基于叶绿体DNA序列和ISSR分子标记的菘蓝不同种质遗传变异分析  

Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker

作　　者：朱田田[1,2,3] 杜弢 晋玲[1,2,3] 王富胜 康舒淇[1] 徐丽 张明惠 林和 ZHU Tiantian;DU Tao;JIN Ling;WANG Fusheng;KANG Shuqi;XU Li;ZHANG Minghui;LIN He(Gansu University of Chinese Medicine,Lanzhou 730000,China;Northwest Collaborative Innovation Center for Traditional Chinese Medicine Co-constructed by Gansu Province&MOE of PRC,Lanzhou 730000,China;Engineering Research Center for Evaluation,Protection and Utilization of Rare Traditional Chinese Medicine Resources,Lanzhou 730000,China;Dingxi Academy of Agricultural Sciences,Dingxi 743000,China;School of Information Science and Engineering,Lanzhou University,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学,兰州730000 [2]西北中藏药省部共建协同创新中心,兰州730000 [3]甘肃省珍稀中药资源评价与保护利用工程研究中心,兰州730000 [4]定西市农业科学研究院,甘肃定西743000 [5]兰州大学信息科学与工程学院,兰州730000

出　　处：《中国实验方剂学杂志》2022年第18期117-126,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：道地药材生态种植及质量保障项目(国中医药科技[2020]153号);中国工程院战略研究与咨询项目(GS2021ZDA06-2);中央本级重大增减支项目(2060302);甘肃省重点研发计划项目(21YF5FA110,20YF8NJ166);定西市科技计划项目(DX2020N10)。

摘　　要：目的对47个菘蓝种质进行序列变异和遗传多样性分析,开展其遗传分化和遗传结构研究。方法采用试剂盒提取法提取菘蓝基因组DNA,用2条叶绿体DNA(cp DNA)序列和5条简单重复序列(ISSR)引物进行扩增并测序,通过软件Chromas、Mega 7.0、DanSP5、GenALEx对得到的序列进行校正、拼接及序列特征分析,利用软件PERMUT、PopGen1.31进行遗传多样性参数和遗传结构分析,最后利用NTSYS软件得到47个菘蓝种质的非加权组平均法(UPGMA)聚类树状图。结果菘蓝47个种质的129个样本被成功扩增和测序,2个cp DNA序列经拼接后长度为1412 bp,共有377个多态性变异位点,单倍型数为36个,Fu and Li's D*检验在P<0.01水平上显著;基于cp DNA的核苷酸多样性(Pi)、平均遗传多样性(H_(S))和总遗传多样性(HT)分别为0.11989、0.787和0.891,遗传分化系数遗传分化系数(Gst)、核苷酸分化系数(Nst)和群体间遗传分化指数(Fst)分别为0.117、0.468和0.488,基因流(Nm)值为0.615;基于ISSR的多态位点百分率(PPB)、香农(Shannon)信息多样性指数(I)、Nei's基因遗传多样度指数(H)和Gst平均值为78.85%、0.3348、0.2186和0.7544,Nm值为0.1628。结论菘蓝在物种水平上遗传多样性较高,具有丰富的单倍型类型,不同种质居群间遗传分化较明显,且基因交流不频繁,遗传变异主要存在于居群间,种群近期积累了较多的低频基因突变,推测其历史上曾经历了显著的区域扩张。Objective To analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials,and carry out the study on the genetic differentiation and structure.Method Genomic DNA of 47 I.indigotica germplasm materials were extracted by kit extraction method.Two chloroplast DNA(cp DNA)sequences and five inter-simple sequence repeat(ISSR)primers were used for amplification and sequencing.Chromas,Mega 7.0,DanSP5,and GenALEx were used to calibrate,splice,and analyze the sequence characteristics.PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure,and NTSYS was used to obtain the unweighted pair-group method with arithmetic means(UPGMA)clustering tree plot of 47 I.indigotica germplasm materials.Result A total of 129 samples from 47 I.indigotica germplasm materials were successfully amplified and sequenced.The length of 2 cp DNA sequences after spliced was 1412 bp,and there were 377 polymorphic variation loci,and 36 haplotypes.Fu and Li's D*test was significant(P<0.01).The values of Pi,H_(S),and HT based on cp DNA were 0.11989,0.787,and 0.891,respectively.The genetic differentiation coefficients of gene differentiation coefficient(Gst),nucleotide differentiation coefficient(Nst),and fixation index(Fst)were 0.117,0.468,and 0.488,respectively,and the gene flow(Nm)was 0.615.The mean values of PPB,Shannon information diversity index(I),Nei's genetic diversity index(H),and Gst based on ISSR were 78.85%,0.3348,0.2186,and 0.7544,respectively,and the Nm value was 0.1628.Conclusion I.indigotica has high genetic diversity and abundant haplotypes at the species level,with abundant haplotypes.Genetic differentiation among different germplasm materials is obvious,and gene exchange is not frequent.Genetic variation mainly exists among populations.The population has accumulated various low-frequency gene mutations recently,suggesting that it has experienced significant regional expansion in the history.

关 键 词：菘蓝 种质 叶绿体DNA(cp DNA) 简单重复序列(ISSR)分子标记 遗传变异 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学] R22R2-031R33