基于流式细胞术和K-mer分析测定两种车前属植物基因组的大小  

Estimation of Genome Sizes of Two Plantago Species by Flow Cytometry and K-mer Analysis

作　　者：梅启明 董斌[3] 王丹 郑明轩 Mei Qiming;Dong Bin;Wang Dan;Zheng Mingxuan(College of Life Sciences,West China Normal University,Nanchong,637009;South China Botanical Garden,Chinese Academy of Sciences,Guangzhou,510650;Guangdong Agriculture Industry Business Polytechnic,Guangzhou,510507;College of Forestry and Landscape Architecture,South China Agricultural University,Guangzhou,510642)

机构地区：[1]西华师范大学生命科学学院,南充637009 [2]中国科学院华南植物园,广州510650 [3]广东农工商职业技术学院,广州510507 [4]华南农业大学林学与风景园林学院,广州510642

出　　处：《分子植物育种》2021年第8期2564-2569,共6页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31600341);广东省普通高校重点科研平台和项目(2020GCZX009,2019KZDZX-2035)共同资助。

摘　　要：车前属(Plantago)植物车前(Plantago asiatica)和大车前(Plantago major)是中国传统药用植物。本研究采用流式细胞术与K-mer分析方法对车前和大车前的基因组大小进行测定,为后续全基因组测序提供参考。实验过程采用木本植物缓冲液(woody plant buffer, WPB)解离并制备车前和大车前嫩叶材料的细胞核悬浮液,以玉米基因组DNA为对照,通过流式细胞仪测定经碘化丙啶(propidium iodide, PI)染色的车前属植物细胞核样品的荧光强度,计算基因组大小。此外,本研究采用高通量测序技术对车前和大车前进行双末端测序,同时利用K-mer法估计基因组大小,并分析杂合率、重复序列和GC含量等信息。结果显示,利用流式细胞术测得车前基因组大小约为2 386.01 Mb;大车前基因组大小约为743.14 Mb。而通过基因组测序,分别获得了78 Gb的车前和58 Gb的大车前基因组数据。K-mer分析的结果表明,车前基因组大小约为2 161.54 Mb,测序深度为36 X;大车前基因组大小约为701.17 Gb,测序深度为80 X,K-mer分布曲线图显示车前和大车前都是低杂合基因组,车前基因组含有较高比例的重复序列(51.72%)。研究结果为揭示车前属植物基因组大小的进化提供了重要证据,也为后续三代全基因高通量测序和组装提供了必要的数据参考。P. asiatica and P. major(Plantago) are two traditional Chinese medicinal plants. In this study, flow cytometry and K-mer survey methods were utilized to estimate the genome sizes of P. asiatica and P. major,providing a reference for subsequent whole genome sequencing. The nucleuses were prepared by woody plant buffer(WPB) and stained by propidium iodide(PI). Then, the genome sizes of P. asiatica and P. major were estimated by fluorescence intensity of flow cytometry and Zea mays DNA was used as standards. Also, two-paired end sequencing using second-generation high-throughput technology was conducted. Then K-mer survey was used to assess the genome sizes, heterozygosities, repeat sequences and GC contents. The genome size of P. asiatica was estimated to be 2 386.01 Mb by flow cytometry and the P. major genome is about 743.14 Mb. Also we obtained78 and 58 Gb data from P. asiatica and P. major respectively by genome sequencing. The genome sizes of P.asiatica was 2 161.54 Mb and the total sequencing depth was about 36 X by K-mer survey;the P. major genome was 701.17 Mb and the sequencing depth was about 80 X. Distribution curves of K-mer showed that the heterozygous rates were low in both species, and the content of repeats was relatively high in P. asiatica(51.72%). This study improve the knowledge of evolution of genome sizes in genus Plantago. Also, our results provide an important database reference for the future work of whole-genome sequencing and assembly based on third-generation high-throughput technology.

关 键 词：车前 大车前 流式细胞 K-mer分析 基因组大小 

分 类 号：S567[农业科学—中草药栽培]