The Genetic Structure And Diversity Analysis In Four Populations Of Misgurnus Anguillicaudatus

The molecular markers of mitochondrial control region (D-loop), mitochondrial cytochrome oxidase subunit I (COI) and internal simple sequence repeat (ISSR) were used to study genetic diversity, genetic differentiation and the relationship between different groups among the four populations of Misgurnus anguillicaudatus in Anhui Huai (HY), Henan Fanxian (FX), Anhui Shucheng (SC) and Anhui Province (CZ). The study want to provide a theoretical basis on the loach genetic diversity, evolutionary relationships analysis, breeding and so on. The main results were gained as follows:1. mtDNA D-loop sequences of the four geographical groups loach were sequenced and analyzed,918-920bp DNA fragment was gained by comparison. We successfully identified three regions includding the termination sequences of areas (ETAS), central conserved domain (CD), and conserved sequence areas (CSB), indicated a number of conserved sequences (CSB-F, CSB-E, CSB-D and CSB1, CSB2, CSB3) of control region.The average content of T, C, A and G in the fragment were31.7%,19.9%,34.2%and14.3%, respectively, the AT content was obvious higher than GC content.71variable sites were found and36haplotypes were defined among the four geographical populations. Among these four populations, Chizhou(CZ) holded the best nucleotide diversity(Pi), the average number of nucleotide differences within the group and the genetic distance, showing that the most abundant genetic diversity, the Shucheng population showed the minimum. Using MEGA4.0software to calculate the four populations through the Kimura2-paramter genetic distance and gained the result which Shucheng and Chizhou populations had the largest genetic distance between populations for the0.01385, Fanxian and Huaiyuan populations had the genetic distance between populations, as0.00856. The analysis of molecular variance (AMOVA) analysis showed that the total genetic differentiation among four populations was Fst=0.3148(P>0.05), within population differentiation was the major genetic differentiation. NJ and UPGMA phylogenetic trees showed that the four populations was clustered into two categories, Chizhou population clustered together and Huaiyuan, Fanxian, Shucheng populations clustered into another group. Tajima’s D and Fu’s neutrality test indicated that the four populations′size remains stable and in the past and didn′t occur the phenomenon that population expansion and sustained growth pattern. 2. Mitochondrial COI gene fragment was amplified and sequenced to obtain597bp nucleotide fragment. Among the fragment138variable sites were found and30haplotypes were defined, insertion or deletion was not observed, base composition obiviously found, transition and transversion exchange ratio of2.65, the gene replacement had not reached saturation. Chizhou population showed the best haplotype diversity, nucleotide diversity, the average number of nucleotide differences and genetic distances within the group, while genetic distance between the average number of nucleotide substitutions per site (Dxy) and each point of the net number of nucleotide substitutions (Da) showed that Chizhou population had the highest genetic variation and the most abundant genetic diversity. Fst coefficient of genetic differentiation between Chizhou population and other populations achieved a high degree and attained the division level. NJ and UPGMA phylogenetic trees showed that the Huaiyuan and Shucheng populations clustered with Fanxian, and finally with the Chizhou populations.Chizhou population has the farther genetic distance than other three populations.3. ISSR molecular marker technique was used to study four genetic diversity and inter-group differences. Fifteen ISSR primers were selected to amplify102individuals polymorphic, which the percentage of polymorphic loci was32.35%ratio. Comprehensive genetic diversity index of all the various populations showed that the level of genetic diversity: Chizhou> Huaiyuan> Fanxian> Shucheng populations. Genetic distance among four populations between0.04022and0.08532, which genetic distance among Chizhou and the other three populations were between0.07988and0.08532. The data indicated genetic distance between Chizhou and the other populations reached the maximum. The total coefficient of genetic differentiation among populations (Gst) was0.364,63.6%of the genetic differentiation occurred within the population, that population genetic differentiation within the main source of variation. The level of gene flow between populations (0.4368) was low, genetic drift caused changes in population genetic structure. NJ and UPGMA cluster analysis, the nearest was the Huaiyuan and Fanxian populations, and then clustered the Shucheng groups, and finally with the Chizhou groups together.