东乡野生稻叶绿体基因组拼接及系统进化分析.docVIP

  东乡野生稻叶绿体基因组拼接及系统进化 分析 *  5 10 15  （浙江大学农业与生物技术学院农学系，浙江省作物种质资源重点实验室，杭州 310058） 摘要：叶绿体基因组序列对于研究植物物种的起源、进化演变及不同物种间的亲缘关系等具 有重要意义。高通量测序技术的快速发展，推动了植物叶绿体基因组的测序工作。但传统的 叶绿体基因组测序方法需要建立在分离纯化叶绿体 DNA 的基础上，操作繁琐，耗时较长。为 了优化叶绿体基因组 DNA 序列的获取和拼接方法，以东乡野生稻（Oryza rufipogon）嫩绿 叶为材料，不需分离叶绿体 DNA，利用高通量测序获得的全基因组短序列（reads）及叶绿 体基因组高度保守的特性，与参考序列进行比对，从而组装拼接出叶绿体 DNA 序列，并同时 利用生物信息学手段和 PCR 扩增进行补洞。最终获得东乡野生稻完整叶绿体基因组序列，大 小为 134537bp，大（LSC）、小（SSC）单拷贝区和反向互补重复区（IR）大小分别为 80585bp、 12346bp 和 20803bp，共注释叶绿体基因 152 个。基于获取的东乡野生稻及其他叶绿体基因 组 序 列 ， 通 过 构 建 进 化 树 分 析 ，结 果 显 示 在 禾本 科 中 水 稻 与 麻 竹 （ Dendrocalamus latiflorus）和黍亚科（Panicoideae）亲缘关系最近，粳稻与中国普通野生稻的亲缘关系 较近，粳稻与籼稻并非同时驯化出现。 关键词：作物遗传学；东乡野生稻；叶绿体基因组；高通量测序；嫩绿叶 中图分类号：S511.9 20 Assembly and phylogenetic analysis of Dongxiang wild rice chloroplast genome LIN Zhangxiang, WANG Yingying, FU Fei, YE Chuyu, FAN Longjiang (Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang Key Laboratory 25 30 35 40 45 of Crop Germplasm, Zhejiang University, Hangzhou 310058, China) Abstract: Complete chloroplast genome sequence is very useful for studying the evolution of species. The rapid development of high-throughput sequencing technology promotes the plant chloroplast genome sequencing. For traditional chloroplast genome sequencing method, it is necessary to isolate and purify the chloroplast DNA before sequencing. Due to low concentration of chloroplast DNA, it is difficult to separate it from nuclear genome DNA. Therefore, chloroplast DNA isolation-based method is tedious and time consuming. This study employed a simple and rapid method for chloroplast genome sequences acquisition without isolation of chloroplast DNA. Based on conservation of chloroplast genomes, the whole genome short reads generated by Illumina Hiseq 2000 were directly used to map against chloroplast reference genomes. Subsequently, the aligned reads were collected and further did de novo assembly. Finally, the chloroplast genome sequence of Dongxiang wild rice was obtained. The chlor