药用菊花SSR-PCR反应体系优化及引物筛选

Optimization and Primer Screening of SSR-PCR Reaction System in Medicinal Chrysanthemum morifolium Based on Orthogonal Design

为进一步开发利用药用菊花种质资源和开展分子标记辅助选择育种,本研究利用L25（56）正交设计对影响药用菊花SSR-PCR反应的模板DNA、Mg2＋、d NTPs、Taq酶、引物等5个因素进行优化,并对SSR引物进行筛选。结果建立了药用菊花SSR-PCR最佳反应体系（20μL）：模板DNA 60 ng,正、反向引物0.25μmol/L,d NTPs 0.3 mmol/L,Mg2＋3.0 mmol/L,Taq酶1.5 U。运用优化后的反应体系,从136对引物中成功筛选出了扩增条带清晰、多态性丰富的SSR引物57对,大多数条带大小集中在100~500 bp,不同引物扩增的条带数为5~15条。优化的SSR-PCR反应体系在多个药用菊花品种遗传多样性研究中得到了验证,获得了稳定性、重复性良好和多态性丰富的扩增图谱。该体系的建立可为今后利用SSR标记对药用菊花种质鉴定、遗传多样性分析、系统发育研究、遗传图谱构建、基因定位和分子标记辅助育种等研究提供了依据。

In order to make a best utilization of medicinal Chrysanthemum morifolium resources and to develop marker-assisted selection breeding,the L25（56） orthogonal design was applied to optimize the five factors at five concentration levels of SSR-PCR（simple sequence repeat-polymerase chain reaction） system and a certain amount of primer pairs were selected.Optimized PCR reaction mixture was as follows：20 μL total volume including60 ng template DNA,1.5 U Taq DNA polymerase,0.25 μmol/L primer,3.0 mmol/L Mg2＋and 0.3 mmol/L d NTPs.In order to facilitate further application and ensure SSR-PCR reproducibility,fifty-seven SSR primers were screened from 136 primers existing in medicinal C.morifolium.Furthermore,the optimal reaction system was further used to verify with multiple medicinal C.morifolium varieties,The result showed that it is excellent stability and repeatability for the above reaction system.Most of the amplified bands were concentrated between 100 bp and 500 bp and different primers generated from 5 to 15 loci.Our results indicate that the optimized SSR-PCR system would provide theoretic and technical support for germplasm evaluation,analysis of genetic diversity,establishment of genetic maps,gene localizations and molecular marker assisted breeding for medicinal Chrysanthemum morifolium.