Microsatellite Loci Isolation And Population Polymorphism Analysis In Sepiella Maindroni

Microsatellites are now widely applied in genetic studies even though they are relatively difficult to obtain. In this experiment, biotinylated magnetic beads are used to enrich microsatellite loci of Sepiella maindroni. DNA pool, which is mixed from genetic DNA of 4 different individuals derived from LiuHeng island of Zhoushan, was digested by restriction endonuclease Sau3AI. After ligation of specific adaptor, digested fragments were amplified via PCR to construct a DNA library, from which microsatellites were enriched using (GT)15 biotin labeled probe and magnetic beads. The captured fragments containing microsatellites were amplified, inserted into pMD-18T vector, transferred into DH5αand then PCR examined. 120 positive clones in total 278 were sequenced and 102 clones (85%) showed the existence of microsatellites. Among them, 64 microsatellite sequences are available to design primers and conduct further researches. At last, 33 microsatellite primers are designed and synthesized.We also searched microsatellites of Sepiella maindroni at the NCBI website. As a result, 10 microsatellite sequences were found to be available. 10 candidate primers were designed for the ten microsatellites, out of which, 4 primers gave specific bands by polyacrylamide gel examination after PCR (As we called a polymorphism test, 6 individuals are randomly selected to test the microsatellite primers). Moreover, the pattern of primer SMC414 showed 4 microsatellite loci and 5 different combinations of loci in totel, while another primer SMC2142 gave 3 microsatellite loci and 3 different combinations of loci, which indicated a possibility of polymorphic amplification in Sepiella maindroni group using these two primers. However, no polymorphism was observed for the other two primers.Using 33 microsatellite primers that we designed to amplify templates from Zhoushan and Xiapu group, 15 of them had polymorphic bands and 13 of them were able to be analyzed. After the data were encoded with 0-1 mode and then transformed to ABCD encoding mode, the software Popgen32 was applied to analyze the data through the aspects such as Allele Number, Allele Frequency, Observed Heterozygosity, Expect Heterozygosity, Effecitve number of alleles,et al. And PIC value of alleles was calculated accoding to Allele Frequency. After inputted with the data of Zhoushan group (86 samples) and Xiapu group (55 samples), the software gave a result of 0.0519 on group genetic distance according to Nei's unbiased measures of genetic distance, which indicated a significant genetic diversity of the two groups.