利用AFLP方法筛选中华绒螯蟹性别相关标记

利用AFLP(amplified fragment length polymorphism)技术筛选中华绒螯蟹性别相关标记,共使用192对引物组合检测中华绒螯蟹雌、雄和雌雄混合3个基因组池的多态性,共扩增出5 376条多态性条带,平均每对引物组合扩增出28条多态性条带,获得88条雌雄差异条带。利用雌雄各10个个体再次进行AFLP验证,共62条差异条带具有性别差异,包括两种情况:(1)有49条条带在雌性的4～6个个体中存在,在雄性全部个体中缺失;(2)有13条条带在雄性4～6个个体中存在,在所有的雌性个体中缺失;其余的26条没有性别差异性。差异条带经回收、克隆、测序,结果发现有些条带包含多个序列,共得到77条DNA序列,其中31条序列与鸟类、两栖类和哺乳类等脊椎动物性染色体DNA部分片段具有同源性,相似区间在22～212 bp不等,其中6条DNA序列与命中序列之间具有生物学相关性。设计特异性的引物在基因组中检测,没有获得性别特异SCAR标记。

Isolation of the sex associated fragments in the Chinese mitten crab (Eriocheir sinensis) using AFLP technique

In the present study,amplified fragment length polymorphism(AFLP) was used for the isolation of sex-associated markers in the genome of Chinese mitten crab,Eriocheir sinensis.By screening the male,female and male/female mix bulks with 192 selective AFLP primer combinations,we obtained 5 376 polymorphic fragments in total,each combination generated 28 fragments on average.A total of 88 sex difference AFLP candidates were retrieved.Subsequently,the candidates were further verified in 10 female and 10 male individuals by AFLP again.Sixty-two sex difference fragments were obtained finally,and they could be classified into two groups:(1) Thirteen bands were present in four to six male crabs and absent in all female crabs;(2) Forty-nine bands were present in four to six female crabs,but absent in all male crabs.No sex difference was found in the other 26 bands.The sex difference fragments were recovered,cloned and sequenced.The results showed that multiple sequences presented in some bands.Seventy-seven DNA sequences were obtained,thirty-one of which shared partial homology with sex chromosomal DNA sequence of birds,amphibians and mammals.The length of identical sequences ranged from 22 to 212 bp.Among them,six sequences had biological relevance with hit sequences.The SCAR(sequence characterized amplified region) primers were designed to identify sex-specific SCAR marker.However,none of the SCAR derived from the presumed sex-associated fragment showed sex specificity.