山东省主要栽培苹果基因组重测序及SNP芯片位点挖掘

SNP Chip Loci Mining and Genome Resequencing of Main Cultivated Apple Varieties in Shandong Province

为促进苹果品种快速鉴定、种质资源评价及选择利用,本研究对山东省31个栽培苹果开展了重测序及SNP位点挖掘研究。样品DNA提取自叶片,并按照Illumina操作手册制备双端文库。经由Hiseq 4000平台对31个苹果基因组进行重测序,共产生了363 Gb的高质量序列,平均每样品12.5 Gb,这代表了苹果基因组大约15.9倍覆盖度。充分满足重测序分析及SNP位点挖掘的需要。错配率比较试验发现随着错配率逐渐升高,比对率逐渐升高至饱和。其中总比对率、成对数据比对率及单端数据比对率与错配率呈现显著相关(P<0.000 1),均符合一元四阶方程(回归系数R^(2)>0.99)。随着错配率提高,比对严谨度降低;基因组覆盖度逐渐升高,杂合位点准确度逐渐提高。采用两种算法所得到的位点,根据‘染色体+位点信息’作为特征值取交集,得到高可靠的单碱基SNP位点数据集:共检测到374 404个变异,平均每隔1 896个位点能够检测到一个变异,桑格验证试验准确度高达98.1%。SNP的功能注释分析结果显示在全部373 763个位点中有143 269个(38.27%)位于基因间区,25 047个(6.7%)位于基因编码区,179 426个(47.92%)位于基因上游-下游的2 kb区域。在所有编码区SNP里,有13 422个是非同义变异位点,11 625个是同义变异位点。两种SNP比率为1.15∶1。进一步利用过滤的4DTV位点,采用邻接算法构建的聚类分析结果符合山东省栽培苹果分类的趋势。

In this study, we carried out genome resequencing and SNP mining for cultivated apples in Shandong Province, for the promote rapid identification, germplasm resource evaluation and selection and utilization of apple varieties. Genomic DNA was extracted immediately from leaves of each sample, and Paired-end Illumina genomic libraries were prepared and sequenced on an Illumina Hiseq 4000 platform following the manufacturer’s instructions. Resequencing of the 31 apple genomes generated a total of 363 Gb high-quality cleaned sequences, with an average of 12.5 Gb per accession that represented approximately 16.29 × coverage of the apple genome. The data volume fully meets the needs of downstream analysis and SNP mining. When we used the nucleotide mismatch parameter, the mapping rate gradually increased to saturation. There was a highly significant correlation(P<0.000 1)between the total mapping rate, mapping rate of pair-end data, and mismatch parameter. Univariate fourth-order equation(Regression coefficient R^(2)>0.99) were predicted. As the mismatch rate increases, the accuracy of mapping decreases;The genome coverage gradually increases, and the accuracy of heterozygous sites gradually increases.In this study, the SNP mining was obtained by the two algorithms, and the intersection was further taken based on the ’chromosome+site information’ as the eigenvalues to obtain a highly reliable single nucleotide variant dataset.A total of 374 404 SNP locus were detected. On average, one mutation can be detected every 1 896 loci. The accuracy of the Sanger verification test is as high as 98.1%. Annotation analysis shows that among the 373 763 SNPs, 25 047(6.7%) are located in the gene coding region, 143 269(38.27%) are located in the intergenic region,and 179 426(47.92%) are located in the 2 kb region upstream or downstream of the genes. Among the coding region SNPs, 13 422 are non-synonymous mutations, and 11 625 are synonymous mutations. The ratio of non-synonymous to synonymous SNP is 1.15:1. Using filtered 4 DTV sites, clustering analysis results constructed by neighbor-joining algorithms are in line with the trend of the classification of cultivated apples in Shandong Province.