Isolation And Genetic Diversity Analysis Of Microsatellite In Exopalaemon Carinicauda

The transferability of Penaeus chinensis SSR markers to Exopalaemon carinicauda was explored. The conservativeness of the side sequences of the microsatellite in species of close genetic relationship has already been discovered.11 polymohic microsatellite primers identified in Penaeus chinensis were employed to amplify in the genome of Exopalaemon carinicauda. The conditions of polymerase chain reaction(PCR) were optimized for the fidelity of DNA synthesis during PCR amplification. It was found that 9 loci failed to be amplified and 2 loci (RSI 101 and 72A64) amplified specific products successfully. The two loci were polymorphism.The SSR markers were developed and characterization in Exopalaemon carinicauda. Enrichment by magnetic beads is a simple and efficient method for rapid isolation of microsatellite DNA. In this experiment, we isolated microsatellite from Exopalaemon carinicauda genome with this method. Exopalaemon carinicauda genomic DNA was extracted and digested with restriction enzyme MboI. Fragments were ligated with artificial linkers(20bp). They were used to create a "whole genome PCR library". This genomic DNA was hybridized with a biotin-labeled microsatellite probe (CA)16. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNA contains the selected microsatellite DNA. The selected DNAs are then amplified using primers designed complementary to the linkers, cloned into the pGEM-T vector and sequenced. In this experiment, we selected 61 positive clones. From these positive clones, we isolated 32 microsatellite DNA sequences and all most (about 93.75%) of them were with corn repeats CA/GT, among which 84.38% microsatellites were perfect repeats,12.5% imperfect repeats and 3.13% compound repeats; 84.38% microsatellites repeated more than 20 times and the biggest repead number was 39. This allowed us to designed 23 pairs of primer with the software Oligo 6.0.Genetic diversity among three populations of Exopalaemon carinicauda distributing in Jiangsu, Zhejiang and Fujian province were studied by using microsatellite analysis technology.10 pairs of microsatellite primers of Exopalaemon carinicauda which were selected and proved to amplify successfully on Exopalaemon carinicauda. Amplified PCR products were imagined by Biolight. Average observed heterozygosity and average expected heterozygosity were calculated by Popgene3.2 software. The results showed that 6 to 12 alleles per pair in the three populations were amplified. The total number of alleles is 89 and the average number of the alleles per pair was 8.90; the average number of the effective alleles per pair was 7.43; the average heterozygosity was 0.863; the average polymorphism information contents(PIC) was 0.843 and all the 10 loci presented high polymorphic(PIC>0.5). The average heterozygosity of the three Exopalaemon carinicauda populations in Jiangsu, Zhejiang and Fujian province were 0.827,0.845,0.805; the average PIC were 0.792,0.815,0.771. The results indicated that the genetic diversity of the three populations was relatively abundance, and the genetic variability was high. The highest genetic similarity index(0.6594) and the lowest genetic distance(0.4164) were found between Zhejiang population and Fujian population, which indicated a closer phylogenetic relationship between these two populations. On the other hand, the lowest genetic similarity index(0.6374) and the highest genetic distance(0.4504) existed between Jiangsu population and Fujian population, which suggested a farthest phylogenetic relationship between these two populations. Phylogenetic relationship of the three populations was analyzed by using UPGMA methods based on their genetic distances and the results showed that Zhejiang population and Fujian population were clustered in the first group, then the Jiangsu population was in the second group.From the results we can predict that in order to get the maximum heterosis, we can choose the two populations of Exopalaemon carinicauda from Jiangsu and Fujian for hybrid breeding.