美洲南瓜SSR-PCR反应体系的优化及引物筛选

摘要

Abstract:The best SSR-PCR reaction system of Cucurbita pepo L.was established by optimizing the main factors of the SSR-PCR reactions according to the single factor test and orthogonal design of L16 (44).The experimental results showed that the SS-PCR reactions were significantly affected by various factors at different levels.The optimal,20 μL Cucurbita pepo L.SSR-PCR reaction system consisted of 1.00 μmol · L-1 primer; 0.150 mmol · L-1 dNTP; O.9 U Taq DNA polymerase; 50 ng DNA template.Sixty eight primer pairs with clear banding and plentiful polymorphism were selected from 300 primer pairs for SSR analysis in Cucurbita pepo L.using the optimized reaction system.%应用单因素实验和L16(44)正交设计对影响美洲南瓜SSR-PCR的主要参数进行优化,建立适合美洲南瓜SSR-PCR反应的最佳体系.结果表明,不同水平的各因素对PCR反应结果均有影响,优化后美洲南瓜SSR-PCR反应体系(20μL)为:1.00μnnol·L-1引物、0.150 mmol·L-1 dNTP、0.9 U Taq酶、50 ng DNA模板.运用优化后的反应体系对美洲南瓜进行多态性引物的筛选,从300对引物中筛选出68对扩增条带清晰、多态性丰富的SSR引物.