睡莲ISSR-PCR体系的优化及后续亲缘关系鉴定  

Optimization of ISSR-PCR System and Subsequent Identification of Phylogenetic Relationship of Nymphaea nouchali

作　　者：杨志娟 李保豫 王京华 金奇江 王彦杰[2,3,4] 徐迎春 YANG Zhi-juan;LI Bao-yu;WANG Jing-hua;JIN Qi-jiang;WANG Yan-jie;XU Ying-chun(Sanya Institute of Breeding and Multiplication,Hainan University,Sanya 572025,China;College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China;Sanya Institute of Nanjing Agricultural University,Sanya 572025,China;Key Laboratory of Flower Biology and Germplasm Creation,Ministry of Agriculture and Rural Affairs,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]海南大学三亚南繁研究院,海南三亚572025 [2]南京农业大学园艺学院,江苏南京210095 [3]南京农业大学三亚研究院,海南三亚572025 [4]南京农业大学农业农村部花卉生物学与种质创制重点实验室,江苏南京210095

出　　处：《江西农业学报》2024年第12期23-30,39,共9页Acta Agriculturae Jiangxi

基　　金：海南省自然科学基金高层次人才项目(321RC670);国家自然科学基金新疆联合基金项目(U1803104、U2003113);海南省自然科学基金面上项目(322MS158);南京农业大学三亚研究院引导资金项目(NAUSY-MS04);南京农业大学三亚研究院成果转化资金项目(NAUSY-CG-YB09)。

摘　　要：采用正交实验对睡莲的ISSR体系进行优化,利用ISSR分子标记技术对延药睡莲(海南)、延药睡莲(印度)、延药睡莲(越南)以及其他从国内外引进的29份睡莲种质资源进行遗传多样性分析。结果表明:总体积为25μL(模板DNA浓度15 ng/μL、2×ES Taq Master Mix用量14μL、引物浓度1.0μmol/L、ddH_(2)O补齐)的睡莲ISSR-PCR体系为最优。使用延药睡莲等8份睡莲种质为材料,从100条ISSR引物中筛选出了10条重复性强、多态性好的引物,并优化了退火温度。使用最优优化体系在32份睡莲种质资源中共扩增出154条带,平均每条引物扩增条带数为15.4条,多态性条带149条,多态性比例为96.9%;平均shannon信息指数(Ⅰ)为0.4582,平均等位基因数Na为2.0000,平均有效等位基因数Ne为1.5064,平均Nei’s基因多样性指数H为0.3001,遗传距离在0.0355~0.7908。基于遗传距离构建聚类分析图,对海南延药睡莲和其他31种睡莲的亲缘关系进行了说明,在进化距离为0.2时可将实验材料分为三类。研究结论阐明了不同种质间的亲缘关系,可为后续睡莲的群体遗传分析奠定实验基础,并为亲缘关系的鉴定和种质资源的保护与利用提供参考。In this experiment,the ISSR system of Nymphaea nouchali was optimized by orthogonal design,and then the genetic diversity of Nymphaea nouchali(Hainan),Nymphaea nouchali(India),Nymphaea nouchali(Viet Nam)and 29 germplasm resources of Nymphaea nouchali were analyzed by ISSR molecular marker technology.The results showed that the optimal ISCR-PCR system was the total volume of 25μL(template DNA concentration 15 ng/μL,2×ES Taq Master Mix dosage 14μL,primer concentration 1.0μmol/L,ddH_(2)O complement).Ten primers with strong repeatability and good polymorphism were selected from 100 ISSR primers using 8 germplasm materials such as Nymphaea nouchali as materials,and the annealing temperature was optimized.A total of 154 bands were amplified from 32 germplasm resources using the optimized system,with an average of 15.4 bands per primer and 149 polymorphic bands(96.9%).The average shannon information index(Ⅰ)was 0.4582,the average allele number Na was 2.0000,the average effective allele number Ne was 1.5064,the average Nei’s gene diversity index H was 0.3001,and the genetic distance was 0.0355~0.7908.Based on the genetic distance,the clustering analysis diagram was constructed.The phylogenetic relationship between Nymphaea nouchali and 31 other Nymphaea species was explained,and the experimental materials could be divided into three types when the evolutionary distance was 0.2.The aim of this study was to clarify their genetic relationships.It laid the experimental foundation for the subsequent population genetic analysis of Nymphaeae nouchali,and provided reference for the identification of genetic relationships and the protection and utilization of planting resources.

关 键 词：延药睡莲 ISSR-PCR体系优化 亲缘关系 

分 类 号：S682.32[农业科学—观赏园艺]