沙棘木蠹蛾AFLP引物筛选及反应体系的建立

该文以沙棘木蠹蛾幼虫为材料，用改进的SDS-蛋白酶K法获得了高质量的符合AFLP分析要求的基因组DNA。通过对AFLP试验过程中的酶切 连接、预扩增、选择性扩增等各关键因素的比较研究，建立了一套优化的沙棘木蠹蛾AFLP分子标记体系，获得了清晰的指纹图谱。研究结果表明:利用EcoRⅠ/MseⅠ双酶切系统以酶切 连接2～4 h, 预扩增产物稀释20倍、Mg 2+浓度为1.5 mol/mL的选择性扩增效果最好。进一步利用该反应体系从80对E+3 /M+3选择性引物中筛选出10对多态性丰富、分辨率高、谱带清晰的引物组合。该研究结果为利用AFLP标记技术开展沙棘木蠹蛾的种群遗传结构、遗传分化等方面的研究奠定了基础。 

Selection of primers and establishment of AFLP analysis system on Holcocerus hippopaecolus

In this study, high quality genomic DNA isolated by improved SDS proteinase K method from larvae of Holcocerus hippopaecolus was obtained and is approved suitable for AFLP analysis. The AFLP reaction system of H. hippopaecolus was established by optimizing enzyme digestion/ligation reaction, pre-amplification and selective amplification. The optimal reaction time for double enzyme digestion (EcoRⅠ/MseⅠ) was 2-4 hours. Products of the pre amplification diluted 20 fold and effects of the selective amplification with 1.5 mol/mL Mg ^{2 +}were the best. Ten pairs of selectiveprimer combinations which revealed sufficient amplified bands with high resolution and polymorphism were selected from 80 pairs of E₊₃/M_{+ 3} primers and clear fingerprints were obtained. The AFLP reaction system can be used tostudy the population structure and genetic diversity in population of H. hippopaecolus.