A 20K SNP liquid phase breeding chip for pea whole genome and its application

Abstract

This invention discloses a pea genome-wide 20K SNP liquid-phase breeding chip and its application. This chip contains 21,659 SNP loci from the pea genome, specifically based on agronomic trait association analysis and whole-genome resequencing data from pea samples, along with resequencing data from various varieties. High-quality, representative SNP loci of 21,659 were selected from these data. This chip can be widely used for pea genotyping, contributing to improved pea breeding efficiency and pea variety innovation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications and relates to a pea whole genome 20K SNP liquid phase breeding chip and its application. Background Technology

[0002] Peas (Pisum sativum L.) are an important food source, animal feed, and industrial raw material, possessing outstanding nutritional and health benefits. In recent years, significant progress has been made in breeding and improving important agronomic traits in peas. However, due to their large and complex genome, genomics-based complex phenotypic design breeding faces numerous challenges, severely limiting the rapid development of basic research and breeding. With the rise of computational breeding and bio-breeding, the breeding gap between peas and staple crops has further widened. To bridge this gap, there is an urgent need to develop breeding chips suitable for peas, which will help scientists more effectively identify and utilize promising genes, improve breeding efficiency, promote the innovation and improvement of pea varieties, and bring new growth points to agricultural production.

[0003] The development and application of gene chips have played a crucial role in accelerating the analysis of crop genetic characteristics, variety identification, resource conservation, and breeding. Through SNP chips, researchers can conduct population genetic structure analysis, identify candidate genes associated with economic traits, and implement various breeding studies such as genomic selection. Therefore, screening for molecular markers related to important economic traits and accelerating the breeding of pea varieties through cutting-edge molecular breeding technologies is essential.

[0004] Genome-wide association studies (GWAS) and genome-wide selection breeding are fundamental to modern breeding efforts. However, the high cost of resequencing and the low site density and flexibility of traditional solid-phase microarrays limit their application in large-genomic species such as peas and wheat. Therefore, developing an efficient, rapid, and low-cost large-scale genotyping tool will promote the development of pea molecular breeding in my country.

[0005] Locus information is fundamental to gene chip probe design, and locus selection is the core and crucial step in chip design, directly impacting the chip's practical value. For a long time, locus selection has primarily been based on criteria such as allele frequency, deletion rate, and heterozygosity. However, with the deepening of pea genomics research, more reference information has become available for locus selection and evaluation in novel chips.

[0006] Therefore, a new type of breeding chip needs to be developed to provide a new tool for pea breeding. Summary of the Invention

[0007] The purpose of this invention is to provide a pea whole-genome 20K SNP liquid-phase breeding chip and its applications to solve the aforementioned existing technical problems. This pea 20K SNP liquid-phase breeding chip can be widely used in the analysis of important pea traits, genetic diversity analysis of germplasm resources, variety identification analysis, assisted selection breeding, population structure analysis, genetic and evolutionary analysis, kinship identification, genome-wide association analysis, or genome selection. It can improve pea breeding efficiency, promote the innovation and improvement of pea varieties, and bring new growth points to agricultural production.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A liquid-phase breeding chip for 20K SNPs in the whole pea genome is characterized in that the genotyping target of the chip includes 21,659 SNP sites on the whole pea genome, as shown in Table 1 below.

[0010] The reference genome for the SNP locus is the pea genome “Pisum sativumcultivar Zhewan1(PeaZW1)”.

[0011] This invention also provides the application of the above-mentioned liquid phase chip in genotyping of pea samples.

[0012] The beneficial effects of this invention are:

[0013] 1. The pea 20K SNP liquid phase breeding chip of the present invention is based on association analysis of 57 agronomic traits and whole genome resequencing data of 237 pea samples. In addition, resequencing data of 77 varieties from previous studies were added. A total of 21,659 high-quality and representative SNP loci were obtained from these data, including 235 published reliable functional loci that are significantly associated with the 57 traits.

[0014] 2. Based on high-throughput sequencing technology, it outputs a large amount of information at one time, and can simultaneously detect nearly a thousand materials. It is suitable for mainstream second-generation sequencing platforms such as Illumina and MGI, and has broad platform adaptability.

[0015] 3. The pea 20K SNP liquid phase breeding chip of the present invention can be widely used in the analysis of important traits of peas, genetic diversity analysis of germplasm resources, variety identification analysis, assisted selection breeding, population structure analysis, genetic and evolutionary analysis, and kinship identification. It can improve the efficiency of pea breeding and promote the innovation and improvement of pea varieties. Attached Figure Description

[0016] Figure 1 This is a map showing the distribution of the SNP sites of this invention on the pea genome;

[0017] Figure 2 This is a diagram showing the number and spacing of SNP sites on each chromosome according to the present invention;

[0018] Figure 3 This is a graph showing the probe capture rate of the pea 20K SNP liquid phase breeding chip sites in 14 representative samples according to the present invention.

[0019] Figure 4 To construct a phylogenetic tree using the microarray genotyping results of 14 pea samples;

[0020] Figure 5 The figures shown are pea 20K SNP liquid phase breeding chip combined with population segregation analysis (BSA) diagrams of the present invention; where Figure (a) is a gene mapping map; and Figure (b) is a resequencing gene mapping map. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to a detailed description of specific embodiments. However, the embodiments described below are merely illustrative of the technical solutions of the present invention and do not limit the technical solutions of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0022] The SNP referred to in this invention is a single-nucleotide polymorphism (SNP), which mainly refers to DNA sequence polymorphism at the genome level caused by a single nucleotide variation in the nucleotide sequence, including single base deletions, insertions, transversions, and transitions.

[0023] Example 1

[0024] Design of a 20K SNP liquid phase breeding chip for peas:

[0025] This invention provides a combination of SNP loci for pea variety identification, comprising 21,659 SNP loci on the whole pea genome. The reference genome for the combination of SNP loci is the pea genome “Pisum sativumcultivarZhewan1(PeaZW1)”, the source of which can be found in the literature Liu N, Lyu X, Zhang X, et al. Reference genome sequence and population genomic analysis of peas provide insights into the genetic basis of Mendelian and other agronomic traits[J]. Nature Genetics, 2024, 56(9):1964-1974.

[0026] The location and variation information of the SNP sites are shown in Table 1.

[0027] Table 1

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] Here, Chr1, Chr2, Chr3, Chr4, Chr5, Chr6, and Chr7 represent the chromosome IDs where the SNP sites are located, and the numbers following the chromosome IDs indicate the positions of the SNP sites on the chromosome.

[0184] The specific preparation process of the liquid-phase breeding chip with the combination of 21,659 SNP molecular marker sites is as follows:

[0185] 1. Germplasm resource collection

[0186] A diversity sample group consisting of 237 varieties was planted at the Yangdu Research Base of the Zhejiang Academy of Agricultural Sciences (120.21551°E, 30.25308°N). These varieties were planted on November 12, 2020 and 2021, respectively. Resequencing data from 77 varieties previously studied were also included. The variety categories are shown in Table 2. Young leaves were collected two weeks after planting and rapidly frozen in liquid nitrogen for DNA extraction. A total of 57 traits were investigated. The experiments were replicated twice in 2020 and 2021. Each trait was measured five times independently, and the average value was calculated. The list of pea varieties used is as follows.

[0187] Table 2

[0188]

[0189]

[0190]

[0191] 2. Data quality control

[0192] Genomic DNA was extracted from fresh leaves using the CTAB method. DNA libraries for Illumina sequencing were constructed using NEBNext Ultra DNA LibraryPrep Kits. DNA was randomly fragmented using a Covaris ultrasonic disruptor, followed by end repair and the addition of A-tailed adapters. The purified DNA was amplified by PCR, and the library was sequenced using a DNBSEQ-T7 in PE150 mode after evaluation. The resulting raw image data files were converted into raw sequencing reads through base identification analysis. The results were stored in FASTQ file format. To ensure the quality of information analysis, the raw reads were filtered using the default parameters of fastp (v.0.12.2) to remove sequencing adapters and low-quality bases, resulting in Clean Reads for subsequent information analysis.

[0193] 3. Design of microarray sites for 20K SNP liquid-phase breeding of peas

[0194] Clean Reads were aligned to the reference genome PeaZW1 using the mem algorithm in BWA (v.0.7.17-r1188) software. After alignment, PCR duplicates were removed using the Picard tool (v.1.123). A single BAM file was generated for each sample for SNP detection. Subsequently, the SAM file format was converted and the BAM files were sorted using Samtools (v.1.9), and alignment quality was filtered using the 'q30' parameter. Raw SNPs were extracted using the HaplotypeCaller module of the Genome Analysis Toolkit (GATK, v.4.1.9.0). Individual SNP extraction was performed using hard filtering with mapping quality ≤20.0, minimum sequencing coverage ≤5, and maximum sequencing coverage ≥200. Variants were then merged into a single variant call file using GATK Combine Variants. Possible false SNPs were further filtered using VCFtools (v.0.1.16) based on the following criteria:

[0195] (1) If a marker is not detected in more than 20% of the samples, the marker is filtered out, i.e., the genotype loss rate is ≤20%, and the parameter is vcftools--max-missing 0.8;

[0196] (2) If a marker has a minimum allele frequency (MAF) of less than 0.01, it is filtered out, i.e., the minimum allele frequency (MAF) is less than 0.01. The parameter is vcftools--maf 0.01;

[0197] (3) Extract 100bp sequences upstream and downstream of SNP markers, perform whole genome sequence BLAST alignment, and if the similarity is greater than 60% and the alignment length is greater than 60% (about 120bp), it is considered to be a copy, and only single copy sites are retained;

[0198] (4) Extract 100bp sequences upstream and downstream of SNP markers, calculate GC content, and retain sites with GC content greater than 40% and less than 60%;

[0199] (5) Extract 100bp sequences upstream and downstream of SNP markers, analyze whether they contain SSR sequences, and retain sites without SSR sequences;

[0200] (6) Analyze the InDel within 100bp upstream and downstream of the SNP marker, and retain the SNPs that have no InDel of more than 5bp within 100bp upstream and downstream.

[0201] (7) Extract 100bp sequences upstream and downstream of the SNP marker, analyze whether the sequence contains N, and retain sequences without N within 100bp upstream and downstream of the SNP site.

[0202] (8) Addition of chip functional sites

[0203] Large-scale GWAS was performed using the filtered SNPs. Association analysis was performed using the Efficient Mixed-Model Association eXpedited (EMMAx) (v.2012-021-0) program with default parameters. The first three principal components were used as covariates. The effective number of independent SNPs and the proposed p-value were estimated using the Genetic type 1 Error Calculator (gec, v.0.2) software. In the mixed model, the proposed p-value was p < 4.63 × 10^-7. A total of 235 reliable SNP functional loci significantly associated with 57 traits were ultimately obtained.

[0204] Through the above methods, 21,659 high-quality and representative SNP loci were finally obtained, including 235 published reliable functional loci significantly associated with 57 traits. The distribution of these loci on the chromosome is shown below. Figure 1 As shown, the number and spacing on each chromosome are as follows: Figure 2 As shown.

[0205] 4. Development of a 20K SNP liquid-phase breeding chip for peas

[0206] The 21,659 SNP loci ultimately selected were used to design and synthesize liquid-phase capture probes by Tianjin Jizhi Gene Technology Co., Ltd. A pea 20K SNP liquid-phase breeding chip was developed using TargetSeq liquid-phase hybridization capture technology. TargetSeq liquid-phase hybridization capture technology is based on a multi-factor algorithm to design capture probes for the target region genome, synthesize effective and specific probes at high throughput, and then perform liquid-phase hybridization with fragmented genomic DNA to capture and enrich the target region sequence. Finally, high-throughput sequencing is performed using mainstream sequencing platforms such as Illumina and Life Technologies.

[0207] Example 2

[0208] The method for genotyping pea samples using the pea 20K SNP liquid phase breeding chip of this invention is as follows:

[0209] First, genomic DNA is fragmented into small fragments of 200-300 bp. Adapters are repaired and ligated to the ends of these fragments. Pre-PCR amplification is then performed on the DNA fragments to amplify the library. Probes are hybridized to the target region, and streptomycin affinity-labeled magnetic beads are used to capture the hybridized probes, enrich the target fragments, elute them, and perform post-PCR amplification. Finally, next-generation sequencing is performed on the target region. The raw image data files obtained from the high-throughput sequencing DNBSEQ-T7 sequencing platform are converted into raw sequencing sequences, called Raw Data or Raw Reads, through base recognition analysis. The results are stored in FASTQ file format, containing sequence information and corresponding sequencing quality information. Because the raw sequencing sequences or Raw Reads obtained from sequencing contain low-quality reads with adapters, they need to be filtered to ensure the quality of information analysis. The filtered Clean Reads are used for subsequent information analysis.

[0210] The main steps of data filtering are as follows:

[0211] (1) Remove reads with adapters.

[0212] (2) Filter reads with N content exceeding 10%.

[0213] (3) Remove more than 50% of reads with a quality value of less than 10.

[0214] Resequencing reads obtained from resequencing need to be repositioned onto the reference genome before subsequent variant analysis can be performed. The bwa software is primarily used for aligning short sequences obtained from the DNBSEQ-T7 next-generation high-throughput sequencing platform with the reference genome. By aligning, the positions of clean reads on the reference genome are located, and information such as sequencing depth and genome coverage for each sample is statistically analyzed, along with variant detection. Subsequently, Samtools (v.1.9) is used to convert SAM files to BAM format, and the alignment quality is filtered using the 'q30' parameter. The HaplotypeCaller module of the Genome Analysis Toolkit (GATK, v.4.1.9.0) is used to extract raw SNPs. Individual SNP extraction uses hard filtering based on mapping quality ≤20.0, minimum sequencing coverage ≤5, and maximum sequencing coverage ≥200. Then, GATK Combine Variants is used to merge variants into a single variant call file. VCFtools (v.0.1.16) is used to further filter for possible false SNPs, with criteria including:

[0215] (1) If a marker is not detected in more than 20% of the samples, the marker is filtered out, i.e., the genotype loss rate is ≤20%, and the parameter is vcftools--max-missing 0.8;

[0216] (2) If a marker has a minimum allele frequency (MAF) of less than 0.01, it is filtered out, i.e., the minimum allele frequency (MAF) is less than 0.01. The parameter is vcftools--maf 0.01;

[0217] Ultimately, the genotyping results of each target SNP in a specific pea material are obtained, achieving high-throughput SNP genotyping.

[0218] Example 3

[0219] To verify the genotyping effect of the pea 20K SNP liquid phase breeding chip, genotyping detection was performed on 14 pea samples using the pea 20K SNP liquid phase breeding chip designed in Example 1, as described in Example 2.

[0220] like Figure 3 As shown, probe capture efficiency analysis of sequencing data from 14 representative materials revealed that 98.46% of the reads aligned to the reference genome, with an average probe capture rate of 44.47%. Off-target reads accounted for 53.98%, indicating that the 20K SNP liquid-phase breeding chip for peas has a high target site detection rate and accurate and reliable genotyping results when performing genotyping on the tested materials. It can be used for SNP genotyping detection of different pea samples.

[0221] Genetic distance matrices were calculated using VCF2Dis software to perform cluster analysis on 14 materials. Phylogenetic trees were then constructed using FastME software, yielding results consistent with the actual groupings. Figure 4 As shown, the loci have high representativeness. Combined with phylogenetic trees, they can be used for research such as genetic diversity analysis of pea germplasm resources, variety identification analysis, population structure analysis, genetic and evolutionary analysis, and kinship identification.

[0222] Example 4

[0223] To verify the effectiveness of the pea 20K SNP liquid-phase breeding chip in gene mapping, important trait analysis, and assisted selection breeding, a mixed segregating population analysis (BSA) targeting pod color traits was performed using the pea 20K SNP liquid-phase breeding chip designed in Example 1. In this analysis, ZWR12, with green pod characteristics, was used as the maternal parent, and ZWNJ004, with yellow pod characteristics, was used as the paternal parent. Thirty plants were selected from the segregating population to form a yellow pod pool (WD_Y1) and a green pod pool (WD_G1), respectively, and genotyping analysis was performed using the methods described in Example 2, along with resequencing. Clean reads obtained by both methods were aligned to the PeaZW1 reference genome using BWA software. After sorting using SAMtools, SNPs were identified using GATK v4.0 and filtered according to predefined criteria. The ΔSNP index and G value were calculated and plotted using the R package QTLseqr.

[0224] The results showed that the polymorphic probes of these populations were mainly located on chromosome 3, specifically between positions 225,461,652-367,063,164 and 386,708,349-552,070,089. Figure 5 As shown in the figure, Figure (a) is the gene mapping map and Figure (b) is the gene mapping map using resequencing; these locations are consistent with the genomic coordinates of next-generation sequencing. This demonstrates the accuracy of the chip in identifying genes associated with specific traits, highlighting its potential for genetic improvement research and development in pea trait analysis, assisted selection breeding, and other areas.

[0225] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A pea whole genome 20K SNP liquid breeding chip, characterized in that, The genotyping object of the chip includes 21,659 SNP sites on the whole genome of pea as shown in Table 1.

2. The pea whole genome 20K SNP liquid breeding chip of claim 1, wherein, The reference genome of the SNP sites is the pea genome "Pisum sativum cultivar Zhewan1 (PeaZW1)".

3. Use of the pea 20K SNP liquid breeding chip as claimed in claim 1 in genotyping a pea sample.

Images