青鱼微卫星标记的开发与特性分析

青鱼(Mylopharyngodon piceus)是中国最为重要的淡水养殖鱼类。开发青鱼的微卫星标记能为青鱼的遗传多样性分析提供更多工具。本研究使用磁珠富集法,利用生物素探针(CA)10和(GACA)6,富集得到青鱼基因组微卫星片段,进一步通过设计微卫星引物检验其在青鱼原种群体中的有效性和多态性水平。结果显示,所构建文库中849个克隆含有微卫星序列,通过利用PCR技术在吴江原种青鱼36个个体中进行多态性筛选,获得了25个多态性微卫星位点。其平均等位基因数(Na)和有效等位基因数(Ne)分别为7.08和3.526,平均观测杂合度(Ho)和期望杂合度(He)分别为0.602和0.619,平均多态信息含量(PIC)为0.568。其中,Mp23、Mp27和Mp35这3个位点极显著偏离哈迪-温伯格平衡(P 0.01)。本研究开发的微卫星标记能为青鱼种质资源的评价和保护等研究提供工具。

Development and Characterization of Microsatellite Markers in Black Carp (Mylopharyngodon piceus)

Black Carp (Mylopharyngodon piceus) is an important aquaculture species in China. Developing microsatellite DNA markers from genome of M. piceus will provide a tool for its genetic diversity analysis. In this study, two biotin probes (CA)10 and (GACA)6 were used to construct two microsatellite enrichment libraries of M. piceus by magnetic beads enrichment. A series of microsatellite primers was designed and their polymorphisms were tested in a population of M. piceus. Softwares GeneMapperv4.0, Cervus3.0 and PopGen32 were used to analyze their genetic diversity information. Microsatellite markers with high polymorphism and rich genetic diversity were screene. Through analysis, 849 sequences containing microsatellite loci were obtained from 993 positive clones. The repeat number of the repeating unit was then counted (Fig. 1). The statistics showed that most numbers of the repeats were between 5 and 21. The repeating units were classified according to the number of nucleotides in each repeating unit. The results showed that the number of dinucleotide microsatellite loci was the most, as high as 1 076. It accounted for 82.45% of the total. There were 217 trinucleotide type microsatellite loci, accounting for 16.63% of the total. In trinucleotide type, only 0.77% of all 39 microsatellite markers were designed and assessed in 36 individuals of black carp from Wujiang population. Results showed that 25 microsatellite loci showed polymorphism (Table 1). The mean number of alleles (Na) and effective number of alleles (Ne) were 7.08 and 3.526, respectively. The mean value of observed heterozygosity (Ho) and expected heterozygosity (He) were 0.602 and 0.619, respectively. The mean value of polymorphic information content was 0.568. Mp23, Mp27 and Mp35 were extremely significant deviating from Hardy-Weinberg equilibrium (P < 0.01) (Table 2). The microsatellite primers developed in this study will provide a tool and theoretical basis for evaluation and protection of germplasm resources, and genetic diversity analysis in M piceus.