半滑舌鳎微卫星标记的开发及其在F_1家系中分离方式分析

Isolation and characterization of microsatellite markers for half-smooth tongue sole(Cynoglossus semilaevis) and an analysis of seg-regation in the F_1

利用富集文库-菌落原位杂交的方法筛选半滑舌鳎（Cynoglossus semilaevis）微卫星标记, 构建富含（AC）和（AG）序列重复的半滑舌鳎微卫星富集文库, 随机从文库中挑选1060个克隆, 筛选得到883个（83.3%）阳性信号, 菌落PCR检测742个阳性克隆片段的长度在500-1 200 bp范围内, 随机挑选其中50个克隆进行测序, 共设计引物33对, 进行退火温度优化、多态性检测后, 共得到20个多态的半滑舌鳎微卫星标记。对其进行分离方式分析, 其中有17个位点符合孟德尔分离定律, 共表现5种分离方式, 可用于半滑舌鳎遗传连锁图谱的构建; 另外有3个位点偏离孟德尔分离定律（P〈0.05）。结果同时证明富集文库-菌落原位杂交是半滑舌鳎微卫星标记大量筛选和高密度遗传连锁图谱构建的一种行之有效的方法。

Microsatellite （or simple sequence repeat, SSR） marker is an important tool for molecular research. We isolated and characterized microsatellite markers for half-smooth tongue sole （Cynoglossus semilaevis） by constructing an enriched genomic library and conducting colony in situ hybridization. The genomic library was enriched for （AC） and （AG） of half-smooth tongue sole. We detected 883 positive signal from 1 060 random clones by colony in situ hybridization （positive ratio was 83.3%）. The majority of positive clones were between 500–1 200 bp in length. We randomly selected 50 clones for sequencing using 33 pairs of primers that were designed with Primer Premier 5.0. Twenty SSRs were polymorphic. A polymorphism analysis of 48 F1 individuals revealed that 17 SSRs were consistent with Mendelian segregation and could be used to construct the linkage map. Conversely, 3 SSRs deviated from Mendelian segregation （P〈0.05）. Our results suggest that our approach was useful for isolating large numbers of microsatellite markers and construction of a high density genetic linkage map.