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对AFLP实验流程中的基因组提取、酶切等关键因素进行优化,建立了芒属植物的AFLP(扩增片段长度多态性)分析体系。本研究分别采用常规CTAB法和改良的CTAB法提取芒属植物基因组DNA,基因组分别用HindⅢ/MseⅠ系统和PstⅠ/MseⅠ系统进行酶切和连接一步反应,连接产物稀释20倍后进行预扩增,预扩产物稀释20倍后进行选择性扩增。结果显示:采用改良CTAB法提取的DNA质量优于常规CTAB法;采用PstⅠ/MseⅠ系统酶切的DNA多态性高于HindⅢ/MseⅠ系统。采用优化后的AFLP体系分析30份供试芒属植物材料,从64对引物中筛选出8对多态性较好的AFLP选择性扩增引物,多态性比率达到100%,表明优化后的AFLP体系适合于芒属植物的分子标记分析,为芒属植物种质遗传多样性的分子研究奠定了技术基础。
AFLP experimental process of genomic extraction, enzyme digestion and other key factors to optimize the establishment of the genus Mount AFLP (amplified fragment length polymorphism) analysis system. In this study, genomic DNA was extracted from the genus Miscanthus by conventional CTAB method and modified CTAB method respectively. The genome was digested and ligated in one step with the HindIII / MseI system and the PstI / MseI system respectively. The products were pre-amplified after 20-fold dilution. Pre-amplified products were diluted 20-fold selective amplification. The results showed that the quality of DNA extracted by modified CTAB method was better than that of conventional CTAB method. The DNA polymorphism of PstⅠ / MseⅠ system was higher than that of HindⅢ / MseⅠ system. The optimized AFLP system was used to analyze 30 M. officinale plants and 8 pairs of AFLP selective amplification primers with good polymorphism were screened from 64 pairs of primers and the polymorphic ratio was 100% AFLP system is suitable for Miscanthus molecular marker analysis, which laid the technical foundation for the molecular study on the genetic diversity of the genus Miscanthus.