| 利用GBS技术研究240份宽皮柑橘的系统演化 |
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| 引用本文： | 王小柯,江东,孙珍珠.利用GBS技术研究240份宽皮柑橘的系统演化[J].中国农业科学,2017,50(9):1666-1673. |
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| 作者姓名： | 王小柯 江东 孙珍珠 |
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| 基金项目： | 国家“十二五”科技支撑计划（2013BAD01B04）、重庆市科委重点项目（cstc2016shms-ztzx80004） |
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| 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,同时也导致宽皮柑橘遗传多样性的增加。另外,一些宽皮柑橘资源中的可疑亲本也通过GBS技术得以准确鉴定。本研究表明沃柑与金诺橘有较近的亲缘关系。【结论】GBS技术用于柑橘种质资源的基因分型高效可靠,建立的系统演化树可以对240份宽皮柑橘进行准确划分,与用植物形态学划分的结果高度吻合。另外,GBS技术用于资源材料的准确鉴定和亲缘关系的研究,可为柑橘植物新品种权的保护提供可靠的技术支撑。
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| 关 键 词： | GBS 宽皮柑橘 系统演化 品种鉴定 |
| 收稿时间： | 2016-11-16 |
Study on Phylogeny of 240 Mandarin Accessions with Genotyping-by-Sequencing Technology |
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| WANG XiaoKe,JIANG Dong,SUN ZhenZhu.Study on Phylogeny of 240 Mandarin Accessions with Genotyping-by-Sequencing Technology[J].Scientia Agricultura Sinica,2017,50(9):1666-1673. |
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| Authors: | WANG XiaoKe JIANG Dong SUN ZhenZhu |
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| Institution: | 1.Citrus Research Institute, Southwest University, Chongqing 400712; ;2.Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing 400712 |
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| Abstract: | 【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 genomic 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 Southern and Northern of China have unique evolutionary route. The wild mandarins distributed in Nanling Mountain and southern China have a closer phylogenetic relationship with sour mandarin (Citrus sunki Hort.ex Tanaka) and Shatang ju mandarin, which are widely cultivated in southern China nowadays, whereas the evolution of mandarins in the northern of China were related to some primitive and old cultivars. In addition, hybrid breeding, long-term artificial selection and domestication led to the subdivision formation and increased the genetic diversity of mandarin. Besides, results of this study showed that GBS has a potential advantage to identify some mandarin accessions with suspicious parents. For example, the phylogenetic tree clearly shows that “Or” tangor has a very close relationship with Kinnow mandarin. 【Conclusion】GBS technology provides an effective and high feasible approach to assistant the taxonomic classification of 240 mandarin accessions, the classification results are in accordance with the conclusion based on morphological method. Meanwhile GBS also is a powerful tool for germplasm identification, and can be applied in the new cultivars identification and intellectual property protection. |
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| Keywords: | GBS mandarin phylogenetic evolution germplasm identification |
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