海湾扇贝微卫星标记开发及其分离方式分析

利用尼龙膜吸附杂交法构建了海湾扇贝的(AC)_(15)、(AG)_(15)微卫星富集文库,随机挑取3 000个克隆,经菌落原位杂交二次筛选共获得阳性克隆1 268个(42.3%).经序列测定和生物信息学分析后得到521个独立的阳性克隆,其中微卫星506个,小卫星15个.微卫星中,完美型248个,占总数的49.0%;非完美型216个,占42.7%;复合型42个,占8.3%;其中AG/TC重复占70.4%(356个),AC/TG重复29.6%(150个).选择其中的55条序列设计引物,筛选出其中的20对引物检测它们在海湾扇贝CC10家系的双亲和94个子代个体中的分离情况,结果表明:(1) 其中6个位点在母本和子代中发生分离,10个位点在父本和子代中发生分离,4个为双亲共享位点;(2) 2个位点存在无效等位基因;(3) 16个位点符合孟德尔遗传定律,4个位点的子代个体基因型频率偏离孟德尔遗传定律.上述结果表明,这些微卫星引物可以用于构建海湾扇贝的遗传连锁图谱,并且所构建的富集文库适用于微卫星标记的大规模开发.

Development of microsatellite markers in bay scallop and their inheritance patterns in an F1 hybrid family

Three genomic libraries enriched for (AC)_(15) and (AG)_(15) were constructed using the method of nylon membrane hybridization in bay scallop (Argopecten irradians). A total of 3 000 clones derived from the enrichment libraries were screened by (AC)_(15) and (AG)_(15) probes, and 1 268 (42.3%) were proved to contain microsatellite repeat sequences. One thousand positive clones were sequenced, 506 independent microsatellites, and 15 minisatellite sequences were obtained. Among the 506 microsatellite repeat sequences, 248 (49.0%) were identified to be perfect microsatellites, 216 imperfect(42.7%) and 42 compound (8.3%). Fifty-five primer pairs were designed and amplified in one bay scallop family to test their polymorphism in this family. Twenty polymorphic microsatellite markers were selected to analyze their segregation and inheritance in parents and their 94 progenies of this bay scallop family, among which 4 were polymorphic in both parents, 6 in female and 10 in male. Two markers were observed to produce null alleles; and when null alleles were considered, there were 4 loci showing segregation distortion at 5% significance level. These results indicated that the microsatellite-enriched libraries constructed in the present study are efficient and suitable to isolate a large amount of microsatellite markers in bay scallop.