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林业科学 ›› 2021, Vol. 57 ›› Issue (2): 72-81.doi: 10.11707/j.1001-7488.20210208

• 论文与研究报告 • 上一篇    下一篇

基于RAD高通量测序的贵州百里杜鹃保护区杜鹃花属分类

黄承玲1,姚刚2,田晓玲3,任永权1,黄家湧4,马永鹏2,*   

  1. 1. 贵州民族大学生态环境工程学院 贵阳 550025
    2. 云南省极小种群野生植物综合保护重点实验室 中国科学院昆明植物研究所 昆明 650201
    3. 贵州民族大学人文科技学院 贵阳 550025
    4. 贵州百里杜鹃科研所 毕节 551614
  • 收稿日期:2020-03-23 出版日期:2021-02-25 发布日期:2021-03-29
  • 通讯作者: 马永鹏
  • 基金资助:
    国家自然科学基金项目"三种杜鹃花的交互自然杂交及种间生殖隔离的维持机制研究"(31770418);贵州省科技厅联合基金项目"百里杜鹃杜鹃属植物亲缘关系与遗传多样性研究"(黔科合LH字[2016]7085)

Phylogenomic Analysis of Rhododendron Species in Guizhou Baili Rhododendron Reserve Based on RAD Sequencing

Chengling Huang1,Gang Yao2,Xiaoling Tian3,Yongquan Ren1,Jiayong Huang4,Yongpeng Ma2,*   

  1. 1. College of Eco-Environment Engineering, Guizhou Minzu University Guiyang 550025
    2. Yunnan Key Laboratory for Integrative Conservation of Plant Species with Extremely Small Populations Kunming Institute of Botany, Chinese Academy of Sciences Kunming 650201
    3. College of Humanities and Sciences, Guizhou Minzu University Guiyang 550025
    4. Guizhou Baili-Rhododendron Scientific Research Institute Bijie 551614
  • Received:2020-03-23 Online:2021-02-25 Published:2021-03-29
  • Contact: Yongpeng Ma

摘要:

目的: 准确界定物种是野生植物资源开发与利用的前提。鉴于叶绿体片段无法区分贵州百里杜鹃区域的杜鹃花属植物,本研究采用基于基因组水平的RAD-seq技术探讨该区域杜鹃花属的系统进化关系,以期为复杂植物类群的物种鉴定提供参考。方法: 对百里杜鹃保护区的34种杜鹃花进行RAD-seq技术测序,统计测序深度、覆盖度及多态性位点等基本指标;用de novo方法获得高质量SNP位点,以Tajima’s D为指标筛选中性的SNPs,最后用中性SNPs通过fastStructure、PCA、GCTA及IQ-Tree软件对其进行基因分型、聚类与系统树构建。结果: 共获得42 083个高质量SNP位点,其中28 983个为中性SNP位点。基于PCA、fastStructure及系统发生树的分析结果,支持目前34种杜鹃花在亚属水平的形态学分类处理,6个亚属中的常绿杜鹃亚属、马银花亚属、羊踯躅亚属和映山红亚属能明显分开,而糙叶杜鹃亚属与杜鹃亚属不能区分开,表明二者亲缘关系较近。物种水平能区分绝大部分的种,相比前期研究有了大幅度提升,但一些形态特征极相似的种如繁花杜鹃与皱叶杜鹃无法区分。结论: RAD-seq数据产生大量的中性SNP位点,可在亚属水平和物种水平区分百里杜鹃保护区杜鹃花属的大部分植物,同时证实RAD-seq技术在复杂植物类群的物种分类方面相比传统分子标记,具有明显优势。

关键词: RAD-seq, 杜鹃花属, 百里杜鹃保护区, 单核苷酸多态性, 系统分类

Abstract:

Objective: Accurate species definition is the premise for development and utilization of wild plant resources. Due to the fact that chloroplast DNA fragments cannot distinguish Rhododendron species of the Baili Rhododendron Reserve in Guizhou reported before, the present study attempted to explore the phylogenetic relationships of Rhododendron in this region based on genome-level RAD(restriction-site associated DNA) sequencing technology, aiming to detect the power of RAD for species definition of complicated plant groups. Method: RAD-seq sequencing was performed on 34 species of Rhododendron from the reserve, and the basic characteristics of the data such as depth, coverage and polymorphic sites were evaluated. De novo method was employed to call SNPs which were subsequently evaluated to be neutral or selective by Tajima's D. Based on these neutral SNPs, fastStructure, PCA, GCTA and IQ-Tree software were finally employed to perform genetic separation, clustering and phylogenetic tree construction. Result: A total of 42 083 SNPs were obtained, among which 28 983 SNPs were detected to be neutral. The analyses of PCA, fastStructure and phylogenetic tree supports the morphological classification of 34 species of Rhododendron at the subgenus level. Subgen. Hymenanthes, Subgen. Azaleastrum, Subgen. Pentanthera and Subgen. Tsutsusi of the 6 subgenera can be distinguished, whereas Subgen. Pseudorhodorastrum and Subgen. Rhododendron cannot, suggesting that the 2 subgenera are closely related. In addition, at species-level most species can be distinguished, indicating a great improvement compared to previous studies, but the species with very similar morphological characteristics are indistinguishable, such as R. floribundum and R. denudatum. Conclusion: RAD-seq data produced a large number of SNPs that can distinguish most Rhododendron species in the reserve at the subgenus and species levels, proving that RAD technology had significant advantages over traditional molecular markers in the classification of complicated plant groups.

Key words: RAD-seq, Rhododendron, Baili Rhododendron Reserve, SNPs, phylogenetic classification

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