长江水系草鱼遗传多样性的微卫星DNA分析
MICROSATELLITE DNA ANALYSIS OF GENETIC DIVERSITY OF GRASS CARP IN YANGTZE RIVER SYSTEM
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摘要: 利用已发表的鲤微卫星引物在草鱼中进行PCR扩增，结果有5对引物(6个座位)能成功扩增并且有较高多态性，等位基因数在3-7个之间。这些异种扩增的草鱼微卫星符合孟德尔遗传规律。测序证明草鱼中的微卫星核心重复序列部分与鲤中的原始核心序列相似，也有一些变化。随后用这6个多态微卫星座位研究了来自长江水系的四个草鱼群体的遗传结构，结果显示每个群体的平均等位基因数在38与48之间，平均观测杂合度(Ho)在04000与05741之间，平均期望杂合度(HE)在04773与06489之间，有多个座位在不同的群体中偏离哈代-温伯格平衡。遗传距离分析表明四川群体与洞庭湖群体遗传距离最远，而嘉鱼群体与鄱阳湖群体遗传距离较近。分子变异分析(AMOVA)表明，群体内遗传变异与群体间遗传变异分别占总遗传变异的9560%与440%，固定系数(FST)为0044，这表明长江水系草鱼目前的群体分化很微弱。Abstract: Grass carp (Ctenopharyngodon idellus) has been one of the most important traditional domestic freshwater aquaculture fish in China. Its habitat includes major rivers and lakes in China, but it is most abundant in Yangtze River and its adjacent lakes. In the present study, the genetic diversity and population structure of grass carp from Yangtze River system were investigated using microsatellite DNA markers. In total 109 individuals were collected from four sites of the Yangtze River System including Yibin, Sichuan; Jiayu, Hubei; Dongting Lake, Hunan and Poyang Lake, Jiangxi. A full-sib family was prepared using artifical fertilization. Genomic DNA was extracted from ethanol-preserved fin tissues by using phenol-chloroform method. Using a set of microsatellite primers originally reported in common carp (Cyprinus carpio), we carried out many cross-species PCR amplifications. PCR products were separated on 7.5% nondenaturing polyacrylamide gels, stained with ethidium bromide in water and visualized with ultraviolet. A successfully amplified locus with clear banding pattern (i.e. one or two bands) was subject to test for genetic segregation in a full-sib grass carp family before it was considered to be used in population analysis. Purified PCR products were cloned into E. coli DH5α strain using pMD-18T vector, and positive clones were selected and sequenced by Shanghai BioAsia Biotech Company for confirmation of repeat motifs. The size of alleles was scored by comparison with pBR322 DNA/Msp I markers. Five microsatellite primers successfully amplified six loci with reasonable polymorphism in grass carp. The number of alleles in these loci ranged from 3 to 7. These six microsatellite loci were inherited with Mendelian mode in a test full-sib grass carp family. Sequencing analysis for the PCR products in grass carp confirmed that all these six loci contained core repeat motifs, and some di-nucleotide motifs were similar to those in common carp (e.g. CA repeat) while others were new in grass carp, especially those tri-nucleotide repeats (i.e. TTA and GGA). Six microsatellite loci in grass carp, i.e. MFW1-1, MFW1-2, MFW5, MFW15, Koi3 and Koi20, were applied to subsequent studies on genetic diversity and possible population structure of grass carp samples from the Yangtze River System including river and its major adjacent lakes. The results showed that the number of alleles of a population ranged from 3.8 to 4.8 on average, and mean observed heterozygosity (Ho) from 0.40 to 0.57, while mean expected heterozygosity (H E) from 0.48 to 0.65. After applying Hardy-Weinberg Equilibrium (HWE) test, several loci were found to be significantly deviated from HWE in some populations in which deficiency of heterozygotes was apparent. Genetic distance analysis showed that the largest distance was between Sichuan and Dongting Lake population and the smallest distance was between Jiayu and Poyang Lake population. However, the analysis of molecular variance (AMOVA) indicated that almost majority of the variance in the grass carp was within populations (95.60%), and only small proportion was among populations (4.40%). The lower level of overall genetic diversity and particularly lower Fixation Index (Fst=0.044) indicated that the grass carp in Yangtze River system had a relatively lower genetic variation and its genetic structure between populations was weak.
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