利用低拷贝基因序列推测人参属多倍体类群的起源

	利用低拷贝基因序列推测人参属多倍体类群的起源
	龙小霞
导师	潘跃芝
关键词	羽叶三七复合群 Panax bipinnatifidus complex 低拷贝核 DNA 片段 Low-copy nuclear DNA fragments 克隆 Clone 多倍体起源 Polyploid origin
摘要	人参属（Panax Linnaeus）植物分布广泛，从林下到高原草甸都有分布，导致营养器官形态差异较大，且存在较多过渡性状，但生殖器官却高度保守。这些特性造成分类和鉴定都存在困难。其中，分布于中国西南及喜马拉雅地区、分类范畴得不到很好确认的类群被归为羽叶三七复合群（Panax bipinnatifidus complex）。羽叶三七复合群中多倍体类群的存在可能是导致该复合群内各物种分类范畴以及系统发育关系得不到很好的确认的原因。基于课题组先前的研究，初步确定该复合群内的分布于云南省普洱市镇沅县和景谷县的姜状三七 P. zingiberensis、湖南省张家界桑植县和湖北省宜昌市五峰县的 P. wangianus、云南省丽江市玉龙县分布的 P. bipinnatifidus、云南省昭通市永善县的 P. bipinnatifidus 为多倍体类群。本研究利用三个叶绿体 DNA 片段（psbA-trnH、psbK-psbI、 rps16）和两个低拷贝核 DNA 片段（ PZ15、PZ8），通过克隆测序、 PHASE 手动分型来获得核 DNA 片段全部的序列类型，与 Zhou（2018）论文中的二倍体类群数据相结合，通过聚类树构建（最大似然树、贝叶斯树）和 STRUCTURE 聚类分析，以揭示羽叶三七复合群内多倍体类群的起源问题。通过分析和探讨这些问题，本研究将有利于人参属植物的分类和物种界定工作的进一步推进，从而为人参属植物合理的资源保护和利用提供科学支持。论文主要结果和结论如下：（1）在核 DNA 片段原有序列构建的矩阵基础上，根据引物设计原则，重新设计引物，提高了扩增率，且引物特异性较高，使后续的克隆实验成功率显著提高。新设计引物序列如下： PZ15 ： F-TGAACAGGCATTATTACTCG ， R-TCTTGAACGGCTTTGTTGTA ； PZ8:F-GGGAAGGAAAAGTTGCTCTG ， R-TCTTCTAGTATAGGAACTGC。（2）姜状三七为异源多倍体，且不是单一起源。越南三七（P. vietnamensis）极大可能参与了姜状三七的多倍体杂交起源，且是作为母系来源参与到其中，而另一亲本可能已经灭绝或未被采集到。（3）桑植县和湖北省五峰县分布的 P. wangianus 为异源多倍体。但是，由于本研究针对在华中地区人参属植物采样覆盖度低，因此本研究结果并未显示桑植县和五峰县的 P. wangianus 的父系、母系的直接来源。但是，研究结果显示该类群与四川省峨眉山云南大学硕士学位论文分布的 P. wangianus、永善县分布的 P. bipinnatifidus 以及云南省马关县分布的 P. wangianus 有着较近的亲缘关系。（4） PZ15 数据的研究显示来自玉龙县的 P. bipinnatifidus 为异源多倍体，三个叶绿体片段的分析结果表明分布于云南省怒江傈僳族自治州贡山县的P. bipinnatifidus可能作为母系来源参与到了该多倍体类群的形成当中，而父系来源尚不明确。但是分析结果同时表明核 DNA PZ8 片段上发生了质同进化，因而在 PZ8 片段上显示出同源多倍体的模式。（5）来自永善县的 P. bipinnatifidus 为异源多倍体，同域分布的二倍体 P. wangianus 可能是父系基因来源的贡献者，二者都拥有小叶片 3 枚，此特征与羽叶三七复合群其他植物 5-7 小叶的特征均不一样，进一步佐证了这一推测。本论文通过三个叶绿体 DNA 片段和两个低拷贝核 DNA 片段，揭示羽叶三七复合群内多倍体类群的起源问题。结果表明，本研究所包含的四个多倍体类群，即姜状三七、桑植县和五峰县的 P. wangianus、玉龙县的 P. bipinnatifidus、永善县的 P. bipinnatifidus 均为异源多倍体。其中，姜状三七的母系来源可能是越南三七，玉龙县的 P. bipinnatifidus 的母系来源可能是贡山县的 P. bipinnatifidus，永善县的 P. bipinnatifidus 的父系来源可能是同域分布的 P. wangianus，其余亲本尚不明确。; Plants of Panax Linnaeus distribute widely across China with diverse habitats, and their vegetative organs are diverse and reproductive organs highly conserved, which results in the difficulty in species classification and identification. The taxa whose taxonomic category and phylogenetic relationships cannot be well confirmed are classified as Panax bipinnatifidus complex. The complex contains diploidy and polyploidy plants, which is probably one of the reasons why the taxonomic category and phylogenetic relationships within this complex are so difficult to be resolved. In our previous research in P. bipinnatifidus complex, that the plants of P. zingiberensis distributed in Zhenyuan County and Jinggu County, Puer Yunnan, P. wangianus in Sangzhi County, Zhangjiajie Hunan and Wufeng County, Yichang Hubei Province, P. bipinnatifidus in Yulong County, Lijiang Yunnan Province, and P. bipinnatifidus in Yongshan County, Zhaotong Yunnan Province were identified as polyploidy groups. In this study, three plastid DNA fragments (psbA-trnH, psbK-psbI, rps16) and two low-copy nuclear DNA fragments (PZ15 and PZ8) were used to clone and sequence, and we obtained all the sequence types of nuclear DNA fragments by cloning and PHASE manual typing. In order to reveal the origin type (autopolyploid or allopolyploid) of these taxa, we conduct the clustering analysis by constructing the phylogenetic trees (Maximum likelihood trees and Bayesian trees) and STRUCTURE analysis using the DNA sequences obtained in this study and the study of Zhou (2018). The results of this study will be beneficial to the classification and species identification of Panax, and provide scientific support for the rational resource protection and utilize of Panax. In addition, we also expect to understand the regularity of polyploidy by exploring the frequently polyploidy events of Panax in the natural environment. The main results are as follows: 1) Based on the sequence matrix constructed with the original sequences of nuclear DNA fragments, the primers were redesigned for the polyploidy taxa to improve the amplification rate. The specificity of the redesigned primers was higher, and the success rate of subsequent cloning was significantly improved. The newly designed primer sequences are as follows: III 云南大学硕士学位论文 IV PZ15: F-TGAACAGGCATTATTACTCG, R-TCTTGAACGGCTTTGTTGTA; PZ8: F-GGGAAGGAAAAGTTGCTCTG, R-TCTTCTAGTATAGGAACTGC. 2) P. zingiberensis is an allopolyploid and not of single hybridization rigin P. vietnamensis is most likely as a maternal source involved in this hybridization origin event. The paternal taxa were not collected yet in this study or it has been extinct in the wild. 3) P. wangianus distributed in Sangzhi County and Wufeng County was tested to be an allopolyploid. However, the result of clustering analysis did not reveal directly the parental sources of this allopolyploid, the reason is probably that we did not sample the parental plants because of the low sampling coverage
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75156
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	龙小霞. 利用低拷贝基因序列推测人参属多倍体类群的起源[D]. 中国科学院大学,2022.

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