用微卫星DNA技术对中国对虾人工选育群体遗传多样性的研究

Genetic diversity of cultured populations of Fenneropenaeus chinensis shrimp using microsatellites

利用微卫星技术对中国对虾人工选育群体第1代和第6代群体的遗传多样性进行了分析。对10个微卫星位点进行了扩增,共产生74个等位基因,每个位点产生的等位基因数从3到13不等。在两个群体中,所观察到的等位基因数都比有效等位基因数多。多态信息含量PIC值0.5567～0.8877,说明这10个微卫星位点在中国对虾中具有较高的信息含量。两个群体的平均杂合度分别为0.6400(CP1)、0.6300(CP6),并通过计算基因型的P值,确定了对Hardy Weinberg平衡的偏离情况。对Fis值的计算表明两个群体内共有5个微卫星位点存在杂合度观察值过剩的现象。两个群体的Shannon多样性指数分别为1.6830、1.7382,整个选育群体(两个群体作为一个群体)的遗传多样性指数为1.7742。从遗传多样性所占的比例来看,96.415%的遗传变异是来自群体内,只有3.585%的遗传变异是来自群体间。两个群体间的相似性系数高达0.9187,彼此间的遗传距离仅为0.0848,体现出人工选育群体的遗传分化程度较低。结果均说明第6代群体还有较大的选育潜力,可以继续保持遗传效应,最终保证选种育种工作的成功。

Selective breeding on growth character and disease-resistant trait of shrimp Fenneropenaeus chinensis has been conducted since 1997, and six successively bred stocks were produced between 1997 and 2002 at breeding base of the Yellow Sea Fisheries Research Institute, Shandong Province, China. Challenge Test has shown their better growth rate and disease-resistant performance than controlled shrimp. Samples of CP1 and CP6 were randomly collected. Tail muscle tissue from shrimp of mostly adults and sub-adults was collected. Total DNA was extracted from tail muscle. DNA quality controls were performed using agarose gel electrophoresis and only samples with good DNA quality were used for microsatellite analysis.Microsatellite technique was used to investigate the genetic variation between the cultured populations (CP1 and CP6) of F. chinensis. Ten microsatellite primers were designed according to small-size fractionated genomic libraries of F. chinensis. Optimal results were obtained by optimizing quantity of reagents and reaction conditions. Under the optimal conditions, PCR was performed for analyzing the genetic diversity of the two selected populations. PCR products were electrophoresed through 8% polyacrylamide gels. Samples were heated to 95℃ for 5 min before being loaded onto the polyacrylamide gels. The gels then were silver-stained. All samples were run next to a known sequence in order to determine size. To avoid inaccuracy in scoring due to differences in gels, a control DNA sample was included in each set of samples for each gel.To analyze the variation in microsatellite loci in the two populations, number of alleles per locus (na), Polymorphism Information Content (PIC), number of genotypes per locus (G) effective number of alleles (αe), observed heterozygosity (Ho) and expected heterozygosity (He) were estimated for each population at each locus. Allele frequencies for each population at each locus were calculated based on the number of alleles provided by the computer software TFPGA. A total of 74 different alleles were found over all ten loci. The total number of alleles per locus ranged from 3 to 13. In the two populations, the effective number of alleles was lower than the observed number. PIC per locus ranged from 0.5567 to 0.8877. The average heterozygosity of the first and sixth selected generation was 0.6400 and 0.6300, respectively. To access departures from HWE, P-values of genotypic frequencies were calculated. Furthermore, five cases of observed heterozygosity excess ( F_(is)<0 ) were observed in the two populations. Shannon genetic diversity index revealed that the average variation level of the two populations was 1.7742, and the variation level of them was 1.6830 and 1.7382, respectively. Partitioning of the genetic variation revealed that 96.415% is distributed within populations and 3.585% among populations. Nei's genetic identity and genetic distance between the two populations were 0.9187 and 0.0848, respectively. In summary, data showed low genetic diversity of F. chinensis and difference over the levels of genetic variation between the two populations. There was great potential of selective breeding for the sixth generation.