松毛虫遗传多样性研究中AFLP反应体系的建立

以油松毛虫为材料,对T4 DNA连接酶不同用量、预扩增中的Mg~(2+)浓度、dNTP浓度、引物浓度以及选择性扩增巾的预扩增产物稀释倍数、Mg2~(2+) 浓度、dNTP浓度、引物浓度和Taq酶浓度进行了比较分析.最终建立了适合松毛虫的AFLP反应体系.用优化的AFLP反应体系,以油松毛虫、赤松毛虫和马尾松毛虫为材料筛选引物,从81对引物组合中筛选出10对多态性高的引物组合.图9参17

Establishment of AFLP Fingerprinting in Genetic Diversity Analysis of Pine Caterpillars

In order to establish AFLP technique system by using the genomic DNA of Dendrolimus tabulaeformis Tsai et Liu as test material, different dosages of T4 DNA ligase, concentration of Mg~(2+) dNTP and primers were optimized in preamplification reaction system, and the dilute multiple of preamplification production and the concentration of Mg~(2+), dNTP, primers and different dosages of Taq DNA polymerase were also optimized in selective amplification reaction systerm. Very steady results were achieved. In the linking reaction, T4 DNA ligase 2 U was added to the ligation system. The preamplification system included Mg~(2+) 1.5 mmol/L, dNTP 0.2 mmol/L, PstI primer and Taql primer 5.0×10~7μmol/L, and 40 dilute multiple of preamplification production. The selective amplification systerm included Mg~(2+) 1.5 mmol/L, dNTP 0.2 mmol/L, PstI primer and Taql primer 5.0×10~7μmol/L, Taq DNA polymerase 1 U. By using D. Tabulaeformis Tsai et Liu, D. Spectabilis Butler and D. Punctatus (Walker) as test materials, ten pairs of informative primers were identified from eighty-one pairs of primers by optimizing the AFLP reaction system. The result established a strong foundation for the AFLP analysis of pine caterpillar. Fig 9, Ref 17