| Channa argus and Channa maculata , which belonged to Perciformes, Channoidei, Channidae, Channa, were important freshwater commercial fish. As for the advantages of delicious meat flavor, be nourishing, non-myenteric thorns, being easy to culture, high growth speed, and so on, it had high value for food., More and more culturists begin to culture them. In the two fish ,the male individuals grow 2-3 times faster the female ones. Thus,the development of all-male stock would be of significant benefit for aquaculture. In the present study,SSR technique was used to identify the sex-specific markers in the two fish .Besides, SSR technique was used to screen the genome DNA of cultured population and wide population and analyze the genetic diversity in order to do something for the breeding of the two fish.At the same time,SSR was used to analyze C.maculata,C.argus and Their Hybrid(C.maculata♀×C.argus♂). Some results were obtained as following:1. Microsatellites combined by bulked segregation analysis were used to screen the gender differences of 96 individuals (48 for male and female each ) from a family of C. argus. Two gene pools were constructed using 24 individuals for male and female each separately, gene pools were used to scan using totally 140 pairs of microsatellite primers and specific DNA fragments of female gene pool were amplified by 3 pairs of primers (HLJWL17, HLJWL59, HLJWL70). Then, the specific DNA fragments amplified by the three pairs of primers were verified for first round in the 48 individuals constructing the gene pools, showing the specific bands of HLJWL17 and HLJWL70 in gene pools only appeared in few individuals, while HLJWL59 was successfully amplified in females. And in the second round verification, the same results were got in the remaining 48 individuals with the HLJWL59. The amplified fragments by primer HLJWL59 of eight different alleles in females were cloned and sequenced. Sequence alignment of the eight individuals using Vector 8.0 multiple confirmed that the bands of each individual are the same sequence, the sequence length of 243bp. the repeat units was TGC, and 51% of GC. Compared by the BLASTn, homologous sequences was not found in the GenBank database. According to the statistics, the probability of different bands appeared in the female C. argus was 62.5%, that is the correct classification rate of the mark for female C. argus was 81.25%. We believe that this microsatellite loci may be associated with C. argus female gender under certain conditions. This study was foundation for culturing pure male hybrid using female-specific DNA fragments in C. argus. At the same time,use the 139 SSR primer combinations of C. argus. to scanning the genomic DNA of male and female groups of C. maculate.Three primer combinations suggested two female gender specific and two male gender specific DNA bands in the genetic pool . But in individuals, these specific bands disappeared.We think these bands were not what we wanted to find.2.Using eight SSR primer combinations to screen the genome DNA of two wild group and two breeding group of C. maculata for the first time. The numbers of alleles detected respectively of four groups are: 12, 20, 16, 20, and the number of alleles in breeding group had relatively decreased significantly compared wild group detected. We found a significant allele of YN(yunan) population at the loci HLJWL115 .In four groups, YN wild population had the highest polymorphism information content for 0.4148;Secondly for wild population HD(huadu) for 0.4028; Then is ZS ( zhongshan ) breeding group, for 0.2014; And FS ( foshan ) the minimum for 0.1523, suggested that in the breeding population the polymorphisms dropped significantly, About genetic distance, FS and ZS had the shortest, for 0.0616; FS and YN had the farthest distance, for 0.3168. Genetic distances and genetic consistency of results are consistent. At the same time, 34 primer-combinations were used to screen the genome DNA of 30 individuals of cultured and 19 individuals of wild C. argus.There were a total of 5 single loci,most of the alleles are belong to the two populations ,but some rare alleles in wile population had vanished in the cultured population . In effective alleles number and polymorphism information content ,the wild group were higher than the cultured group. From a certain extent,it shows that the cultured population genetic diversity has dropped.3. SSR technique was used to identificate C.maculata,C.argus and Their Hybrid(C.maculata♀×C.argus♂). A total of 85 individuals (30 from C.maculata, 30 from C.argus, 25 from the hybrid) were analyzed by 16 out of 139 SSR primers . 7 were "parents complementary",7 were "partial parent type",2 were "hybrid model". It suggested that SSR technique has wide prospective in hybrid test. |
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