刨花润楠SSR-PCR体系优化及天然种群遗传多样性研究  

Optimization of SSR-PCR Reaction System and Study on Genetic Diversity of Natural Populations of Machilus pauhoi

作　　者：朱芹 李培 周鹏 张俊杰 阙青敏 惠文凯 陈晓阳 ZHU Qin;LI Pei;ZHOU Peng;ZHANG Jun-jie;QUE Qing-min;HUI Wen-kai;CHEN Xiao-yang(School of Life Science, Jiaying University, Meizhou 514015, Guangdong, China;College of Forestry and landscape Architecture,South China Agricultural University, Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm,State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangzhou 510642, Guangdong, China;College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083,China)

机构地区：[1]嘉应学院生命科学学院,广东梅州514015 [2]华南农业大学林学与风景园林学院,广东省森林植物种质创新与利用重点实验室,亚热带农业生物资源保护与利用国家重点实验室,广东广州510642 [3]北京林业大学生命科学与技术学院,北京100083

出　　处：《林业科学研究》2019年第4期70-78,共9页Forest Research

基　　金：国家“十二五”科技支撑项目(2012BAD01B04);广东省林业科技创新项目(2011KJCX002)

摘　　要：[目的]建立刨花润楠SSR-PCR最佳反应体系,从樟科树种SSR引物中筛选多态性高的引物,并对刨花润楠遗传多样性进行分析。[方法]对影响PCR反应体系的5个因素设置7个水平,利用正交设计L 16 (4 5)进行优化,确定最佳体系,并用该体系从187对候选引物中进行引物筛选。利用软件POPGENE1.32、PowerMarkerv3.25、FSTAT、GenAlex6.5、Structure2.3和POPTREE分别计算观测等位基因数目(Na)、观察杂合度(Ho)、期望杂合度(He)、多态性信息量( PIC )、基因流(Nm),进行群体遗传结构分析和UPGMA法聚类分析。[结果]刨花润楠最佳体系(20 μL)为: Taq 酶1.0 U、dNTPs 0.25 mmol·L -1 、Mg^ 2+ 1.25 mmol·L ^-1 、引物0.5 μmol·L^-1 、模板50 ng。最终筛选出12对具有多态性高的SSR引物。 Na、Ho、He和PIC 的平均值分别为15.083、0.576、0.751和0.722,表明种群具有较丰富的遗传多样性;Nm 平均值为1.500,种群间存在频繁的基因交流;UPGMA将24个种源聚为3类,与Structure分析结果一致。[结论]本研究成功优化了刨花润楠SSR-PCR反应体系,并获得12对适用于刨花润楠的SSR引物,刨花润楠种群遗传多样性丰富,种群间存在频繁的基因交流,24个种源可聚为3类。[Object] To establish SSR-PCR reaction system of Machilus pauhoi, screen the high polymorphic primers from the SSR primers of Lauraceae, and to investigate the genetic diversity of M. pauhoi.[Method] In order to establish the optimum SSR-PCR reaction system of M. pauhoi, seven concentration levels were set for each of five factors to determine the suitable concentration range by using L 16 (4 5) orthogonal design. The system was used to filter primers from 187 candidate primers. The software of POPGENE1.32, PowerMarkerv3.25, FSTAT, GenAlex6.5, Structure2.3 and POPTREE were used to calculate the observed allelic number ( Na ), observed heterozygosity ( Ho ), expected heterozygosity ( He ), Polymorphism Information Content ( PIC ), gene flow ( Nm ), the genetic structure and cluster analysis based on the unweighted pair group method of arithmetic averages (UPGMA).[Result] The optimal reaction system was established, the optimum concentration of five factors are as follows: Taq polymerase, dNTPs, Mg^ 2+, primers and template DNA were 1.0 U·μL^-1 , 0.25 mmol·L ^-1 , 1.25 mmol·L^-1 , 0.5 μmol·L^-1 and 50 ng in a total volume of 20 μL, respectively. Twelve pairs of SSR primers with high polymorphism were screened. The average Na, Ho, He and PIC were 15.083, 0.576 , 0.751, and 0.722, respectively, which means higher genetic diversity in M. pauhoi. The average Nm was 1.500, which means high gene flow existed among the provenances. The analysis with UPGMA was consistent with Structure software which indicated that the twenty-four provenances could be divided into three groups.[Conclusion] The study optimizes the SSR-PCR reaction system successfully, twelve primers are screened out from 187 candidate primers using the optimal reaction system .There is higher genetic diversity in M. pauhoi population. The limited gene flow exists in these provenances, and the twenty-four provenances can be divided into three groups.

关 键 词：刨花润楠 SSR PCR 体系优化 种群 遗传多样性 

分 类 号：S718.46[农业科学—林学]