利用CottonSNP63K芯片构建棉花品种的指纹图谱

Construction of Cotton Variety Fingerprints Using CottonSNP63K Array

【目的】利用SNP位点的单拷贝特性,结合陆地棉TM-1参考基因组序列信息,筛选基因组特异的SNP。【方法】以719份遗传背景来源广泛的陆地棉种质资源为材料,采用Illumina公司开发的Cotton SNP63K芯片,利用Genome Studio软件对芯片扫描所获得原始数据进行基因型数据质量控制,获得待测样品SNP位点的基因型数据。根据已公布的陆地棉TM-1基因组的两个版本——中国农业科学院棉花研究所版本Gossypium hirsutum(AD1)genome BGI v1.0与南京农业大学版本G.hirsutum(AD1)genome NBI v1.1为参考序列,对Cotton SNP63K芯片(63 058个SNP)各位点的侧翼序列分别进行全基因组Blast比对分析,以筛选具有单拷贝特性的特异SNP位点并用于样品指纹图谱的构建。【结果】利用Cotton SNP63K芯片对719份材料进行SNP位点基因分型,主要表现为无检出信号的SNP位点、无多态性的SNP位点、具有多态性的SNP位点,而具有多态性的SNP位点的分型结果又可分为单位点SNP(基因组特异SNP)、双位点SNP和多位点SNP。通过对两个已公布的陆地棉TM-1参考基因组序列Blast比对结果表明,中国农业科学院棉花研究所TM-1基因组版本比对获得基因组特异SNP标记为5 474个,而南京农业大学TM-1基因组版本比对获得基因组特异SNP标记仅为1 850个,两者共有的特异SNP为1 594个,进一步通过分型效果、检出率及多态性3个评价指标,筛选score值≥0.7,call frequency值≥0.95,且MAF值≥0.2的SNP位点,获得471个分型效果理想,检出率高且多态性较高的特异SNP位点。在471个SNP位点中,430个位于染色体上,41个位于scaffold片段上。考虑到标记间的连锁程度,剔除连锁标记37个,最终获得393个核心SNP位点。利用393个核心SNP构建了719份品种资源的特征DNA指纹图谱,除个别材料之间遗传背景高度相似、基因型完全一致外,97%的材料均能实现准确有效的鉴别。【结论】筛选出393个基因组特异的SNP,并利用这些核心SNP构建了719份资源材料的特征DNA指纹图谱,为SNP分子标记应用于棉花重要性状遗传改良提供了参考。

【Objective】 The objective of this study is to screen genomic-specific SNPs, considering the SNP with single loci and the reference genomic sequence information of upland cotton TM-1. 【Method】 Based on 719 natural germplasm resources with plentiful genetic backgrounds, using the Cotton SNP63 K array developed by Illumina, quality control of the original genotyping data obtained from chip scanning was performed by Genome Studio software, the genotyping data of the SNP locus of the tested samples were obtained. According to the two published Gossypium hirsutum TM-1 genome versions, including the G. hirsutum（AD1） genome BGI v1.0 of Cotton Research Institute of Chinese Academy of Agricultural Sciences and the G. hirsutum（AD1） genome NBI v1.1 of Nanjing Agricultural University, the genome-wide BLAST analysis was performed on the flanking sequences of each SNP with Cotton SNP63 K（63 058 SNPs） array to screen specific SNP loci with single copy, and these SNPs were used for the construction of fingerprints. 【Result】 SNP genotyping of 719 materials using Cotton SNP63 K was classified into SNP locus without detectable signal, SNP locus without polymorphism and the polymorphism SNP loci. The polymorphism SNP loci could be divided into double-site SNP, multiple-site SNP and single-site SNP（genomic-specific SNP）. The results of the Blast analysis with the two upland cotton TM-1 reference genome sequences showed that there were 5 474 specific SNP markers in the BGI v1.0 version, while NBI v1.1 TM-1 version only 1 850, and the common specific SNPs of both were 1 594. Further considering the three evaluation indexes including the genotyping effect, call rate and polymorphism, the SNPs with scores≥0.7, call frequency≥0.95 and MAF≥0.2 were screened, and 471 specific SNPs with high call rate and high polymorphism were obtained. Among these SNP loci, 430 were on chromosomes and 41 were on scaffolds. Taking into account of the degree of linkage between the markers, finally 393 core SNP loci were obtained after removing 37 linkage markers, and the DNA fingerprints of 719 resource materials were constructed by using 393 core SNPs. The results showed that 97% of the materials could be accurately and effectively identified except for some accessions with similar genetic background. 【Conclusion】 A total of 393 genomic-specific SNPs were screened out, and these core SNPs were used to construct the DNA fingerprints of 719 resource materials. This study will provide a reference for application of SNP molecular markers in genetic improvement of important cotton traits.