光皮桦AFLP分子标记体系的建立

Establishment of AFLP Molecular Labeling Technique System for Betula luminifera

为了建立光皮桦AFLP分子标记体系,利用SDS法、常规CTAB法和CTAB-硅珠法提取光皮桦(Betula luminiferaH.Wink.)嫩叶DNA,并进行检测比较。结果显示,CTAB-硅珠法更适合光皮桦基因组DNA的提取,所得在基因组DNA纯度高OD值在1.8左右,适用于AFLP分析。利用AFLP分子标记技术,采用MseⅠ-EcoRⅠ酶切组合,从64个引物组合中筛选出51个带型分布均匀、多态性高且分辨能力强的引物组合。同时,通过对比实验确定了光皮桦AFLP反应的最佳模板DNA用量为300ng、酶切时间4h和预扩增产物稀释倍数30倍等,优化了相关AFLP反应体系,为今后利用AFLP分子标记技术研究光皮桦野生居群的遗传多样性分析和分子遗传图谱的构建打下坚实的基础。

For Establishing AFLP molecular labeling technique system of Betula luminifera, we using the method of CTAB - SiOx, CTAB and SDS were used to extract total genomic DNA from tender leaves of Betula luminifera. The results indicated that the DNA extracted by CTAB - SiO2 was clean and integrated. The method of CTAB - SiO2 was useful for amplified flagnent length polymorphism （AFLP） of Betula luminifera,whose OD are almost at 1.8.By the AFLP technique, using EcoR 1 and Mse Ⅰ enzyme- cut combination, fifty- one pairs of primers with high polymorphism and powerful distinctiveness were selected from sixty - four pairs of primers, and the best DNA amount is 300ng, enzyme - cut time is about 4h and dilution multiple of pre - expending product that is 30 times which were determined by comparison of experiments. Our results tentatively established the basis for the research of the genetic diversity analysis and genetic mapping.