利用GBS技术研究240份宽皮柑橘的系统演化  

Study on Phylogeny of 240 Mandarin Accessions with Genotyping-by-Sequencing Technology

作　　者：王小柯[1] 江东[1,2] 孙珍珠 

机构地区：[1]西南大学柑橘研究所,重庆400712 [2]中国农业科学院柑橘研究所,重庆400712

出　　处：《中国农业科学》2017年第9期1666-1673,共8页Scientia Agricultura Sinica

基　　金：国家"十二五"科技支撑计划(2013BAD01B04);重庆市科委重点项目(cstc2016shms-ztzx80004)

摘　　要：【目的】GBS(genotyping-by-sequencing)是一种高效而经济的SNP(单核苷酸多态性)发掘和基因分型技术。采用GBS技术对240份宽皮柑橘进行基因分型,以阐明一些野生宽皮柑橘和地方品种的遗传背景,为其起源和演化研究提供更可靠的证据。【方法】选用国家柑橘种质重庆资源圃保存的具有广泛遗传多样性和地理起源的240份宽皮柑橘作为材料,利用Eco R I限制性内切酶消化基因组DNA后构建GBS文库;然后进行Illumina HiSeqPE150二代测序获得短读序列,通过BWA软件将序列映射到克里曼丁参考基因组上,再利用SAMTOOLS软件鉴定SNP位点。依据SNP的基因分型结果,采用邻近法构建系统演化树,并进行主成分分析。【结果】利用GBS简化基因组测序技术对240份宽皮柑橘进行测序,共获得96.3 Gb的测序数据,平均每个样本测序数据为401.26 Mb,经过测序深度为4X、Miss0.2、次要等位基因频率(MAF)>0.01的筛选条件过滤,最后共获得了114 200个高质量的SNP位点。主成分分析结果显示240份宽皮柑橘被分为4大类,其中温州蜜柑亚群、野生宽皮柑橘亚群可明显区分于其他宽皮柑橘。利用系统演化树可将240份宽皮柑橘划分到11个类群中。系统演化树和主成分分析都揭示了不同地理来源和特定形态的宽皮柑橘在遗传水平上存在明显的差异,比如来源于日本的温州蜜柑、欧美的克里曼丁橘及其杂种后代,以及中国南方的野生宽皮柑橘由于地理分布不同而形成了较为独特的类型,彼此间能够相互区分开。进化树结果表明中国南、北不同地域的宽皮柑橘可能存在不同的演化路径,南岭山脉及南方地区的野生宽皮柑橘、酸橘和目前南方地区栽培的砂糖橘存在较近的起源演化联系,而北方宽皮柑橘的演化却与宽皮柑橘中的古老地方品种存在紧密联系。人工杂交育种、长期的人工选择和驯化形成了不同类型的宽皮柑橘,同时�[Objective] Genotyping-by-sequencing （GBS） is an economic technique to discover SNP and genotype myriad of crop germplasms in an effective way. The aim of this study is to clarify the classification and evolution of 240 mandarin germplasms by using GBS. [Method] A total of 240 mandarin germplasms conserved in the National Citrus Germplasm Repository in Chongqing, with widely genetic diversity and geographic origin, were selected as trial materials. GBS library was constructed with genomie DNAs after digested with EcoR I restriction endonuclease and sequenced on Illumina HiSeq PE150, then the sequences were mapped to the clementine （Citrus clementina hort. ex Tanaka） reference genome by using BWA, and SNPs were called with the SAMTOOLS pipeline. With the SNPs genotyping data, a phylogenetic tree was built by using Neighbor-joining method and a principal component analysis （PCA） was carried out. [Result] By using GBS, a total of 96.3 Gb of sequences were generated from the 240 mandarin germplasms, and each sample produced 401.26 Mb in average. After screening with parameter of dp4, miss0.2 and minor alleles frequency （MAF）〉0.01, a total of high quality 114 200 SNP sites were retained. The PCA analysis showed that the 240 mandarin germplasms could be divided into 4 groups, in which satsuma sub-group and wild mandarin sub-group could be clearly separated from other mandarin accessions. With phylogenetic analysis, the 240 mandarin germplasms could be divided into 11 groups. Both the phylogenetic analysis and PCA suggested that the genetic variations were presented in mandarin germplasms with different geographical origins and morphological characteristics. For example, satsuma mandarin （Citrus unshiu Macf.） derived from Japan, clementine （Citrus clementina Hort.ex. Tanaka） and its offspring from Europe and America, as well as wild mandarins from China could be clearly distinguished based on phylogenetic tree, moreover the phylogenetic tree showed that the mandarin germplasms derived from the

关 键 词：GBS 宽皮柑橘 系统演化 品种鉴定 

分 类 号：S666[农业科学—果树学]