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.
出处
《中国农学通报》
CSCD
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)
关键词
胡椒
SRAP
分子标记
PCR扩增
black pepper
SRAP
molecular marker
PCR amplification
