix and cluster analysis using Plink software show that the 463 cowpea materials can be divided into two subgroups, G1 and G2.

[0102] (4) The SNP sites screened out in step (3) were used to screen SNP markers suitable for designing probes using a perl program script, and ultimately 46,494 SNP molecular markers were obtained.

[0103] The screening principle is as follows: bedtools software is used to extract SNP sites and sequences 100 bp upstream and downstream thereof, and GC content between 40-60% is retained, and there are no SSR sequences, no insertions or deletions of more than 5 bp, and no N-base SNP sites in the sequences; blastn software is used to align the extracted sequences to the cowpea IT97K-499-35 genome, and only single-copy (similarity greater than 60% is considered to be one copy) SNP sites are retained.

[0104] (5) Collect and organize relevant published literature, explore loci and genes related to cowpea traits such as 100-grain weight, flowering period, resistance to bean weevils, drought, fruit length, and resistance to aphids, and obtain 6,829 SNP markers suitable for designing probes based on the corresponding loci and gene intervals. These were integrated with the aforementioned 46,494 SNP molecular markers, and duplicate loci were removed to finally obtain 53,131 SNP molecular markers. All loci are evenly distributed on the 11 chromosomes of cowpea, with an average spacing of 8.9 kb and an average PIC (polymorphic information content) value of 0.22. The loci information is shown in Table 1, and the density distribution of the loci on the cowpea genome is shown in Figure 3 .

[0105] (6) Using the 53,131 SNP loci obtained in step (5), a population structure analysis was performed on 463 cowpea resources. The results are shown in Figure 4 and Figure 5 . Figure 4 is the PCA result, Figure 5is the cluster analysis result. It can be seen that the analysis results of 53131 SNPs are basically consistent with those of 237329 SNPs, and both can separate the two populations. This shows that the 53131 SNP marker combination selected is representative and can be used for the analysis of cowpea population structure.

[0106] (7) Based on the 53131 SNP molecular markers obtained in step (5), a liquid phase probe for detecting each SNP molecular marker is designed to construct a cowpea 50K liquid phase chip that can capture 53131 SNP sites at one time for subsequent capture sequencing.

[0107] Example 2

[0108] A method for performing cowpea genotyping using the cowpea 50K liquid phase chip described in Example 1 specifically includes the following steps:

[0109] (1) Genomic DNA extraction

[0110] The cowpea sample leaves were collected, stored with ice packs and transported back to the laboratory in time, and the total DNA of the samples was extracted using the magnetic bead method.

[0111] (2) Genomic DNA library construction and hybridization capture

[0112] The DNA sample was digested with fragmentase, the digested ends were repaired, and the A base was added to the 3' end. The fragment size was detected by agarose gel electrophoresis. The sequencing adapter, index, and P5 and P7 sequences were connected using T4 ligase, and the connection products were purified using magnetic beads. The concentration of the purified products was detected by Qubit fluorescence quantification instrument, and the fragment size was detected by agarose gel electrophoresis. The purified connection products were PCR amplified, and the amplified products were screened for fragments using magnetic beads. 750ng of the constructed library was taken, and after adding probes and hybridization reagents, it was incubated at 50℃ overnight to complete the hybridization reaction. The target segment was captured using streptavidin magnetic beads, and the captured products were washed with cleaning solution to remove non-specific binding fragments, and PCR amplification was performed after capture. 1μL of the library was taken, and the library concentration was detected using Qubit fluorescence quantification instrument. The library concentration was about 1-20ng / μL; the fragment length was determined using Agilent2100 system. After the concentration and fragment size were qualified, the sequencing library construction was completed.

[0113] (3) High-throughput sequencing

[0114] The library obtained after capture and amplification in step (2) was subjected to high-throughput sequencing using the MGI DNBSEQ-T7 sequencing platform to obtain DNA sequencing results, and the obtained data was subjected to basic cleaning processing.

[0115] (4) Bioinformatics analysis

[0116] The cleaned sequencing data were aligned to the cowpea reference genome IT97K-499-35 using the bwa software, and the SNP genotyping data of the cowpea varieties were obtained using the GATK software; the fingerprint analysis of the cowpea varieties was completed using the SNP genotyping data.

[0117] Example 3

[0118] An application of a cowpea SNP molecular marker combination and a 50K liquid phase chip in cowpea resource population structure analysis specifically includes the following processes:

[0119] (1) Experimental Materials

[0120] A total of 263 cowpea germplasm resources from a wide range of sources were selected. Sample leaves were collected, refrigerated and transported to the laboratory in a timely manner, and the total DNA of each sample was extracted using the magnetic bead method.

[0121] (2) Liquid chip capture rate evaluation

[0122] The 50K liquid phase chip provided by the present invention, which contains 53,131 SNP molecular marker combinations of cowpea, was used to detect DNA of 263 cowpea materials, and the target site genotyping was extracted. The average detection rate of the samples was 98.98%, indicating that the SNP chip disclosed by the present invention has a high detection rate of target sites and the typing results are accurate and reliable.

[0123] (3) Population genetic analysis

[0124] Based on the genotyping data of 53131 SNP markers, population genetic analysis was performed on 263 cowpea materials. Plink software was used to analyze the PCA components of the test materials and construct a scatter plot. Plink software was used to calculate the genetic distance matrix, perform cluster analysis on the test materials, and construct a phylogenetic tree. The results are shown in Figure 6 and Figure 7 . Figure 6 PCA scatter plot of 263 cowpea test samples based on 53,131 SNP loci; Figure 7 The cluster analysis results of 263 cowpea test samples based on 53131 SNP loci. From the results, both principal component analysis and cluster analysis can divide all materials into two subgroups, and the clustering results are consistent with the actual clustering. The PCA and phylogenetic tree results are consistent with expectations, proving that 53131 SNP markers can better distinguish different cowpea materials.

[0125] The specific raw materials involved in the embodiments of the present invention are all existing substances and can be directly purchased from the market.

[0126] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. A cowpea SNP molecular marker combination, characterized in that: The molecular marker combination consists of 53131 SNP molecular markers, and the physical positions of the 53131 SNP molecular markers are determined based on the comparison with the cowpea reference genome IT97K-499-35. The physical positions of the SNP molecular markers are shown in Table 1 of the specification. The positions and variation information of the SNP molecular markers are expressed in the form of chromosome_physical position: reference genotype / variant allele type in Table 1.

2. A cowpea SNP chip, characterized in that: The chip comprises probes for detecting the cowpea SNP molecular marker combination according to claim 1.

3. The SNP chip according to claim 2, characterized in that: The SNP chip is a liquid phase chip.

4. Application of the SNP chip according to claim 2 or 3 in identification of new cowpea varieties, gene mapping, molecular marker-assisted selection breeding, whole genome selection or population genetic diversity analysis.

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