Validation Of QTLs Associated With Important Economic Traits Of Sea Cucumber(apostichopus Japonicus)

Seacucumber?Apostichopusjaponicus?belongstoHolothuroidea,Echinodermata as one of the important treasures in Northern China.In recent years,the culture of sea cucumber expanded rapidly and got 30 billion yuan outputs,which has became the aquaculture species with the largest value in China.The sea cucumber aquaculture has prompt great importance in the structure adjustment of aquaculture in China and provided approach for fisherman in increasing production and benefits.While with expansion of the industry,many problems appeared such as the genetic degradation,low growth rate,and the frequently breakout of disease,which has limited the development of aquaculture industry.Marker assisted selection?MAS?is an effective method to solve above problems.With the development of molecular techniques,genetic linkage map construction and marker assisted selection has become the hotspots in genetic breeding of aquatic animals.Validation of QTL is the precondition of MAS.In this research,QTL associated with important economic traits based on high-density genetic linkage map has be selected and validated,then the markers were applyied for breeding practice.Thirteen SSR loci were used to parentage identification.All of these data could be used in marker assisted selection.All the results are showed as follows:1.Screen of SNPs associated with important economic traits in sea cucumberIn previous study,high-density genetic linkage map of sea cucumber were constructed in previous with 4002 SLAF labels.The associated analysis of SLAF labels with the 5 economic trait value?body length,body width,body weight,pallet number,and disease resistance?of the 142 offspring revealed 9 quantitative trait locus?contains 81 SLAF labels?.One hundred and eight-nine SNPs has be selected from841 SNP in 81 SLAF which associated with traits.Further association using the genotype selected 79 candidate SNPs,26 of them can be genotyping use high resolution melting.Loci number associated with body length,body width,body weight,pallet number,and disease resistance were 8,14,9,8,18,respectively?15sites associated with multiple traits?.Fourteen pairs of primers were designed successfully using sequence of SNP which can be used for small fragment of HRM.Genotyping analysis showed 13 SNP could be genotyped successfully using HRM.2.Validation of SNPs associated with important economic traits in large populationThirteen candidate SNPs associated with important economic traits were genotyped and analyzed of phenotypism in expand population to validate QTLs.Result showed 3 loci were monomorphic sites and the other 10 were polymorphic site,Minor allele frequency?MAF?of the 10 polymorphic sites ranged from 0.013?SNP113?to 0.322?SNP160?with an average of 0.173.Observed heterozygosity?Ho?ranged from 0.031?SNP113?to 0.818?SNP9?with an average of 0.433.Expected heterozygosity?He?ranged from 0.03?SNP113?to 0.834?SNP160?with an average of 0.402.The polymorphism information content?PIC?value ranged from 0.030 to0.393 with an average of 0.284.Six loci depart from Hardy-Weinberg equilibrium.The results of QTL verification indicated that locus SNP40 and SNP160 associated with growth traits?body length,weight,body width?with the dominant genotypes SNP40?CC?,SNP160?AA?.SNP88,SNP112 and SNP126 are associated with disease resistance.The dominant genotypes is SNP88?CC?,SNP112?AA?,and SNP126?TT?respectively.Diplotypes were constructed based on the five SNPs and association analysis revealed that S₁?CC AA?,S₃?CC AC?are dominant diplotypes for growth traits,K₁?CC AA TT?is best for disease resistance.3.Validation of SNPs associated with important economic traits in selecting breedingParental sea cucumbers were genotyped and classified using the 5 SNPs associated with growth traits or disease resistance which has been validated in large population.Parental sea cucumbers carried quick growth or low growth SNP markers and disease-resistant or disease susceptible SNP markers were selected and using to construct potentially faster growth breeding?marked A?,slower growth breeding?marked B?,disease resistant breeding?marked C?and disease susceptible breeding?marked D?.The body weight of breeding A and B were measured monthly from5-month old to 8-month old.The survival rate of breeding C and D challenged with Vibrio splendidus were tested at 8-month old.Results showed that:the proportion of genotypes?CC AA?and?CC AC?were 74%and 26%respectively in breeding A,4genotypes were detected in breeding B with the proportion of?CT CC?,?CT AC?,?CC AC?and?CC CC?were 32%,20%,10%,and 32%respectively.The average weight of breeding A are higher than breeding B with 24.32%,65.69%,304.40%,and182.16%higher at 5-month,6-month,7-month and 8-month respectively.All genotypes of the offspring in breeding C was?CC AA TT?.Five genotypes were got in offsprings of group D constructed by?CT AG TG?×?CT AG TG?parents.The proportion of the 5 genotypes?CT AG TG?,?CT AG GG?,?TT AG TG?,?TT AG GG?and?CT GG TG?were 15%,15%,11%,9%and 12%respectively.The cumulative survival rate of breeding C was 47.06%higher than that of group D after challenged by high concentration of Vibrio splendidus.All the results suggested that the seedlings bred using dominant genotypes parents could got performant performance in terms of growth traits or disease resistance.4.Application of SSR markers in parentage identification in sea cucumberTwenty-three parental sea cucumbers?13 females and 10 males?were selected and used for seed breeding.Thirteen SSR markers were used for parentage identification in sea cucumbers.72 individuals were randomly selected for genotyping and paternity.Allele frequency analysis showed that there are totally 121 alleles in 13loci with an average of 9.31.Ho,He,PIC values ranged from 0.281⁰.719,0.316⁰.87,0.304⁰.853 with the mean valued were 0.564,0.683,0.648 respectively.There were 9 higher polymorphic sites and 4 medium polymorphic site of 13 loci.Loci AJ09 depart from Hardy-Weinberg equilibrium,loci AJ11 depart from Hardy-Weinberg equilibrium significantly.The value of the total exclusionary power of 13SSR loci were 0.997,0.999 and 0.999,respectively.Sixty-eight offsprings could found its most like parents at 95%confidence level using simulations analysis.The successful rate of parentage identification with 94.4%.As deleting sites following PIC value from high to low,exclusion probability revealed the minimum loci that needed for the identification were 7-9 loci and 6 loci respectively,when single parent were known and both parents were known.As deleting sites following PIC value from low to high,3-4 loci and 2-3 loci were needed when single parents were known and both parents were known.Exclusion probability increased with PIC value increased.Based on the data of the 68 succeed identified offspring,at least 7 SSR loci were needed for complete paternity test.All the results could provide basic data for the identification of parentage and selection breeding.