胡椒SRAP反应体系的建立和优化  

Establishment and Optimization of Black Pepper SRAP Reaction System

作　　者：姜艳[1,2] 刘进平[1] 

机构地区：[1]海南大学热带作物种质资源保护与开发利用教育部重点实验室/海南大学农学院,海口570228 [2]云南省德宏热带农业科学研究所,云南瑞丽678600

出　　处：《中国农学通报》2012年第31期141-145,共5页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金项目"海南野生胡椒种质资源遗传多样性分析和保护利用"(31060204)

摘　　要：建立并优化胡椒SRAP分子标记体系,为海南胡椒属植物亲缘关系和遗传多态性分析、物种和品种鉴定等打下技术基础。利用单因素随机试验对胡椒SRAP-PCR反应体系中各组分(TaqDNA聚合酶、dNTP、模板DNA、引物和Mg2+)的浓度进行优化,同时筛选SRAP-PCR反应的循环数和最适退火温度。通过实验确定了SRAP-PCR反应体系为:反应总体系为20μL,其中引物0.35μmol/L,TaqDNA聚合酶1.0U,dNTP0.6mmol/L,Mg2+1.5mmol/L,模板DNA25～200ng,同时通过梯度PCR试验,确定引物最佳退火温度;最佳SRAP-PCR反应程序为:94℃预变性5min;94℃变性30s,35℃退火30s,72℃延伸45s,5个循环;然后94℃变性30s,48℃退火30s,72℃延伸45s,40个循环;最后72℃延伸7min,4℃保存。SRAP-PCR体系适为胡椒属植物遗传多样性分析奠定了基础,并成功地应用于海南胡椒属植物亲缘关系和遗传多态性分析。The aim of the experiment was to establish and optimize black pepper SRAP molecular marker system,lay a technical foundation for the analysis of genetic relationship and polymorphism,species and variety identification of Piper plants in Hainan.The conditions of SRAP-PCR reaction（concentrations of Taq DNA polymerase,dNTP,primer,Mg 2 ＋ and template DNA,the number of thermal cycles and optimum anneal temperatures） were optimized using the method of single-factor experiment respectively.The optimal ISSR-PCR amplification was established as follows：20 μL PCR mixture consisted of 1.0 U Taq DNA polymerase（TaKaRa Biotechnology）,6× PCR buffer,0.6 mmol/L dNTP,0.35 μmol/L primer,1.5 mmol/L Mg 2＋,and 25-200 ng template DNA.Thermal cycling（Biometra T1 Thermocycle） started with 5 min at 94℃ for initial denaturing,and 5 cycles of 30 s at 94℃,30 s at 35℃,and 45 s at 72℃,followed by 40 cycles of 30 s at 94℃,30 s at 4℃ and 45 s at 72℃.The last cycle was followed by a 7 min extension at 72℃.This SRAP-PCR system was suitable for the analysis of genetic diversity of Piper,and was successfully applied to the analysis of genetic relationship and genetic polymorphism of Piper spp.in Hainan Island.

关 键 词：胡椒 SRAP 分子标记 PCR扩增 

分 类 号：S59[农业科学—作物学] S573.9