磁珠富集法筛选虾夷扇贝微卫星序列

Microsatellite enrichment by magnetic beads in japanese scallop,Patinopecten yessoensis

采用生物素—磁珠吸附微卫星富集法,筛选虾夷扇贝(Patinopecten yessoensis)微卫星分子标记,并用同位素法进行二次筛选。结果在筛选的192个菌落中获得136个阳性克隆,经测序分析,获得微卫星序列179个,其中完美型占50.8%,非完美型43.0%,混合型占6.1%。除探针中使用的CA重复外,还得到TC、AG、ACA、CTAT的重复序列。用引物设计软件Primer Premier5.0设计引物85对。

Japanese scallop（Patinopecten yessoensis） is one of the most important cultured seashells in the world. It is distributed widely over the cold seas along the coastline of the northern islands of Japan, the northern part of the Korean Peninsula, Sakhalin, and the Kuril Islands, and was introduced to China from Japan more than 20 years ago. Along with nearly 20 years＇ artificial culture, available DNA markers are needed for its genetic research. Microsatellites recently have become an extremely popular marker in a wide variety of genetic investigations. In this research, genomic DNA was extracted from muscle of Japanese scallop by normal methods and digested with restriction enzyme Sau 3AI. Fragments of 400-900 bp were isolated and then ligated short linkers（20 bp）. They were to create a ＂whole genome PCR library＂. Microsatellite isolated from genomic DNA was hybridized with a biotin-labeled SRS（simple repeat sequence） probe （CA） 15, and the hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SRS fragments, the eluted single-stranded DNA contains the selected microsatellite DNA. The selected DNA are then amplified using primers designed complementary to the linkers, cloned into the pGEM-T vector and transform into competent E. coli DHSa. Then the genomic library was secondscreened for microsatellite DNA using（CA）is probes labeled with 732p-ATP at 5＇ end the probes hybridized with bacterial colonies. As a result, 136 positive clones were identified from 192 clones, and 179 microsatellites were found. Among the 179 microsatellites, 50.8 % were perfect, 43.0 % were imperfect and the rest were compound type （ 6.1% ）. Except the biotin-labeled SRS probes CA, there were TC, AG, ACA and CATA in the microsatellites. Finally, 85 microsatellite primers were designed by software Premier Primer 5.0,and 40 pairs were screened, 34 pairs were amplified. They made positive contribution to exploring genomes, and developing genetic linkage maps, as well as made possible the systematic search for quantitative trait loci （QTL） of the Japanese scallop.