Genetic Studies On The Sea Cucumber (Apostichopus Japonicus)

1 Identification and characterization of microsatellite markers derived from expressed sequence tags (ESTs) of the sea cucumber Apostichopus japonicusEleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Apotichopus japonicus cDNA libraries. The numbers of alleles ranged from three to ten, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and 0.077 to 0.690, respectively. Significant deviations from Hardy-Weinberg expectations were observed at eight loci, due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of A. japonicus.2 Microsatellite genetic variation in wild and hatchery populations of A. japonicusThe farming of the sea cucumber Apostichopus japonicus has started 20 years ago and is still in rapid expansion in China. In order to assess the genetic status of both wild and cultivated stocks of this species, we used eight microsatellite markers to estimate the level of genetic diversity within five hatchery stocks and two wild populations of A. japonicus, and compared the degree of genetic differentiation between them. High levels of polymorphism were observed over all loci. The mean alleles and expected heterozygosities over the seven stocks were 10.4-12.3 and 0.735-0.783, respectively. The results of the microsatellite survey provide no evidence to show that hatchery practice of the sea cucumber in China to date has significantly affected the genetic variability of the cultured stocks. Significant differentiation was found between most pairs of the hatchery stocks and wild populations (Fst range: 0.008-0.036), and no obvious difference was detected between the wild populations (Fst = 0.008). The information on the genetic variation and differentiation obtained in this study can be applied for future genetic monitoring of A. japonicus aquaculture stocks and will be useful for future genetic improvement by selective breeding, and for designing suitable management guidelines for these genetic materials. 3 Construction of a linkage map for A. japonicusThe linkage maps of A. japonicus were constructed with AFLP and SSR markers. 21 microsatellite loci and 37 primer combinations of AFLP markers which were genotyped in the parents and 88 progeny of the mapping family.Two genetic linkage maps were constructed using markers segregating in the female or the male parent, excluding the distorted segregating ones. The female framework map was composed of 141 markers in 17 linkage groups, covering 1291 cM with an average interval of 10.4 cM. The male framework map contained 125 markers in 18 linkage groups, spanning 945.7 cM with an average marker density of 8.8 cM. The observed coverage was 77.6% for the female map and 74.4% for the male map.4 Inheritance mode of mitochondrial DNA of A. japonicusThe inheritance mode of mitochondrial COI gene of A. japonicus was analyzed by DGGE method. We selected 5 pairs which have difference in parents in the end. The results showed that all the progeny have the same haplotype with female parent. This means the mitochondria inheritance mode of A. japonicus used in this experimentation is matemal inheritance.5 Sequence analysis of mitochondrial 16S rRNA and COI gene and molecular phylogeny of four species of sea cucumber500 bp 16S rRNA and 540 bp COI fragment were obtained by PCR method from mitochondrial DNA of four species of sea cucumber, Apostichopus japonicus, Holothuria fuscogilva, Cucumaria frondosa and Mensamaria intercedens. Variable sites, nucleotide diversity, average number of nucleotide differences and average number of nucleotide substitutions per site were calculated to analyze sequence difference, and phylogenetic trees were constructed by the NJ and ME method. As a result, the biggest difference was found between Changdao and Japan of A. japonicus while the least between Changdao and Rongcheng. Differentiation among A. japonicus and other three species was higher than that within A. japonicus. NJ and ME phylogenetic trees based on 16S rRNA and COI gene showed that Apostichopus and Parastichopus was closely related, this means the two genus may be have a common origin. Holothuriidae didn't cluster with Stichopodidae but with Cucumariidae and Phyllophoridae, which is different from the result of morphology taxon.