Isolation of the sex-associated fragments in the Chinese mitten crab(Eriocheir sinensis) using AFLP technique
Isolation of the sex-associated fragments in the Chinese mitten crab(Eriocheir sinensis) using AFLP technique
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发表时间：
2012
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通讯作者：
Qiu Gao-feng
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作者：
Qiu Gao-feng
本研究采用扩增片段长度多态性（AFLP）技术分离中华绒螯蟹（Eriocheir sinensis）基因组中的性别相关标记。利用192个选择性AFLP引物组合对雄、雌、雌雄混交群体进行筛选，共获得5 376个多态性片段，每个组合平均产生28个多态性片段。共检索到88个性别差异AFLP候选者。随后，再次用AFLP对10名女性和10名男性个体进行进一步验证。最终获得62个性别差异片段，可分为两类：(1)4 ~ 6只雄蟹中存在13个条带，所有雌蟹均不存在；(2) 4 ~ 6只雌蟹有49个条带，雄蟹无条带。在其他26个波段中没有发现性别差异。对性别差异片段进行了恢复、克隆和测序。结果表明，部分条带存在多个序列。共获得77条DNA序列，其中31条与鸟类、两栖动物和哺乳动物的性染色体序列具有部分同源性。相同序列长度在22 ~ 212 bp之间。其中6个序列与命中序列具有生物学相关性。SCAR（序列特征扩增区）引物设计用于鉴定性别特异性SCAR标记。然而，来自假定的性别相关片段的SCAR均未显示出性别特异性。
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.