大白菜SSR检测体系的优化

Optimization of SSR-PCR System for Identification in Chinese Cabbage (Brassica campestris L.ssp.pekinensis)

以不同地区栽培的5份大白菜品种为试材,从PCR反应组成、扩增程序、电泳检测等环节对SSR技术进行了优化,建立了一套适用于大白菜品种鉴定的SSR-PCR体系。即10μL反应组成为:1×buffer;2.50mmol/LMgCl2;0.10mmol/LdNTPs;0.35μmol/LSSR引物;1.00ng/μL模板DNA和0.40UTaq酶。适宜的扩增程序为72℃热启动3min,94℃预变性2min后进行20个迫降循环:94℃变性60s,68℃退火60s,72℃延伸45s(-1℃/2cycles);再进行20个循环:94℃变性60s,58℃退火60s,72℃延伸45s,72℃延伸10min。应用SDS-聚丙烯酰胺凝胶(银染)电泳检测并取得很好的效果。选用108对芸薹属SSR引物,对这5份品种的基因组进行扩增,筛选出具有2条以上特异扩增条带的SSR引物22对。用这些引物对5份品种进行扩增,初步分析了SSR标记的多态性及用于大白菜指纹分析的潜力。

In this study, we optimazed SSR-PCR system including amplification reaction components, procedure program and electrophoresis detection. A stable SSR-PCR system was established for identification of 5 Chinese cabbage cultivars from 5 different cultivating regions which including modifying protocols as following： 1 0 μL reaction solution contained 1× buffer, 2.50 mmol/L MgCl2, 0. 10 mmol/L dNTPs, 0.35 μmol/L SSR primers, 1.00 ng/μL DNA template, 0.40 U Taq polymerase. PCR amplifying program was divided into two steps. After one hot starting step of 3 min at 72℃ and one denaturing step of 2 min at 94℃, DNA was denatured for60 s at 94℃ , annealing temperature was 68℃ for 2 cycles and subsequently was dropped 1℃ every 2 cycles until a final temperature of 58℃ was reached. For the last 20 cycles of the amplification, an annealing temperature of 58℃was employed. After all the cycles finished, amplification was ended for 10 min at 72℃. The SDS-polyacrylamide gel electrophoresis and silver staining were employed for detecting and scoring the banding patterns. In this research 5 genomic DNA extracted from the tested 5 Chinese cabbages were used as template DNA amplified by 108 SSR （simple sequence repeats） primer pairs designed from Brassica spp. 22 SSR primer pairs can revealed two bands or more in 5 Chinese cabbage cultivars. The polymorphisms for Chinese cabbage revealed by SSR marker and the potentiality of cultivar identification using SSR fingerprint were discussed also in this paper.