短丝木犀SSR-PCR反应体系优化及基于DNA混合池的引物快速筛选  

Optimization of SSR-PCR Reaction System in Osmanthus serrulatus and Rapid Primer Selection Based on DNA Mixing Pool

作　　者：钱慧蓉 陈林[1,2] 杨国栋 潘婷婷[1,2] 王贤荣 Qian Huirong;Chen Lin;Yang Guodong;Pan Tingting;wang Xianrong(Co-Innovation Center for the Sustainable Forestry in Southern China,Nanjing Forestry University,Nanjing,210037;College of Biology and Environment,Nanjing Forestry University,Nanjing,210037)

机构地区：[1]南京林业大学南方现代林业协同创新中心,南京210037 [2]南京林业大学生物与环境学院,南京210037

出　　处：《分子植物育种》2019年第2期525-530,共6页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31300558);江苏省基础研究计划项目(BK20130972);江苏省高校优势学科建设工程资助项目(PAPD);江苏高校品牌专业建设工程资助项目(TAPP);南京林业大学高学历人才基金项目(GXL201308)共同资助

摘　　要：本研究采用L9(23)正交试验设计确定短丝木犀的SSR-PCR最佳反应体系,同时设置12个不同的温度梯度优化引物退火温度;利用优化后的体系以50对候选SSR引物为对象,对6个短丝木犀基因组DNA样品等量混合,进行PCR扩增筛选引物。结果表明,第一,短丝木犀SSR-PCR最优反应体系为:1.0μL50ng/μL模板DNA,1.0μL 100μmol/L引物,12.5μL 2×Taq PCR Master Mix,补ddH2O至25μL;第二,DNA混合池方法比常规的筛选方法具有效率高,实验资源消耗低的优点,可用于短丝木犀SSR引物的大量筛选;第三,最终筛选出10对具有高多态性、高稳定性且重复性好的SSR引物。本研究旨在建立适合短丝木犀的SSR-PCR最佳反应体系,并以DNA混合池为模板进行引物筛选,为短丝木犀引物的大量开发提供了一种新的途径。In this study, SSR-PCR optimum reaction system of Osmanthus serrulatus was established by using L9(2^3) orthogonal experimental design, and 12 different temperature gradients were set to optimize the annealing temperature of primers. Using the optimized system of SSR-PCR, 50 pairs of candidate SSR primers were mixed into 6 genomic DNA samples of O. serrulatus by equal quantity, and the primers were screened by PCR amplification. The results showed that firstly, the optimal SSR-PCR reaction system was as follows: 1.0 μL 50 ng/μL genomic DNA, 1.0 μL 100 μmol/L primer, 12.5μL 2×Taq PCR Master Mix, and replenishing ddH2 O to 25 μL. Secondly, compared with conventional screening method, the DNA mixing pooling method could greatly shorten experimental period and significantly reduce the consumption of experimental resources, which would be useful for screening substantive SSR primers in O. serrulatus. Thirdly, 10 pairs of SSR primers with high polymorphism,high stability, and good repeatability were finally screened out. The purpose of this study was to establish an optimal SSR-PCR reaction system suitable for O. serrulatus, and to screen primers by using DNA mixing pool, which would provide a new way for the massive screening of SSR primers in O. serrulatus.

关 键 词：短丝木犀 SSR-PCR 优化 引物筛选 基因组DNA混合池 

分 类 号：S685.13[农业科学—观赏园艺]