决明DNA提取、SRAP-PCR优化及引物筛选

DNA Isolation,Optimization of SRAP-PCR System and Primers Screening of Cassia obtusifolia L.

以决明幼叶为试验材料,对6种DNA提取方法进行比较,并通过单因子梯度比较试验,对SRAPPCR扩增体系中DNA和引物浓度进行优化,在此基础上再对引物进行筛选,随后对不同方法提取的DNA运用优化的SRAP-PCR体系再次进行扩增验证。结果表明改良CTAB法是决明DNA提取的最佳方法;20μL的SRAP-PCR体系中模板最佳质量浓度为2.5mg/L,引物浓度为0.4μmol/L;运用优化体系从180对引物组合中筛选出扩增条带清晰、多态性丰富的SRAP引物72对。

Cassia obtusifolia is an herb of Leguminosae, which seeds is a traditional Chinese medicine used to cure wind-heat cold, flu and acute conjunctivitis. However, the genetic information for this medicinal plant is still unknown, though it is closely associated with the quality control of medicinal materials. Therefore, six DNA extraction methods were evaluated by using young leaves as samples. Through comparing every factor, both concentrations of DNA template and primer pair were opti- mized in SRAP-PCR system. On the basis of optimization, primers were then screened, and the six DNA samples extracted from six methods were verified again. The results showed that the improved CTAB method was the best one. Both 2.5 mg/L DNA template and 0.4μmol/L primer pair were the most suitable concentrations that should be added to the 20 μL total volume of SRAP-PCR system. A total of 72 primer pairs with clear and polymorphic bands were chosen from 180 pairs by using opti- mized PCR system. The optimized SRAP-PCR and screened primers provide the basis for those re- searchers to study the genetic diversity and genetic structure of C. obtusifolia populations.