安徽某淡水龙虾养殖群体分类的分子标记筛选

Screening of Molecular Markers for Classification in Cultured Population of Freshwater Crayfish in Anhui, China

为筛选可用于淡水龙虾养殖群体物种分类的分子标记,对安徽省某水产养殖有限公司采集的淡水龙虾样本进行了线粒体COⅠ、16S rDNA、D-loop、Cyt b及12S rDNA基因的测序分析,并与GenBank中30个滑螯虾属(Cherax)物种的相应基因序列比对,同时,进行上述5种分子标记在种内个体间、属内种间、科内属间和目内科间的同源性变异范围比对分析。结果显示,本研究所采集淡水龙虾样本与四脊滑螯虾(C. quadricarinatus)的相似性最高,其中,1 113 bp的Cyt b相似性100%,COⅠ、16S r DNA及12S rDNA相似性都在99%以上;在以克氏原螯虾(Procambarus clarkii)为外类群构建的邻接系统发育树中,本研究样本与四脊滑螯虾聚为一支,然后与其他物种聚合构成系统树,确定本研究采集的淡水龙虾样本为四脊滑螯虾;通过同源性变异范围比对分析,在淡水龙虾养殖群体的分子分类工作中,16S rDNA、Cyt b及12S rDNA比COⅠ和D-loop这2种分子标记具有较强优势。本研究结果可为今后淡水龙虾养殖场中物种的分类鉴定工作提供参考。

An experiment was conducted to screen molecular markers that can be used for classification of populations of cultured freshwater crayfish samples collected in Anhui, China. After genome extracting, PCR amplifying and sequencing of COⅠ, 16 S rDNA, D-loop, Cyt b and 12 S rDNA of the samples were performed, and the neighbor-joining phylogenetic tree and genetic distance in MEGA6.06 were used to compare the samples with 30 species recorded in GenBank. The reliability of neighbor-joining phylogenetic tree was tested by 1 000 times bootstrap and the genetic distance was calculated based on kimura-2-parameter nucleotide substitution model. In the neighbor-joining phylogenetic tree(Fig. 1) constructed with Procambarus clarkii as an outgroup, the samples first clustered together with red claw crayfish(Cherax quadricarinatus) and then constituted a relationship to other species, strongly showing that the samples should be classified as C. quadricarinatus. The genetic distance range(Table 3) showed that the average genetic distances of 16 S rDNA, Cyt b and 12 S rDNA were increasing from intraspecific individual to intra order interfamily, and were not at the same level, so that they can probably be better used in cultured population. The results from this paper will benefit the people working in the crayfish farm, and provide reference for classification of freshwater crayfish in the future.