Isolation Of Microsatellite Markers Of Channa Argus And Channa Maculata And Their Application Of Identification Of F₁ Generation And Analysis Of Population Genetic Diversity

Channa argus and Channa maculata, which belonged to Perciformes, Channoidei, Channidae, Channa, were important freshwater commercial fish. Recently, some problems presented in aquaculture of Channa argus and Channa maculata, such as over fishing, lack of effective management, and the smaller individuals related to the reduction of genetic variation in these cultured populations. Information on genetic diversity and population structure is important in assessing, utilizing and conserving the genetic resources of these species. In this study, microsatellite DNA was isolated and enriched from genome of Channa argus and Channa maculata by magnetic beads. And using these markers, we studied primary identification of Channa argus, Channa maculata and their reciprocal hybrids analyzed the genetic diversity and population genetics. And our aims were to provide important theoretical basis for further exploitation, utilization and protection of the species. Our studies are as follows:1. Development of Microsatellites MarkersThe microsatellite-enriched library was constructed using magnetic bead hybridization selection method and PCR selection method, and the microsatellite DNA sequences were analyzed in Channa argus and Channa maculata. Genomic DNA was extracted from mantle of Bullacta exarata and digested with a restrication enzyme MseI. Fragments between 200-500bp were enriched by hybridization with (CA)15 probes. The DNA product was ligated into pGEMT vector, transformed E.coli DH5αcompetent cells.A total of 274 white clones were screened using a PCR-based technique. According to Weber (1990), the microsatellite sequences in Channa argus obtained could be categorized structurally into perfect repeats (76 %), imperfect repeats (34%), and compound repeats (4%). The microsatellite sequences in Channa maculata obtained could be categorized structurally into perfect repeats (65 %), imperfect repeats (21%), and compound repeats (14%). Besides the motif of CA contained in the oligoprobe, other 7 types of microsatellite repeats were also found including TG、AG、TAG、GTT、TCTG、AAAG and CTGG. Among the microsatellite repeats, the arrays more than 10 repeats were accounted for 87.3 %. The largest length of microsatellites was 59 repeats. Finally, the microsatellite primers were designed by software Primer 3.0 or Primer 5.0.2. Primary Identification of Channa argus, Channa maculata and their Reciprocal HybridsSixty-two pairs of microsatellite primers of C. argus and C. maculata were designed based on the flanking sequence of the microsatellite loci with software Primer 3.0 or Primer 5.0. Ten primer pairs failed to amplify. Among the 52 primer pairs which could effectively amplify products, 49 universal primer pairs could effectively amplify in F1 generation. Loci CHA16 showed monomorphic in C. maculata and polymorphic in C. argus and loci CHM23 was opposite. In addition, CHM24 showed monomorphic in C. argus and failed to show in C. maculae. Moreover, 22 specific loci between C. argus and C. maculata were screened out, and 18 specific loci could identify C. argus, C. maculata and the reciprocal hybrids alone effectively. Combining part of the other 4 specific loci can also effectively identify C. argus, C. maculata and the reciprocal hybrids. But he reciprocal hybrids [C.argus(♀)×C. maculata(♂), C. maculata(♀)×C. argus(♂)]could not be identified by this research.3. The Microsatellite Analyse of Channa argus, Channa maculata and their Hybrids[C. maculata(♀)×C. argus(♂)]10 microsatellite primers could be used to analyse the population of Channa argus, Channa maculata and their Hybrids[C. maculata(♀)×C. argus(♂)]. The higest mean of number of alleles, expected heterozygosity, observed heterozigosity and polymorphism information content is Channa argus, and their hybrids is next, and Channa maculata is lowest. The analysis of Hardy-Weinberg equilibrium showed that 78% alleles departed from or extremely departed from Hard-Weinberg equilibrium significantly. According to the results of genetic distance, we construct UPGMA dendrogram. The results showed that the hereditary character of their Hybrids[C. maculata(♀)×C. argus(♂)]were more partial to Channa maculata.4. Analysis on genetic diversity and genetic structure 10 microsatellite makers were developed and applied to investigate the polymorphism in 4 wild stocks: Weishan Lake of Shandong (SD), Poyang Lake of Jiangxi (JX), Dongting Lake of Hunan (HN), Qiantang Jiang River of Zhejiang (ZJ). The number of allele of each locus ranged from 3 to 26. The expected heterozygosity ranged from 0.4805 to 0.9482, and the observed heterozygosity ranged from 0.4333 to 1.0000, and the PIC ranged from 0.0017 to 1.0000 in four stocks. The analysis of Hardy-Weinberg equilibrium showed that 7.5% alleles departed from Hard-Weinberg equilibrium significantly. According to the results of genetic similarity, genetic distance, UPGMA dendrogram, it was found that the stocks of HN and ZJ are closer, and the stocks of SD and JX are closer. This research is significant for understanding the genetic structure character and resource status of Channa argus in China.