| Apostichopus japonicus microsatellite DNA technology to select microsatellite markers and population analysis. First Genomic DNA of different tissues from Apostichopus japonicus was extracted through CTAB method, SDS method and Segama kit method respectively. High quality genomic DNA was isolated from six kinds of sea cucumbers tissues:tube feet, body wall, longitudinal muscles, intestinal and respiratory tree. Compare to the other two methods, Segama kit got clearer DNA band with no degradation and was the ideally method; Longitudinal muscle tissue of Apostichopus japonicus get the best quality of genomic DNA from three methods contrast to the other different tissues, so it is the best tissue to extraction of genomic DNA. At the same time, sea cucumbers tentacles and tube feet extracting genomic DNA from live body were used to extract DNA and also got high-quality genomic DNA. This provide data to support non-invasive sampling of Sea Cucumber. And this also proof it is possible to ensure the parents to survive and remain healthy, and reduce the body of the test sample damage caused by sampling in the process of Sea Cucumber family building in the future.We use FIASCO technology to screen microsatellite markers.58 were obtained with microsatellite sequences,6 of which the same sequence. The results show that the three microsatellite sequences containing two or more micro-points,52 more than the number of repeat sequences containing microsatellite repeat sequences of 5,2 and four nucleotide sequence repeat 4 more times, primers can be developed; 5 series repeats in the 5 microsatellite DNA sequences of the following or a break, not suitable for designing primers. Comprehensive analysis of 56 core sequence of microsatellites,52 microsatellite sequences have been submitted to NCBI database. According to Weber Microsatellite DNA sequences proposed classification criteria, microsatellite DNA can be divided into the perfect, imperfect and compound 3 class. The resulting microsatellite core sequence of 56, the perfect total of 30 microsatellite DNA, accounting for 53%; not perfect total of 14 micro satellite DNA, accounting for 25%; compound 12 microsatellite DNA, accounting for 21%. Obtained in the microsatellite core sequence repeat DNA sequences in the 5 to 43 9 times, accounting for 76%; repeated 10 times,7 to 19, accounting for 12.5%; Repeat 20-29 times There are 2,3.5%; repeated 30 times over a four sequences, accounting for 7.1% gain in the microsatellite DNA sequence repeats at most between 1 to 9.The frequency and density of 2-6bp simple sequence repeats (SSRs) have been analyzed from the bee EST (expre ssed sequencetag) database comprising 6632 sequences by microsatellite searching software. The result shows that 416 microsatellite sequences which accounting 6.48% of the whole ESTs database were obtained. The dinucleotide repeats (65.86%) motifs appeared to be the most abundant type in Apostichopus japonicus ESTs, and trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeats are 26.68%,1.68%,0.24%, and 5.53%, respectively.21 primer pairs were designed with Primer software and manual-searching method from EST sequences containing microsatellites.13 primers had polymorphic PCR products in 60 Chinese shrimp samples by electrophoresis analysis. In these 13 microsatellite loci, the alleles numbers distributed from 3 to 8, and the range of these alleles were between 175 to 382bp. The observed heterozygosity and expected heterozygosity distributed from 0.158-0.650 and 0.198-0.841, respectively, which showed that these 13 microsatellite loci were suitable for genetic analysis.Genetic diversities of a wild and two generations of selectively bred populations of the sea cucumber, Apostichopus japonicas, were examined using the microsatellite DNA techniques.43 bands of alleles amplified using 9 primers were obtained from these three populations and 2-7 alleles were detected from each microsatellite locus. Average observed heterozygosities (Ho) of these populations were 0.7164,0.6486 and 0.5968, respectively; and the average values of polymorphism information content were 0.6654,0.5929 and 0.5275, respectively. The results showed that the heterozygosities of two generations of selectively bred A. japonicus were reduced, compared with the wild population, and genetic diversity declined as the breeding generations increased. Hardy-Weinberg equilibrium chi-square test and F-test data showed that selective breeding had changed the genetic structure of the populations, including decreased heterozygous sites, increased loci deviated from the equilibrium, and decreased coefficient of genetic differentiation between the adjacent generations. FST value (0.0443) suggested the breeding population had weak genetic differentiation and illustrated that the main genetic variation existed among individuals within population rather than between populations. Genetic distance and genetic identity indicated that divergence became smaller with increasing breeding generations and the genetic similarity between the adjacent generations became greater, even though they had not reached the theoretical value, thus it was still suitable for further selective breeding. |
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