利用AFLP技术筛选锯缘青蟹性别差异DNA片段

采用高盐和酚氯仿异戊醇 (PCI)结合法提取DNA ,利用AFLP技术 ,应用 5 2个引物组合 ,检测了锯缘青蟹 (Scyllaser rata)雌雄基因组DNA的多态性 ,筛选与锯缘青蟹性别相关的分子标记。实验中共扩增出 4 312条带 ,筛选出候选差异DNA片段 74 8条。这些差异DNA片段的获得 ,为研究锯缘青蟹性别的分子标记奠定了基础

Screen on sex-differential DNA fragments in mud crab Scylla serrata using AFLP

Genomic DNA was extracted from muscle of female and male in mud crab Scylla serrata using high concentrated salty-PCI(Phenol, Chloroform, Isoamylalcohol). Equal genomic DNA from 14 female or male individuals was mixed as genomic DNA pool of female and male. Using amplified fragment length polymorphism (AFLP) technique, which involves three steps : restriction of the DNA and ligation of oligonucleotide adapters, pre-amplification and selective amplification of sets of restricted fragments, and gel analysis of the amplified fragments, genomic DNA polymorphisms of female and male in Scylla serrata were detected with 52 primer combinations. The molecular markers related to sex locus in Scylla serrata were screened. 4 312 fragments were amplified and 748 candidate differential DNA fragments were obtained. These differential segments from different primer pair were re-amplified by PCR. Further work is that the differential fragments showld be confirmed by DNA chip and Southern blot. These markers DNA are important to the development of the sex specific PCR primers or probes that could be used in the sex identification of Scylla serrata.