大黄鱼(Larimichthys crocea)fAFLP方法的建立

以大黄鱼为材料,探索及优化了影响荧光标记AFLP(fAFLP)分析体系的主要因素.结果表明:内切酶EcoRⅠ与MseⅠ各0.35 U,双酶切350 ng大黄鱼基因组DNA,依次反应4 h可得最佳效果;T4DNA连接酶2.5 U,16℃过夜连接3μL酶切产物与接头时效果最佳;以1μL连接产物作底物,EcoRⅠ与MseⅠ预扩引物分别为0.3μmol/L与1.5μmol/L时预扩效果最佳;预扩产物稀释超过100倍作为选择性扩增的模板时致使某些样品的电泳条带缺失.使用该体系分析了一个野生大黄鱼群体的AFLP片段的多态水平,结果证明该方法简便有效,可用于群体遗传多态性分析.

Exploration of fAFLP Method in Large Yellow Croaker (Larimichthys crocea)

The major influence factors on fluorescence labeled AFLP （fAFLP） were explored and optimized for large yellow croaker Larimichthys crocea in the present study. The results showed that the digestion reaction could be performed the best when 350 ng genomic DNA were cleaved by 0.35 U EcoR I for 4 hours and then by 0. 35 U Mse I for 4 hours. In the ligation step, the ideal result could be made by 2. 5 U T4 DNA ligase and 3 μL digested product together with reacting overnight at 16 ℃. The pre-amplification had excellently performance when the PCR consisted of 1 μL ligation product, 0. 3 ℃mol/L EcoR I primer, and 0. 15 ℃mol/L Mse I primer. However, more than 100 times dilution of pre-amplification product could lead to the absence of some bands in selectively amplified fingerprints. This fAFLP method was employed in analyzing the AFLP fragments diversity of wild population of L. crocea , and showed a perfect electrophoresis pattern with clearly discernable bands on it.