| DNA molecular marker can be used as the level directly reflect of genetic polymorphism, microsatellite marker is an ideal marker for population genetic polymorphism analysis with many advantages.There were many researches related on population genetics analyzing of Eriocheir sinensis using microsatellite markers.However, we’ve had no information on the population genetic status of Eriocheir sinensis from Yangtze River, Yellow River and Liao River, further germplasm resources evaluation work should to be performed. In order to solve the above mentioned issues, the three elements as promoting our researches including genome DNA extraction method, genotyping of microsatellite DNA, and what’s more, Based on the Yangtze river, Yellow River and Liao River three drainage of wild Eriocheir sinensis population genetic structure analysis, we can provide basic data to the genetic breeding of Eriocheir sinensis.1. To optimize genome DNA extraction method of Chinese mitten crabs, genome DNA was extracted from gills, muscle, blood and bristles of Chinese mitten crabs using phenol-chloroform method and DNA extraction kit, and the quality of these DNA samples was evaluated in four aspects: the purity, concentration, fragment completeness and microsatellite marker PCR amplification effect. The results showed that, compared with the method of DNA extraction kit, the DNA extracted by phenol-chloroform method was much more complete, had higher concentration,but lower purity. Both the extraction methods showed that the purity of DNA in bristles and blood were higher than that in the gills and muscle, and the descending order of DNA content was gill, muscle, bristles, blood, while the uncutting bristleswere failed to extract genomic DNA. Microsatellite PCR electrophoresis results showed that the DNA extracted from four different tissues by two dissimilar DNA extraction methods could meet the requirements of the application of microsatellite markers.In conclusion, the present study established a convenient noninvasive sampling method and a procedure of DNA extraction that could promote genetic breeding and molecular marker research of Eriocheir sinensis.2. 360 PCR products of 6 loci among 60 Chinese Mitten Crabs(Eriocheir sinensis)were analyzed by capillary electrophoresis with fluorescent-labeled primers and polyacrylamide gel electrophoresis(PAGE) respectively. Total 129 alleles were obtained and 21.5 alleles were detected for every pairs of primers by capillary electrophoresis system, while aggregate 174 and average 29 alleles were detected with PAGE. The repetition rates were at 100% when locus Esin67 was detected for the second time by capillary electrophoresis method, while for PAGE the rates were getting really close to 0, which confirmed that capillary electrophoresis is more reliable than PAGE significantly. More ever, analysis showed that capillary electrophoresis’ s material costs and efficiency is higher than PAGE. Establish a single PCR multiple capillary electrophoresis technology is an effective way to improve efficiency and reduce cost.3. Twenty-three microsatellite markers were used to analyze the genetic diversity and genetic structure of three Eriocheir sinensis wild populations from three river(Yangtze River, Yellow River, Liao River).Total 745 alleles were detected, the average amount of alleles is 31.23 for each microsatellite loci, genetic diversity analysis showed that the average polymorphism information content of PIC = 0.894, 22 of them are high polymorphism loci(PIC> 0.5).All locus’ s observed heterozygosity was0.736 on average, the average expected heterozygosity was 0.903, more than one gene locus were deviated from Hardy-weinberg equilibrium in every groups, on the whole,manifested as heterozygote deficiency.All the three groups showed a higher level of genetic diversity(HO = 0.725-0.752, I = 2.729-2.769); the order of genetic diversity magnitude of 3 populations showed by Shannon information index(I) is: Yellow River> Yangtze River >Liao River.AMOVA analysis results showed that 99.76% ofthe variation is derived from theindividual internal and within populations, population genetic differentiation index FST = 0.0022- 0.0056, the genetic differentiation among groups were at low level(FST < 0.05). The UPGMA clustering based on the Nei’s genetic distance, showed that populations from Yellow River and the Liao River was the first cluster, then with the population of Yangtze River to be the second cluster.Bottleneck analysis showed that all groups have recently experienced the declining of effective population size. Population genetic structure analysis failed to differentiate the optimal theory group, proved that the three wild populations had suffered from mix of variety.Above all, the wild Eriocheir sinensis from three major river systems has high genetic diversity at present, it is suitable for genetic improvement;Genetic differentiation and geographic distance has certain correlation; Three groups with low genetic differentiation and mix of variety, hinted that a large-scale and cross-regional introduction of farming activity as well as escape of the breeding populations, both might be the cause of cross-contaminated of natural Eriocheir sinensis germplasm resources. |
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