萝卜EST-SSR标记PCR反应体系的建立与优化

摘要

In this paper, we optimized the EST-SSR maker PCR system of radish by using L16 (45) orthogonal design. The results showed that the best PCR system in 20 μL was as follows: 65 ng/20 μL DNA template, 0.8 mmol/L Mg2+, 0.25 mmol/L dNTPs, 0.8μmol/L primer, 0.4 U/20μL Taq DNA polymerase, and the greatest factor for this system was the concentration of Mg2+. The system had been verified by 10 pairs of EST-SSR primers, and the newly established EST-SSR PCR system for radish was fully repeatable with high reliability, clear bands and rich DNA polymorphism, which could be applied in the genetic evaluation of radish germplasm resources and in the molecular assisted selection breeding of radish.%　　采用L16(45)正交试验设计优化了萝卜EST-SSR标记的PCR反应体系,建立了适于20μL PCR技术体系的最佳浓度：DNA模板65 ng/20μL,Mg2+0.8 mmol/L,dNTPs 0.25 mmol/L,引物0.8μmol/L,Taq DNA酶0.4 U/20μL,且Mg2+对该体系的影响最大.利用10对EST-SSR引物对该体系的验证结果表明,本研究建立的萝卜EST-SSR PCR体系重复性好、条带清晰、多态性丰富,对萝卜的种质资源的鉴定与评价、分子标记辅助选择育种等多方面都有重要作用.