梨ISSR-PCR反应体系的优化及在SRAP、SSR标记中的通用性研究

为获得最佳的梨ISSR-PCR反应体系,以及验证在其他标记上具有通用型,以‘新世纪梨’ב崇化大梨’杂交F1代为试验材料,采用L25（56）试验正交设计法及单因素方法,对反应体系中的模板DNA量、dNTPs、引物、TaqDNA聚合酶这4个单因素进行优化。结果表明,梨ISSR-PCR最优的20μL体积的反应体系中,含模板DNA3 ng/μL,dNTPs 0.20 mmol/L,引物0.5μmol/L,TaqDNA聚合酶0.03 U/μL。采用优化后的反应体系对试验材料进行了SRAP、SSR标记方法的扩增,结果显示该反应体系也适用于梨属的SRAP、SSR分析。为梨属在以后的多标记的遗传多样性分析、种质资源评价和遗传图谱的构建等提供理论基础,并在试验中减少费用和提高效率。

Optimizing of ISSR-PCR System in Pyrus and General Research in SRAP and SSR Markers

The study aims to optimize ISSR-PCR reaction system in Pyrus and verify its universality in other markers. Taking‘Shinseiki＇בChonghuadali＇F1hybrid generation as the experimental material, the L25（56）orthogonal design was used to optimize 4 factors in ISSR-PCR amplification, including DNA template, primer,dNTPs and Taq polymerase. The result of PCR was analyzed, the most suitable ISSR-PCR system for Pyrus was established, which was 20 μL reaction system, containing DNA 3 ng/μL, dNTPs 0.20 mmol/L, primer 0.5 μmol/L and TaqDNA polymerase 0.03 U/μL. Optimization of the reaction system also applied to SRAP and SSR markers. The study can provide theoretical basis for future genetic diversity analysis, evaluation of germplasm resources and genetic map construction in Pyrus, and cut the cost and raise the efficiency of the experiment.