三角梅SRAP-PCR反应体系的建立及引物筛选

摘要

利用L9(34)正交试验设计,对影响PCR反应的TaqDNA聚合酶量、dNTP浓度、Mg2+浓度和引物浓度4个因素以及模板DNA浓度进行了三角梅SRAP-PCR扩增反应条件优化研究,并对引物进行了全面筛选.三角梅SRAP-PCR优化反应体系结果为:2.5 μL 10×PCR buffer、60 ng模板DNA、TaqDNA聚合酶1.0U、dNTP 0.25 mmol/L、Mg2+ 2.5 mmol/L、引物0.3μmol/L,总体积25 μL.运用该结果从208对引物组合中共筛选出扩增条带清晰,多态性丰富的SRAP引物27对.优化体系的建立及其引物的筛选为今后利用SRAP标记技术进行三角梅遗传分析、图谱构建、基因定位与种质资源鉴定奠定了技术基础.%Bougainvillea is one of most important ornamental trees and shrubs in the world, but the related SRAP-PCR application on it has been hardly reported at present. Based on the L9(34) orthogonal experimental design, the optimum SRAP-PCR reaction system for Bougainvillea was established as follows: 2. 5 μLl0×PCR Buffer, 60ng template DNA, 1. 0 U TaqDNApolymerase, 0. 25 mmol/L dNTP, 2.5 mmol/L Mg2+ and 0. 3 μmol/L primer, the total reaction volume was 25 μL. 27 primer pairs were screened out from 208 SRAP primer pairs combinations based on their stable amplification, clear banding patterns and good polymorphism by utilizing the SRAP reaction system. The optimal SRAP-PCR reaction system was established and primers were screened, which would provide the basis for genetic analysis, map construction, gene localization and i-dentification of germplasm resources for Bougainvillea.