Development And Application Of Molecular Markers In The Sea Cucumber (Apostichopus Japonicus)

The sea cucumber, Apostichopus japonicus, is a common species distributed inshallow waters along northern Asian coasts in China, Japan, Russia and Korea, and isregarded as one of the most valuable aquaculture species due to its potential as adelicacy and traditional medicine. In order to achieve sustainable development of A.japonicus culture industry, selection of bloodstocks with the desired traits, such asrapid growth and disease resistance are urgently needed. It is generally accepted thatmarker-assisted selection (MAS) could provide valuable information to efficientlyimprove desirable traits and accelerate progress in selective breeding. In the presentstudy, we developed and characterized a panel of SSR (Simple Sequence Repeat) andSNP (Single Nucleotide Polymorphism) markers, and also studied their application inparentage assignment and genetic mapping of A.japonicus.1. SSR and SNP mining and marker development for A.japonicusEST (Expressed Sequence Tags) database mining was used to develop novelmicrosatellite markers for A. japonicus. A total of730ESTs containing SSRs wereidentified. Primers were designed for212EST-SSRs according to the flankingsequences using Primer Premier5program. Polymorphism of these loci wereevaluated in48individuals collected from the coast of Shandong province, China.The results showed that expected products were amplified with167of the212primerpairs, of which137loci were polymorphic. The number of alleles ranged from2to7,and the observed and expected heterozygosities varied from0.0667to0.6670andfrom0.1769to0.7200, respectively. Twenty-six of the137loci significantly deviatedfrom the Hardy-Weinberg equilibrium after Bonferroni correction. Gene annotation(BLASTX comparison, e-value threshold of1e-6) indicated that twelve of137polymorphic SSR-bearing ESTs matched to genes with known functions, while otherseither have no significant matches or matched to uncharacterized genes. Throughanalyzing the A. japonicus transcriptome, a large number of putative SNPs were previously identified, among which678were selected for marker development usinghigh-resolution melting (HRM) method. In the analyzed population,249SNP lociwere polymorphic. The observed and expected heterozygosities varied from0.050to0.833and from0.073to0.907, respectively. Thirty-three loci significantly deviatedfrom the Hardy-Weinberg equilibrium. Seventy of249polymorphic SNP-bearingESTs matched to genes with known functions.2Application of SSR markers in parentage assignment of the sea cucumberA.japonicusWe first conducted computer simulation analyses using the developed EST-SSRmarkers to establish guidelines for effective parentage analysis. Real mixed familieswere also utilized for evaluation of the performance of SSR-based parentage analysisin practice. Simulation results demonstrated that the exclusion probability of theselected nine microsatellite markers was0.99which was still satisfactory comparedwith12microsatellites. To evaluate the performance of parentage analysis in a realcase, a total of180larvae from distinguished nine families were genotyped for thepreviously-selected9microsatellite loci. Eventually all the larvae were accuratelyassigned to each family. Then1000offsprings from mixed families were assigned forparentage assignment for9microsatellite loci. The exclusion probability of progenyto their parental couple was81.0%.3Construction of genetic linkage map and identification of quantitative trait loci forthe sea cucumber, A.japonicusWe constructed the genetic linkage map in two F1outbred families of A.japonicususing pseudo-testcross strategy. The sex-specific linkage maps for two mappingfamilies were constructed separately using313microsatellites and249SNPs.22linkage groups (LGs) were identified, which were consistent with the haploidchromosome number of A. japonicus. A significantly higher recombination rate wasobserved in the female map. Significant family-specific differences in recombinationratios were also observed for some LGs between two female maps and two male mapssuch as（LG2-M， LG11-M，LG9-F， L13-F，L18-F）. The sex-averaged mapconsisted of215markers (121microsatellite markers and94SNPs) covering1498.9 cM with an average inter-marker spacing of7.0cM. The length of the linkage groupsranged from44.4cM to121.4cM and the number of markers varied from5to18perlinkage group. On the basis of the expected genome lengths, genome coverage for theconsensus, female, male maps including the triplets was79.2,74.8and72.8%. Geneannotation (BLASTX comparison, e-value threshold of1e-7) indicated that16ESTscontaining markers in maps matched to genes with known functions, while otherseither have no significant matches or matched to uncharacterized genes. Tblastxcomparisons revealed that24hits of the134blasted sequences were significantlyconserved between A. japonicus and sea urchin. Preliminary QTL mapping wereconducted for the growth traits of A.japonicus but no significant results have beendetected. It is possibly due to phenotypic errors caused by measurement.