Diversity Analysis Of Different Loach Populations In The YantZe River Basin By PCR-RFLP

Loach (Misgurnus anguillicaudatus) is a small freshwater economic fish, which is widely distributed in Vietnam, Korea, Japan and most parts of southern China. It has become a special breeding species in China as to its tender meat and delicious, nutrient-rich. In recent years, Loach’s market demand is growing with the improvement of living standard. Germplasm resources of wild loach gradually decline as for the over-fishing, a large number of artificial breeding and water environment deteriorating. Such as:slow growth, sexual maturity early, smaller individuals and the deterioration of disease resistance. Yangtze River as the main distribution area for loach, It is very important to study the diversity of the loach in its main water body and to Properly assess the situation of wild germplasm loach and genetic differences. In this study, we used of fish mitochondrial DNA ND-5/6 segments universal primers to amplify the section, then we analysised the genetic diversity of the region by PCR-RFLP marker.The results obtained are as follows:(1) We found diploid and tetraploid wild loach in Yangtze River and other times of polyploid loach were not found. The result of the amplifying mitochondrial DNA ND-5/6 segments of diploid and tetraploid were about 2.2kbp length. Amplified products were smaller than the carp under the same primer conditions of the amplification.Then we did a further study of diploid and tetraploid loach from Yueyang and Wuhan by PCR-RFLP.66 haplotypes were detected by 6 enzymes (HaeⅢ, DraⅠ, RsaⅠ, TaqⅠ, HinfⅠ, MspⅠ) restriction enzyme. The nucleotide diversity within diploid populations were higher than the tetraploid.The nucleotide diversity (π) between populations was 0.02588-0.04144, with an average 0.031737±0.000005. In which nucleotide divergence between diploid and tetraploid loach from Wuhan were the highest (0.01617), the nucleotide divergence between diploid loach from Yueyang and tetraploid loach from Wuhan were the minimum (0.00462). Monte Carlo simulation showed that the haplotype frequencies between the four populations were significant differences (P<0.0001). UPGMA cluster analysis showed that tetraploid loach from Wuhan and Yueyang and diploid loach from Yueyang were clustered into one branch, and diploid loach from Wuhan was a separate one.(2) We studied the diversity of 219 diploid loach from 7 wild populations on theYangtze River Basin by PCR-RFLP technology. The results showed that the amplified product is about 2.2kbp long, no amplification length polymorphism were found. Screening from 13 kinds of enzymes (BamHⅠ, DraⅠ, EcorⅠ, HindⅢ, PstⅠ, MspⅠ, HaeⅢ, HinfⅠ, TaqⅠ, RsaⅠ, MvaⅠ, PvuⅠ, HhaⅠ), we found 11 kinds of them (BamHⅠ, DraⅠ, EcorⅠ, HindⅢ, PstⅠ, MspⅠ,HaeⅢ,HinfⅠ, TaqⅠ,RsaⅠ, MvaⅠ) has good polymorphism. By restriction analysis, we got a total of 91 haplotypes.The haplotype diversity in Nanjing population was the highest (0.8927), followed by Yueyang population (0.8627), Sichuan population was the lowest (0.2603). The average haplotype diversity index of the 7 loach populations was 0.7410±0.00693 (self),0.7302±0.00671 (non-self). Average nucleotide diversity index was 0.078624±0.0000092. The nucleotide divergence among populations was from 0.085330 to 0.012712, with an average 0.043122±0.0000142. The nucleotide divergence between Yueyang and Wuhan populations was the minimum (0.01617), and that between Wuhan and Leshan populations was the maximum (0.085330).Monte Carlo simulation showed that the haplotype frequencies of the seven populations were significant differences (P<0.0001). UPGMA and NJ cluster analysis showed that the relationship between Yueyang population and Wuhan polulation was close. They got together with the population from Nanjin、Anqing and Yichang. Genetic distance between Sichuan polulation and other populations were very far. This indicates that there is great genetic differentiation between the diploid loach populations from Upstream of Yangtze River and the Yangtze River Basin.Through the study to the diversity of different populations of loach, we found that the diversity of the wild loach from Yangtze River Basin is rich. They have good breeding potential. The distribution of polyploid loach was common, but there is no obvious pattern of their distribution.The current experimental data is relatively scarce, in order to provide the basis for breeding loach, we need to do further study in future. In addition, to protect the genetic diversity of its wildlife resources, when we developed and used of the germplasm resources of wild loach, some necessary measures should be taken.