曼氏无针乌贼微卫星DNA富集文库构建与鉴定

Construction and Identification of DNA Libraries Enriched for microsatellite Repeat Sequences of microsatellite Repeat Sequences of Sepiella maindroni Sepiella maindroni

曼氏无针乌贼是我国四大海产之一,开发其微卫星标记具有重要的科研与应用价值。本研究采用FIASCO法（Fast Isolation by AFLP Sequences Containing repeats）构建了曼氏无针乌贼的（CA）n微卫星富集文库,通过PCR检测出640个阳性克隆,占所有克隆的87%,从阳性克隆中随机选取118个进行测序,序列分析发现,103个克隆含有7次以上的重复序列,其中完全的为67（65%）个,不完全的23个（22.3%）,复合的为13（12.7%）个,重复次数范围为7~59次,平均为38次。在103个序列中共42条可以设计引物,所筛选出的引物可以用于评估曼氏无针乌贼种质资源遗传多样性、遗传结构、亲子鉴定以及放流效果评估等。

Sepiella maindrorti is one of four famouse marine product in China, it is very significant to develop the microsallite markers for research and utilization value. In this study, the microsatellite enriched-libraries of the S. maindroni was constructed according to the FIASCO （Fast Isolation by AFLP Sequences Containing repeats） protocol. Finally, this experiment obtained 640 clones containing microsatellite sequence, of 640 （87%） positive clones, 103 sequences contained repetition that repeated no less than 7 times, according to Weber＇ s classification rules, these sequences were divided into three categories： perfect sequences （65% of total）, imperfect repeat sequences （22.3%）, and compound repeat sequences （12.7%）, the repeat time ranged from 7 to 59 with anaverageof 38. Of the 103 sequence, there are 42 sequences can be designed the primers and will be used to design primer and test polymorphism in further research. This study provides a base for molecular breeding and assessment of germplasm resources, genetic constructure, paternity test and evaluate the effect of enhancement and releasing of the S. maindroni.