基于SAMPL的一种微卫星筛选方法在中国明对虾中的尝试

Preliminary isolation of microsatellite based on SAMPL technique in shrimp Fenneropenaeus chinensis

微卫星的发展主要受限于微卫星及其旁侧特异引物区的分离,在中国明对虾(Fenner-openaeus chinensis)中建立了选择性扩增多态微卫星位点(SAMPL,selective amplified mic-rosatellite polymorphic loci)技术体系,并初步尝试了基于SAMPL的一种微卫星筛选方法。通过5′端锚定引物引入微卫星序列,对传统SAMPL技术加以改进并以期富集简单重复序列。从测序胶上随机选择5条SAMPL条带(分别命名为S11,S13,S14,S22,S24)克隆测序,大小分别为161,157,147,152,298。其中S11,S13,S14,S22四个片段两端引物分别为AFLP选择性引物和SAMPL引物,S13和S22含微卫星序列重复,S24两端引物均为传统的AFLP引物,在此片断中有一(AG)39重复。

The main difficulty in developing microsatellite is to isolate the short tandem repeat -flanking sequences. A method to screen microsatellite based on SAMPL（selective amplified microsatellite polymorphie loci） was tested in shrimp Fenneropenaeus chinensis . 5＇ anchored primer was introduced into traditional SAMPL analysis, and short tandem repeat sequences were expected to be enriched. Five randomly selective bands （nominated as Sll ,S13,S14,S22,S24 respectively）were cut from the silver staining polyacrylamide gel, cloned and sequenced, and 161, 157, 147, 152, 298 bp sequences were obtained respectively. Microsatellites were found in S13,S22. A single sequence repeat of （GA）39 was also found in S24, which comprises AFLP primers only and is different from the other ones that comprise both primers. No mierosatellite was found immediately adjoining to the 5＇ anchored primer, so it seems that 5＇ anchored primer, in this study, did not work as expected. More work is needed for further microsatellite isolation.