板栗SSR和RAPD技术体系的建立

主要从板栗DNA提取、PCR条件的优化等环节对SSR和RAPD的反应条件进行了优化。结果表明,采用PVP和β-巯基乙醇联合除酚,用多次洗涤和高盐相结合进行除糖提取高质量板栗基因组DNA,所获得的DNA完整,无降解,分子量在23kb以上,可扩增、可酶切,D260nm/D230nm在2.0以上,D260nm/D280nm为1.8～2.0,产率为40～300μgDNA/g叶组织,完全满足SSR、RAPD、AFLP等分子标记技术分析的需要。采用单因素对PCR反应体系中各个影响因素进行优化,结果表明对PCR结果影响最为明显的是退火温度和Mg2+浓度的变化。对退火温度和Mg2+浓度进行优化,确定RAPD的PCR反应体系Mg2+浓度为2.0mmol/LMgCl2,退火温度38℃;SSR反应体系Mg2+浓度为2.0mmol/L,退火温度56℃。建立了一套完善的适用于板栗的SSR和RAPD分析的稳定技术体系。

Establishment of SSR and RAPD technique system in Castancea mollissima

SSR (Simple Sequence Repeat) and RAPD (Random Amplified Polymorphic DNA) have been extensively used as techniques in research in fruit crops for different purpose. However, there are seldom reports about these techniques used in chestnut. To establish SSR and RAPD techniques adapted to chestnut, the methods for extraction of high quality DNA were developed. PVP and 2-mercaptoethanol were added to lyses buffer in order to inhibit oxidation of polyphenol. High concentration of salt solution and elution for many times were used to remove polysaccharide. High quality DNA with molecular mass above 23 kb, D260 nm/D280 nmbetween 1.8 and 2.0 and D260 nm/D230 nm over 2.0 was obtained which can be amplified by PCR and digested by restriction enzyme. The reaction conditions of PCR (Polymerase Chain rReaction) for SSR and RAPD with chestnut genomic DNA as template were optimized. The results showed that the annealing temperature and Mg2+ concentration were the most important factors. Stable reaction systems for SSR and RAPD analysis in chestnut were established, i.e. 38℃ as annealing temperature and 2.0 mmol/L MgCl2 for RAPD and 56℃ and 2.0 mmol/L MgCl2 for SSR, respectively.