Mapping Of Metamorphosis-Related Locus And Genomic Selection Of Disease Resistance In Chinese Tongue Sole (Cynoglossus Semilaevis) And Japanese Flounder(Paralichthys Olivaceus)

Flounder fish,commonly known as flatfish,have a flat body shape and always have a benthic life at the near sea bottom.Because of the characteristics like docile habitual nature,low trophic level and limited range of motion,flatfish have become important factory farming fish species.Chinese tongue sole(Cynoglossus semilaevis)and Japanese flounder(Paralichthys olivaceus)as two models of flatfish are widely cultured in Chinese coastal waters.Malformation and high frequency of diseases during the flatfish culture seriously restrict the development of flatfish aquaculture industy.In this study,we chose molecular breeding technologies to research the shape variation and disease resistance of Chinese tongue sole and Japanese flounder,in order to provide the theoretical foundation and technical support for flatfish breeding.1.Detection and analysis for albinism related loci in Chinese tongue soleTo dissect the molecular basis of albinism,the present study used an albinism Chinese tongue sole family to construct 3 SSR linkage groups,by which a preliminary linkage map related to albinism was drafted After WinQTL Cart 2.5 mapping,18 alibinism-related loci were detected under two models,containing 2,407 genes when compared with the Chinese tongue sole genome.One of them,tyrosinase related protein(tyrp2),was reported as an important gene in regulating eumelanin and phaeomelanin levels,which may cause albinism in mammal.By bioinformatic analyses,35 GO terms and 14 KEGG pathways were annotated.Among them,one GO term named as protein tyrosine kinase activity containing 10 genes,was suggested to take part in fish albinism.All these results proved foundation for the in-depth study on albinism in Chinese tongue sole.2.Eyes deflection related locus mapping and candidate gene analysis in Chinese tongue soleAn exclusive family of abnormal deflection of eyes was chosen from Chinese tongue sole families constructed by our lab to study the genetic basis of flatfish metamorphosis,including 178 specimens that were albino but had normal eye migration,23 specimens with both the albino phenotype and abnormal eye migration(eyes on both sides of head)and 32 normal specimens.In this study,we used genetics to map a metamorphosis related-locus(q-10M)in the male linkage group(LG10M),a small interval of 0.9 cM corresponding to a 1.8M bp physical area in chromosome 9 in the Chinese tongue sole.Combined with single marker analysis,ribosomal protein S6 kinase 2(rps6kb2),a member of the family of AGC kinases,was identified as a novel metamorphosis-related candidate gene.The full-length cDNA sequence was 1827 bp with an open reading frame(ORF)of 1413 bp encoding 470 amino acids(aa).The phylogenetic analyses of rps6kb2 showed that C.semilaevis formed a monophyletic clade with P.olivaceus.The rps6kb2 expression patterns of larvaes from C.semilaevis and P.olivaceus during metamorphosis was determined by quantitative RT-PCR.The results showed that the gene was highly expressed in the larvaes at metamorphic climax stage.The whole-mount in situ hybridization analysis showed that the rps6kb2 gene distributed in all the tissues transforming during metamorphosis,including tail,jaw,eye and skin of larvae from C.semilaevis and the signal was widely distributed at metamorphic climax stage.The results suggest that rps6kb2 has a general role in tissue transformations during flatfish metamorphosis including tail changes,skull remodeling,eye migration and asymmetrical pigmentation.3.Genomic selection for resistance against Edwardsiella tarda in Japanese flounderWe took the disease resistance to E.tarda as the phenotype of genomic selection in Japanese flounder.The challenge experiment was last for 3 years from 2013 to 2015 to collect the reference population.Meanwhile,DNA of parent of Japanese flounder families in 2015 was quantified and genotyped by whole genome resequencing using HiSeq 2000 lllumina.As a phenotype used in the genomic selection calculation,the disease resistance to E.tarda was translated to 0-1 trait.The BayesC? method provided by R-package BGLR was used to proform the genomic selection.The genomic estimated breeding value(GEBV)of selection candidate was calculated as the sum of all marker effects according to its marker genotypes.To verify the accuracy of genomic selection,correlation analysis between GEBV of differnet groups and the real preference has been done.The correlation coefficient was 0.81,which proved the BayesC? method was feasible.The cross validation showed that the computation of genomic selection was repeatable.In addition,by the comparison between GEBV and the survival rate of offspring in the challenge experiment,correlation coefficient of male parent fish was 0.65 and the female parent fish was 0.73.The GEBV of male and female parents with high disease resistance was significantly higher than the average of the candidate groups.All these above indicated that the screening of candidate groups by the GEBV calculated from genomic selection can effectively improve the speed and veracity of the breeding process.The conduction and application of a method of genomic selection in Japanese flounder on disease resistance to E.tarda will provide a guidance for "Ping You No.2"breeding and a new technology for disease resistant breeding in flatfish.4.Genomic selection for resistance against Vibrio harveyi in Chinese tongue soleWe chose 107 Chinese tongue sole families infected with V.harveyi that constructed in 2014 by our lab to collect the reference population,and genomic DNA was quantified and genotyped by whole genome resequencing using HiSeq 2000 Illumina.Based-on the sequencing result and the phenotype of reference population,the correlation coefficient between GEBV of diffemet groups and the real preference was 0.99 caculated by the BayesC? method.Afterwards,we chose the male fish from higher GEBV families in reference population as parent fish to conduct some Chinese tongue sole families in 2015 and use them as selection candidate.In addition,by the comparison of parent fish between the genetic selection and normal group,the survival rate in the challenge experiment and in the cultivation process was increased by 24.6%and 17.0%,respectively,which increased the disease resitance to V harvey.