| Apostichopus japonicus (Selenka,1867), as a representative species of echinoderms, have become one of the most important aquacultural species with greatly economic value. As the expansion of the farming area of sea cucumber aquaculture, it has been one of the pillar industries of Chinese aquaculture industries. In this research,2b-RAD (2b-restriction site-associated DNA) was engaged to investigate SNP markers in a high-throughput way for F1 individuals of Apostichopus japonicus. A high density, high resolution genetic linkage map of Apostichopus japonicus has been constructed, the relationship between SNP loci and body weight of sea cucumber has been studied on the fundamental of the linkage map. This research has successfully laid the theoretical foundation and supplied plenty of information for whole genome sequencing and other genomic researches. On the other side, it has facilitated the further study of the mechanism for sea cucumber growth and marker assisted selection in breeding projects both theoretically and technically.1. Construction of mapping family and 2b-RAD sequencing librariesIn this research made use of double pseudo-testcross strategy to construct the mapping family. Individuals with better growth traits were selected and in total of nine sub sib families were constructed which shared a same male parent and nine different female parents. Samples of progenies were obtained at the age of one year and family determination was carried out. Finally the mapping family was determined.In this research, genome DNA samples of two parents and 100 progenies were digested by using Bali to obtain the targeted 33bp DNA fragments, which have a random distribution in the genome; the target fragments were then ligated to the adaptors, which has induced selective bases to control the amount of target tags from the two parents and 100 progenies; Barcode sequences were imported into the specific sequencing by two rounds of PCR amplifications to distinguish libraries from different individuals. After the construction several libraries were randomly chosen for cloning sequencing, and resequencing of two parents and 10 progenies were carried out by reconstructing sequencing libraries and resequencing to test the stability and repeatability of the technology and experimental operation.2. Construction of high-density linkage map in Apostichopus japonicusIn this research, after data processing including tags gathering, reference loci building, mapping tags from progenies against reference loci, genotyping rate and Mendel segregation rate filtering, in total of 11,306 SNP loci were obtained to construct the genetic linkage map of Apostichopus japonicus.6311 and 5517 SNP markers were used for female and male specific map construction separately, among which 522 SNP markers were parent shared loci (p-value=0.05). Joinmap 4.0 were selected to construct linkage maps. But due to the limitation of marker number, a special constructing progress were designed as follows:linkage groups dividing, preliminary calculation of genetic distance of SNPs by using MSTMap software; making SNPs with the same genetic distance clusters, selecting the most informative SNP as the unique loci to represent corresponding clusters; constructing male and female specific linkage map by calculating SNP data without clusters using Joinmap 4.0; integrating of the sex specific maps using the MergeMap software; refiling of clusters into the constructed maps; using MapChart 2.2 to draw the male and female linkage map and the consensus map.According to this research, the consensus map of Apostichopus japonicus contained 7839 SNP loci and 22 linkage groups, with an average marker interval of 0.47 cM. the length of the genetic map was 3706.6 cM, covering 99.57% of Apostichopus japonicus genome. The linkage map obtained in this research was to our knowledge has the highest marker density among the maps constructed in echinoderm before.3. Linkage analysis and association analysis of growth trait in Apostichopusjaponicus, and significant candidate genes locating On the foundation of constructed genetic linkage map of Apostichopus japonicus, linkage analysis and association analysis of growth trait were carried out. The results showed that a QTL region in linkage group 5 were captured to reach the LOD threshold value calculated by a 1000 cycle permutation tests (LOD=12.3). Three SNP loci were located in the region of 79.63-80.01 cM in linkage group 5, accumulate explaining 19.8% growth trait variation in the mapping family.The result of association analysis showed the same tendency between growth trait and SNP loci.Taking both of the result of QTL analysis and GWAS analysis into consideration, a linkage region with a width of 5 cM in linkage group 5 was recognized to have a significant correlation with the growth trait. We extracted the tags of corresponding SNP loci in this region from the sequencing data, and mapped the tags against the genome database (unpublished data). Then the unique match sequences were chosen as the sequence of each SNP loci. After that we made gene annotation for the sequences and obtained some protein sequences. At last the NCBI website was used to make BlastP analysis to examine the function and catalog of the corresponding sequences. In this research, we located a gene named RBBP5 (retinoblastoma-binding protein 5) on the map. RBBP5 has been formally proved to regulate the cell proliferation.The genetic linkage map constructed of Apostichopus japonicus in this study showed significant advantage in large scale QTL mapping and marker-assisted selection (MAS) breeding, and will facilitate karyotype analysis and whole genome sequence assembly in the future. |
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