摘要
采用SSR结合BSA技术对1个家系96个乌鳢个体(雌雄各48个)进行性别差异标记的筛选。首先构建了雌雄基因池(各24个个体),然后用140对微卫星引物对其进行扫描,发现3对引物(HLJWL17,HLJWL59,HLJWL70)在雌雄基因池间扩增出差异条带,且都出现在雌性基因池中。用构建基因池的48个个体对3对引物进行第一轮单个体验证,HLJWL17和HLJWL70在基因池中扩增出的差异条带仅在极个别个体中出现,而HLJWL59的差异条带在绝大多数的雌性个体中被成功扩增出来,雄性个体皆无。用其余的48个个体对HLJWL59进行第二轮单个体验证,得到同样结果。对HLJWL59在8个雌性个体中扩增出来的差异基因片段进行克隆并测序。将8个个体的测序结果用Vector 8.0进行多序列比对,证实各个个体得到的条带是同一序列,该序列长度为243 bp,以TGC为重复单位,GC含量为51%。通过BLASTn比对,发现在GenBank数据库中无同源性序列存在。经统计,此差异标记在雌性乌鳢中出现的概率为62.5%,即该标记对该家系乌鳢性别的正确判别率为81.25%。
Microsatellites combined with bulked segregation analysis（BSA）were used to screen the gender differences of 96 individuals（48 for male and female each）from a family of Channa argus.Two gene pools were constructed using 24 individuals for male and female each separately,gene pools were used to scan using totally 140 pairs of microsatellite primers and specific DNA fragments of female gene pool were amplified by 3 pairs of primers（HLJWL17,HLJWL59,HLJWL70）.Then,the specific DNA fragments amplified by the three pairs of primers were verified for the first round in the 48 individuals constructing the gene pools,showing that the specific bands of HLJWL17 and HLJWL70 in gene pools only appeared in few individuals,while HLJWL59 was successfully amplified in females.And in the second round verification,the same results were obtained in the remaining 48 individuals with the HLJWL59.The amplified fragments by primer HLJWL59 of eight different alleles in females were cloned and sequenced.Sequence alignment of the eight individuals using Vector 8.0 multiple confirmed that the bands of each individual are the same sequence,with the sequence length of 243 bp.The repeat units were TGC,and 51% of GC.Compared by the BLASTn,homologous sequences were not found in the GenBank database.According to the statistics,the probability of different bands that appeared in the female C.argus was 62.5%,that is,the correct classification rate of the marker for female C.argus was 81.25%.We believe that this microsatellite loci may be associated with C.argus female gender under certain conditions.This study may serve as a basis for culturing pure male hybrid using female-specific DNA fragments in C.argus.
出处
《水产学报》
CAS
CSCD
北大核心
2011年第2期170-175,共6页
Journal of Fisheries of China
基金
国家公益性行业(农业)科研专项(200903045)
关键词
乌鳢
微卫星
性别差异
混合分离群体法
Channa argus
simple sequence repeats
gender-differences
bulked segregation analysis
