A combination of SNP molecular markers in cowpea, an SNP chip and its application

Abstract

This invention discloses a cowpea SNP molecular marker combination, belonging to the field of molecular breeding technology. The cowpea SNP molecular marker combination consists of 53,131 SNP molecular markers. The physical locations of these 53,131 SNP molecular markers are determined based on alignment with the cowpea reference genome IT97K-499-35, as shown in Table 1 of the specification. Compared with existing technologies, this SNP molecular marker combination is evenly distributed across the 11 chromosomes of cowpea, exhibiting high polymorphism and detection rate. This invention also provides an SNP liquid phase chip for detecting the cowpea SNP molecular marker combination, providing theoretical and data support for cowpea new variety identification, gene mapping, molecular marker-assisted selection breeding, genome-wide selection, and population genetic diversity analysis, possessing significant theoretical value and application significance.

Description

Technical Field

[0001] This invention relates to a combination of SNP molecular markers in cowpea, an SNP chip, and their applications, belonging to the field of molecular breeding technology. Background Technology

[0002] Cowpea (Vigna unguiculata L. Walp) is an important legume vegetable crop in my country. Rich in lysine and low in sulfur-containing amino acids, it has high nutritional value and can be complemented by cereal crops to improve the protein quality in the national diet. China is considered a secondary center of origin for cowpea and possesses abundant germplasm resources. Currently, my country has collected and preserved over 5,000 cowpea germplasm resources from both domestic and international sources. Multiple research institutions in China 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 to major crops, cowpea is a minor crop. Although its whole genome sequence has been published, most studies still use traditional markers such as SSR and RAPD, which have limited throughput and accuracy, failing to meet the precise and efficient needs of modern bio-breeding. The diverse varieties used in production, insufficient resource inventory, lagging genetic research, and limited information on resource genetic diversity and phylogenetic relationships significantly hinder the genetic improvement of cowpea and the development of the cowpea industry in my country.

[0003] The rapid development of high-throughput sequencing technology and the continuous reduction in sequencing costs have made it possible to accurately and efficiently obtain effective genetic information from germplasm resources. Genotyping platforms, as key supports for genomics, genetics, and molecular breeding, have been successfully 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 features high throughput, low cost, wide applicability, and flexible application. It has been developed and utilized in more than 20 major crops, including rice, corn, and wheat, and is widely used in germplasm resource evaluation, molecular biological breeding, variety rights protection, and seed quality monitoring. However, no liquid-phase detection chip product for cowpeas has yet been launched. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing a cowpea SNP molecular marker combination and SNP chip. This SNP marker combination is evenly distributed on the 11 chromosomes of cowpea, with high polymorphism and detection rate. The liquid-phase probe chip based on this SNP marker has the characteristics of high marker density, strong automation, and high detection throughput, and can be used for cowpea new variety identification, gene mapping, 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 solutions:

[0006] A cowpea SNP molecular marker assemblage, comprising 53,131 SNP molecular markers, wherein the physical locations of the 53,131 SNP loci are determined based on alignment with the cowpea reference genome IT97K-499-35. The specific SNP molecular marker information is shown in Table 1. The location and variation information of the SNP loci are represented in Table 1 in the form of chromosome_physical location:reference genotype / variant allele.

[0007] Table 1 SNP molecular marker information

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[0082] Based on whole-genome resequencing data from 463 cowpea germplasm resources collected in this invention, after filtering and analysis, 53,131 SNP molecular markers were screened out, which were evenly distributed on the 11 chromosomes of cowpea, with high polymorphism and detection rate.

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

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

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

[0086] The beneficial effects of this invention are:

[0087] This invention discloses a cowpea SNP molecular marker combinatorial system, an SNP microarray, and their applications. The SNP molecular marker combinatorial system consists of 53,131 SNP molecular markers. The physical locations of these SNP molecular markers on the cowpea reference genome IT97K-499-35 are shown in Table 1 of the specification. This SNP molecular marker combinatorial system was obtained by aligning whole-genome resequencing data from 463 cowpea resource materials to the cowpea reference genome IT97K-499-35, followed by filtering, analysis, and screening. This SNP molecular marker combinatorial system is evenly distributed across the 11 cowpea chromosomes, exhibiting high polymorphism and detection rate. This invention also provides an SNP liquid-phase microarray for detecting cowpea SNP molecular marker combinatorial systems, providing theoretical and data support for cowpea new variety identification, gene mapping, molecular marker-assisted selection breeding, whole-genome selection, and population genetic diversity analysis, and has significant theoretical and practical value. Attached Figure Description

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

[0089] Figure 2 Cluster analysis results based on 237,329 SNP sites for 463 cowpea resource samples;

[0090] Figure 3Density distribution of 53,131 SNP markers on the genome;

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

[0092] Figure 5 Cluster analysis results based on 53,131 SNP sites for 463 cowpea resource samples;

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

[0094] Figure 7 The results of cluster analysis based on 53,131 SNP sites were used to analyze 263 cowpea samples. Detailed Implementation

[0095] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0096] Example 1

[0097] A method for screening SNP molecular marker combinations and designing SNP chips based on whole-genome resequencing data from 463 cowpea resources, specifically including the following steps:

[0098] (1) Genomic DNA was extracted from leaf tissues of 150 cowpea germplasm resources collected by magnetic bead method. After the DNA quality inspection was qualified, a whole genome resequencing library was constructed using a DNA library preparation kit. Fragments in the range of 260-360bp were selected, enriched and amplified, and the sample concentration was detected by Qubit fluorescence quantitative instrument. The sample fragment length was detected by Agilent 2100 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 6.5Gb.

[0099] (2) The clean data obtained from sequencing in step (1) was integrated with the clean data of the 313 published cowpea resequencing data. Using the cowpea IT97K-499-35 genome as the reference genome, the clean data of all 463 cowpea genomes were aligned to the reference genome using BWA software. The variant sites were detected and filtered using GATK software 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 screen for binary SNP sites with heterozygosity < 0.2, site deletion rate < 0.2, minimum allele frequency > 0.01, and sequencing coverage depth > 5×, resulting in a total of 237,329 high-quality SNP sites.

[0101] Population structure analysis was performed on 463 cowpea accessions using 237,329 SNP loci. The results are shown in […]. Figure 1 and Figure 2 . Figure 1 This is a scatter plot of PCA (Principal Component Analysis) obtained using Plink software. Figure 2 The results show the genetic distance matrix calculated and cluster analysis performed using Plink software. It can be seen that the 463 cowpea accessions can be divided into two subpopulations, G1 and G2.

[0102] (4) The SNP sites selected in step (3) were further screened using a Perl script to select suitable SNP markers for probe design, resulting in 46,494 SNP molecular markers.

[0103] The screening criteria are as follows: SNP sites and their upstream and downstream 100bp sequences are extracted using bedtools software, and SNP sites with GC content between 40-60%, no SSR sequences, no insertions or deletions of more than 5bp, and no N bases are retained. The extracted sequences are aligned to the cowpea IT97K-499-35 genome using blastn software, and only single-copy (similarity greater than 60% is considered as one copy) SNP sites are retained.

[0104] (5) Published literature was collected and organized to identify loci and genes related to traits such as cowpea seed weight, flowering period, resistance to bean weevils, drought resistance, fruit length, and aphid resistance. Based on the corresponding loci and gene regions, 6829 SNP markers suitable for probe design were obtained. These were integrated with the aforementioned 46494 SNP molecular markers, and duplicate sites were removed, resulting in 53131 SNP molecular markers. All loci were evenly distributed across the 11 cowpea chromosomes, with an average spacing of 8.9 kb and an average PIC (polymorphism information content) value of 0.22. Locus information is detailed in Table 1, and the density distribution map of loci on the cowpea genome is shown in [reference needed]. Figure 3 .

[0105] (6) Using the 53,131 SNP loci obtained in step (5), population structure analysis was performed on 463 cowpea resources. The results are shown in […]. Figure 4 and Figure 5 . Figure 4 For PCA results, Figure 5The results are from the cluster analysis. It can be seen that the analysis results for 53,131 SNPs are basically consistent with those for 237,329 SNPs, both of which can separate the two populations. This indicates that the selected 53,131 SNP marker combinations are representative and can be used for cowpea population structure analysis.

[0106] (7) Based on the 53,131 SNP molecular markers obtained in step (5), a liquid phase probe for detecting the SNP molecular marker is designed for each SNP molecular marker, forming a cowpea 50K liquid phase chip that can capture 53,131 SNP sites at once for subsequent capture sequencing.

[0107] Example 2

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

[0109] (1) Genomic DNA extraction

[0110] Cowpea leaf samples were collected, stored with ice packs, and promptly transported back to the laboratory. Total DNA was extracted from the samples using the magnetic bead method.

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

[0112] DNA samples were digested using a fragmentation enzyme, the digested ends were repaired, and an A base was added to the 3' end. Fragment size was detected by agarose gel electrophoresis. Sequencing adapters, indexes, and P5 and P7 sequences were ligated using T4 ligase, and the ligation products were purified using magnetic beads. The concentration of the purified products was detected using a Qubit quantitative PCR instrument, and fragment size was detected by agarose gel electrophoresis. PCR amplification was performed on the purified ligation products, and fragments were screened using magnetic beads. 750 ng of the constructed library was taken, probes and hybridization reagents were added, and the mixture was incubated overnight at 50°C to complete the hybridization reaction. Target regions were captured using streptavidin magnetic beads, and the captured products were washed with washing buffer to remove non-specific binding fragments. Post-capture PCR amplification was then performed. 1 μL of the library was taken, and the library concentration was detected using a Qubit quantitative PCR instrument; the library concentration was approximately 1-20 ng / μL. Fragment length was determined using an Agilent 2100 system. Once the concentration and fragment size were satisfactory, the sequencing library construction was complete.

[0113] (3) High-throughput sequencing

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

[0115] (4) Bioinformatics Analysis

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

[0117] Example 3

[0118] The application of a cowpea SNP molecular marker combinatorial system and a 50K liquid phase chip in the analysis of cowpea resource population structure includes the following steps:

[0119] (1) Experimental materials

[0120] A total of 263 cowpea germplasm resources from a wide range of sources were selected. Leaf samples were collected, refrigerated, and promptly transported to the laboratory. Total DNA was extracted from each sample using the magnetic bead method.

[0121] (2) Evaluation of liquid phase chip capture rate

[0122] Using the 50K liquid phase chip containing 53,131 SNP molecular marker combinations of cowpea provided by this invention, the DNA of 263 cowpea materials was detected, and the genotypes of the target loci were extracted and classified. The average detection rate of the samples was 98.98%, which shows that the SNP chip disclosed in this invention has a high detection rate of target loci and the classification results are accurate and reliable.

[0123] (3) Population genetic analysis

[0124] Based on genotyping data from 53,131 SNP markers, population genetic analysis was performed on 263 cowpea accessions. The PCA composition of the accessions was analyzed using Plink software, and scatter plots were constructed. Genetic distance matrices were calculated using Plink software, and cluster analysis was performed on the accessions to construct phylogenetic trees. Results are shown below. Figure 6 and Figure 7 . Figure 6 PCA scatter plot based on 53,131 SNP sites for 263 cowpea test samples; Figure 7 Cluster analysis of 263 cowpea samples based on 53,131 SNP loci was performed. The results showed that both principal component analysis (PCA) and cluster analysis could classify all materials into two subgroups, and the grouping results were consistent with the actual grouping. The PCA and phylogenetic tree results were also in line with expectations, demonstrating that the 53,131 SNP markers can effectively distinguish different cowpea materials.

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

[0126] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. However, it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A cowpea SNP chip, characterized in that, The chip includes probes for detecting cowpea SNP molecular marker combinations, which consist of 53,131 SNP molecular markers. The physical locations of these 53,131 SNP molecular markers are determined based on alignment with the cowpea reference genome IT97K-499-35v1.

0. The physical locations of the SNP molecular markers are shown in Table 1 of the specification. The location and variation information of the SNP molecular markers are represented in Table 1 in the form of chromosome_physical location:reference genotype / variant allele.

2. The SNP chip as described in claim 1, characterized in that, The SNP chip is a liquid phase chip.

3. The application of the SNP chip as described in claim 1 or 2 in the identification of new cowpea varieties, gene mapping, molecular marker-assisted selection breeding, whole genome selection, or population genetic diversity analysis.

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