大黄鱼微卫星标记的富集与筛选

Enrichment of large yellow croaker genome microsatellite markers using magnet beads

根据生物素与链霉亲和素的亲和原理,采用生物素-磁珠富集微卫星,与传统放射性同位素杂交法相结合,构建筛选大黄鱼的微卫星文库。用生物素-微卫星捕捉单链限制性酶切片段(含有接头和大黄鱼微卫星序列),经PCR扩增单链目的片段形成双链,然后连接至T载体上,转化感受态细胞。将移至硝酸纤维素膜的重组菌用32P标记的放射性同位素探针5′-[γ-32PATP(CA)15筛选出阳性克隆菌。测序结果发现,阳性克隆率为71.9%,105个微卫星位点。其中选取设计合成30对并筛选出22对可用引物。说明所建大黄鱼微卫星文库是一个高质量的文库,可为大黄鱼基因组结构分析、大黄鱼精密微卫星连锁图谱构建、分子进化和系统发育研究、分子标记辅助育种以及经济性状的QTL定位提供大量的微卫星标记。

Microsatellite, as a kind of molecular markers, have many advantages in construction of genetic linkage map, QTLs analysis, kinship identification and so on. This study reports the isolation and characterization of 22 microsatellite markers from large yellow croaker. Construction the first microsatellite-enriched library of large yellow croaker according to the strong affinity between biotin and streptavidin was carried out. A biotin-labelled（CA）lsoligonuclcotide probe was used to enrich for homologous sequences. The library was screened for microsatellite repeats with 5＇-[γ-^32P]ATP（CA）15 using standard hybridization and wash conditions. Of 146 positive clones identified, 105 were sequenced. Among 105 clones containing microsatellite arrays, 30 unique clones with sequences of suitable length of flanking regions were selected for primer design. Among 30 primer pairs designed, 8 failed to amplify and 22 yielded scorable amplification products. The results showed that this method is very efficient to isolate microsatellite markers.