Construction And Application Of High Density SNP Genetic Linkage Map For Apostichopus Japonicas

As an important aquaculture species in China, Apostichopus japonica enjoyshigh edible and medicinal value. It has become one of the pillar industries inaquaculture. Superior seed will be required to develop Apostichopus japonicusaquaculture healthily and sustainably, while the reliable molecular marker-ssistedselection (Lien, Gidskehaug et al.) can greatly accelerate the breeding process. Ourresearch used2b-RAD technology to construct the first high density genetic linkagemap for Apostichopus japonicas, which will facilitate the identification of thequantitative trait loci (QTL) and molecular marker assisted breeding.1. Construction of the Apostichopus japonicas library and quality controlWe adopted2b-RAD method in this research. The research was conducted byusing two parents and100F1individuals as construction group, extracting muscletissues DNA as material. Through BSA-XⅠ digestion, we obtained33bp DNAfragments, ligating specificity adapters, flexible controlling parents and offsprings tagnumber. Two rounds PCR amplification leaded in special barcodes, and102librarieswere constructed after purification. Next clone sequencing ensured the qualifiedlibraries. We adopted HiSeq2000platform to predict the qualified libraries beforeusing the machine, then we came to the high throughput sequencing process.2. Construction a high density SNP genetic linkage map for Apostichopus japonicasand the QTL analysis of weight characterAt the beginning, we used small fragments removal, quality screening to processthe raw data, and then we utilized our own RADtyping software to perform thesequence clustering and genotyping. There are85%male reads and98%female reads,and91%progeny average high-quality reads. After clusting, we obtained3915co-dominance markers and9455dominance markers, which conformed to the Mendelian ratios(P≥0.5).In the co-dominance markers, there were1573male-specific markers（ll×lm）,1782female-specific markers（np×nn）and560shared markers.To successfully build a high density SNP genetic linkage map for Apostichopusjaponicas, we used pseudo-testcross strategy. On the basis of3915co-dominancemarkers we have got through clusting, we optimized filter conditions to screening themost reliable dominance markers and add them to the map. Firstly, we constructedfemale-specific and male-specific linkage map. The female map contained22linkage groups, which had3,156markers with3705.34cm span and1.20cm averagemarker interval. While the male map contained22linkage groups, which had2442markers with1529.45cm span and0.74cm average marker interval. The sex-specificmaps were further integrated using anchor markers that were heterozygous in bothparents by MergeMap Online platform. Construct the consensus A.japonicus linkagemap comprised22linkage groups,5,328markers and3697.02cm span with aresolution of0.57cm. The size of the linkage groups ranged from93.9cm to293.9cmand the number of loci per linkage group varied from132to349with an average of242. It can be calculated that the expected map length is3727.66cm, so the genomecoverage was99.2%. Finally MapCharp software was used to complete graphical map.The research uses the completed Apostichopus japonicas SNP markers geneticlinkage map to record the weight traits of100F1progenies. Through MapQTL5.0software and GWAS, analyzing the possible QTL locus. Eventually, the possible QTLlocus that associated with Apostichopus japonicas weight traits were found only inchromosome level.