中国沙棘SSR-PCR反应体系的优化及引物开发  

Optimization and Primer Development of SSR-PCR Reaction System in Hippophae rhamnoides subsp.sinensis

作　　者：刘青青 马玉花[1] 李雄杰 马福林 高佩 马亚琼 杨开宇 LIU Qingqing;MA Yuhua;LI Xiongjie;MA Fulin;GAO Pei;MA Yaqiong;YANG Kaiyu(College of Agriculture and Animal Husbandry,Qinghai University,Xining,810016;Qinghai Academy of Animal Science and Veterinary Medicine,Qinghai University,Xining,810016)

机构地区：[1]青海大学农牧学院,西宁810016 [2]青海大学青海省畜牧兽医科学院,西宁810016

出　　处：《基因组学与应用生物学》2023年第10期1038-1049,共12页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31660071);青海省科技厅项目(2017-ZJ-734)。

摘　　要：本研究旨在建立适宜中国沙棘(Hippophae rhamnoides subsp.sinensis)SSR-PCR的反应体系,以期为中国沙棘遗传多样性分析、种质资源鉴定等相关研究奠定基础。本研究以来自青海不同地区的中国沙棘野生种的150个植株为试验材料,采用单因素试验和L16(45)正交实验对影响中国沙棘SSR-PCR反应体系的因素进行优化。正交试验极差分析和方差分析结果均显示,影响中国沙棘SSR-PCR反应体系扩增效果的5个因素的主次顺序为缓冲液>dNTPs>引物>模板DNA>Taq DNA聚合酶。结合直观分析的评分结果,确定适用于中国沙棘的最佳SSR-PCR反应体系为0.8 U Taq DNA聚合酶、 2 mmol/L缓冲液、 1.5 mmol/L dNTPs、 0.5μmol/L引物、 25 ng模板DNA,加双蒸水至20μL;筛选出15对条带清晰、多态性丰富且重复性好的SSR引物。本研究可为中国沙棘的亲缘关系鉴定和分子遗传育种等研究提供科学依据。The aim of this study is to establish a response system suitable for SSR-PCR of Hippophae rhamnoides subsp.sinensis,and laying a foundation for the analysis of genetic diversity and germplasm resource identification of Hippophae rhamnoides subsp.sinensis.In this study,150 plants from wild species of Hippophae rhamnoides subsp.sinensis from different regions of Qinghai were used as ex-perimental materials,and univariate experiments and L16(4^(5))orthogonal experimental designs were used to optimize the factors affect-ing the SSR-PCR reaction system of H.rhamnoides subsp.sinensis.The results of orthogonal test range analysis and ANOVA showed that the primary and secondary order of five factors affecting the amplification effect of H.rhamnoides subsp.sinensis SSR-PCR reaction system was buffer>dNTPs>primers>template DNA>Taq DNA polymerase.Combined with the scoring results of intuitive analysis,the optimal SSR-PCR reaction system suitable for H.rhamnoides subsp.sinensis was determined:0.8 U Taq DNA polymerase,2 mmol/L buffer、1.5 mmol/L dNTPs,0.5μmol/L primers,25 ng DNA,add dd H_(2)O to 20μL of the reaction system.Fifteen pairs of SSR primers with clear bands,rich polymorphisms and good repeatability were screened.This study provides a scientific basis for the i-dentification of kinship and molecular genetic breeding of H.rhamnoides subsp.sinensis.

关 键 词：中国沙棘 SSR-PCR 单因素试验 正交试验 

分 类 号：S793.6[农业科学—林木遗传育种]