Development, Characterization And Application Of Microsatellite Markers In The Sea Cucumber (apostichopus Japonicus)

Sea cucumber, Apostichopus japonicus Selenka 1867, is a common species in the Western Pacific Ocean along the coast of China, Japan, Korea and Russia. It has long been regarded as an Asian delicacy and been consumed due to their good flavour and medicinal value. In recent years, sea cucumber aquaculture has developed rapidly along the northern coast of China. It is an effective approach to cultivating the good strains with the performances of disease-resistance and fast growth for the sustainable development of sea cucumber fishery. The marker-assisted selection (MAS) provides the fundamental theories and applied experiences for this task not only in better utilizing the available resources but also increasing production in aquaculture. In the present study, we isolated and characterized a panel of microsatellite markers, and also studied their application in the MAS of A. japonicus.1. Isolation and characterization of microsatellite markers for A. japonicusTwo methods, EST database mining and enrichment-colony hybridization were used to develop novel microsatellite markers for A. japonicus. Primers were designed and synthesized using these microsatellite-containing sequences, the polymorphisms of these loci were assessed using 48 A. japonicus individuals collected from the sea coast of Rongcheng City in Shandong Province, China. The results showed that 276 novel loci were polymorphic, containing 59 EST-derived microsatellites and 217 genomic microsatellites. Of these EST-SSRs, the number of alleles ranged from 2 to 10 with an average of 4.8 alleles per locus, and the values of observed and expected heterozygosities (Ho and He) varied from 0.000 to 0.900 and from 0.202 to 0.809, respectively. On the other hand, the 217 genomic SSR markers revealed a total of 1615 alleles, the number of alleles ranged from 2 to 15 with an average of 6.8 alleles per locus, and the values of observed and expected heterozygosities (Ho and He) varied from 0.000 to 1.000 and from 0.180 to 0.948, respectively. These novel polymorphic microsatellite markers presented in this study will be useful to analyze population structures, reproductive ecology analysis, phylogenetics and comparative genomics studies and construct genetic linkage maps for A. japonicus.2. Performance of whole-genome amplified DNA isolated from A. japonicus larvae using analysis with microsatellite markersSmall larval stages pose a problem for molecular analyses because limited amounts of DNA template are available. Isothermal methods for faithfully copying DNA have the potential to revolutionize studies of such organisms. We evaluated the fidelity of multiple displacement amplification (MDA) for amplifying DNA extracted from A. japonicus larvae. The quality, purity and concentration of DNA samples was determined by agarose gel electrophoresis and UV spectrophotometry. High molecular weight genomic DNA was isolated by the method and most of the DNA remained in the well when ran out on an agarose gel. And the typical yield of illustra GenomiPhi kit was about 1000ng in a final reaction volume. Fifty microsatellite loci were used for the 30 F1 larvae, giving a total of 1500 F1 genotypes. Because these larvae were obtained from a cross between a male and female A. japonicus of known genotypes, we were able to predict the alleles present in the progeny and quantify the genotyping error rate. Just 7/1500 (0.47%) genotypes showed patterns that differed from those expected under Mendelian segregation. These genotyping errors included 4 additional alleles, 2 missing alleles and 1 mutation. We concluded that many hundreds of microsatellites or other genetic markers can be accurately genotyped from a single larva using this method, provide a useful tool for population genetics, pedigree analysis, linkage map construction and marker-assisted selection (MAS) of A. japonicus.3. A microsatellite marker tool for parentage assessment in A. japonicusUsing several pairs of microsatellite markers, we studied the microsatellite paternity testing accuracy by separate breed and the microsatellite paternity appraisal ability by mixed breed. Paternity inference was initially performed using an exclusion-based approach starting from the genotypes at 12 loci. We look for mismatches between parents and offspring using the matrix outputs from the software CERVUS 3.0. The combined exclusion power for the 12 microsatellite loci was higher than 0.999 both for Excl 1 (unknown parent) and Excl 2 (other parent known). With a subset of the seven most polymorphic loci, the figures were still above 99% (99.016% and 99.912% respectively for Excl 1 and Excl 2). Using this microsatellite tool, paternity could be traced back unambiguously in 264 of the 270 offspring analyzed using a strict exclusion strategy, and the parentage analysis conducted on 10×10 outbred family showed that the microsatellite paternity appraisal ability was 93.8%. In this study, a suitable microsatellite-based tool for parentage assignment in A. japonicus was obtained. The high polymorphism of 7 selected loci ensured a theoretical exclusion power for paternity inference higher than 99%. This set of loci could be used with confidence following an exclusion approach to obtain similar actual values, especially if the broodstock is organized according to minimum parentage criteria.4. Construction of microsatellite-based Linkage map for A. japonicusTo better facilitate the molecular-based analyses such as quantitative trait loci (QTL) mapping and molecular marker-assisted selection (MAS), we constructed genetic linkage map of A. japonicus using a total of 509 microsatellite markers typed in 270 larvae from nine F1 outbred families sharing the same male. Two hundred and ninety eight markers were polymorphic and segregated in the shared male and could be used to construct the map. The male linkage map contained 193 loci ordered on 22 linkage groups and spanned 1905cM, with an average interval 10.0cM. The size of the linkage groups ranged from 36.9 to 182.4 cM (mean: 100.5 cM) and the number of markers per linkage group varied from 4 to 12, with an average of 8.7. This map is the first microsatellite linkage map of A. japonicus and will be useful for further genetic analyses such as QTL mapping and MAS.