Application Of The Liquid Chip "Yellow Sea Chip No.1" In The Selective Breeding Of Resistance On AHPND In Litopenaeus Vannamei

Litopenaeus vannamei is a widely cultured shrimp species all around the world,with a huge production per year.However,diseases in the farming process have become more and more threatening.Acute Hepatopancreatic Necrosis Disease（AHPND）is one of the main diseases.In1990,the disease first appeared in China,and then the global direct economic loss of shrimp shrimping by AHPND was more than 10 billion a year.It often occurs during the early stage of culture.AHPND has the characteristics of rapid spread and high mortality,so it is difficult to fundamentally restain the infection of Vp _AHPND through culture management.Therefore,improving the resistance to AHPND of Litopenaeus vannamei by selective breeding is an important way to reduce the impact of the disease on industry.However,only half of additive genetic variation is used in the traditional methods based on the sib testing,which seriously effects the selection accuracy of resistance to AHPND.It has been reported that genomic selection（GS）is an effective method for improving disease resistance.For aquaculture species like shrimp,the key benefit is that GS enables prediction of individual GEBVs for non-phenotyped shrimp,and hence exploits with-family genetic variation to further increase the genetic gain of disease resistance traits.In this study,the self-developed liquid chip“Yellow Sea Chip No.1”（40K）was used to high-throughput sequence of Vp _AHPND infected individuals,and resistance to AHPND was evaluated by ss GBLUP and ss BR using both pedigree and genomic information,which further improved the selection accuracy of AHPND resistance.The main works of this thesis are listed as follows.（1）It was analyzed that AHPND difference in resistance and genetic diversity among different populations of Litopenaeus vannamei.To evaluate the application potential of SNP chip in the construction of the base population for breeding,three different populations（EE,PP and SS）of Litopeneause vannamei were challenged by Vibrio parahaemolyticus(Vp _AHPND),and the genomic information of 146 samples was obtained by using the independently developed 40K SNP liquid chip“Yellow Sea Chip No.1”.When the overall mortality was more than 50%,the survival rate of PP population was 12.9%and 11.6%higher than that of EE and SS populations,respectively.The phylogenetic tree was constructed by using 38148 SNP markers after quality control.The result showed that the individuals of the same family first clustered together,and then the multiple families of the same population clustered together.Principal component analysis and genetic structure analysis also implied that the 146 individuals could be accurately divided into 3 groups,which was consistent with the phylogenetic tree.The analysis of genetic diversity for three populations showed that the average value of observed heterozygosity（H_o）was 0.25-0.29.The average value of polymorphic information content（PIC）was 0.20-0.23.The differences in the above three parameters between three populations were very significant（P<0.01）.The coefficient of genetic differentiation(F_ST)among the three populations was 0.11-0.21,which indicated that there was medium-high genetic differentiation among the three populations.Genomic inbreeding analysis showed that the average inbreeding coefficients of EE,PP and SS populations were-0.05±0.06,0.20±0.09 and 0.37±0.07,respectively.Some individuals of PP and EE populations showed a high level of inbreeding.Using ss GBLUP-MF（Single Step Genomic BLUP with Metafounders）model with genotype,phenotype and pedigree information,the estimated heritability of survival after Vp _AHPND infection was medium（0.24±0.07）,indicating that the trait has a high breeding potential.Taken together,the efficiency of constructing and evaluating the base population can be further enhanced by using the liquid chip"Yellow Sea Chip No.1".（2）Analysis of selection accuracy of resistance to AHPND in a reference population of Litopenaeus vannamei.A total of 1200 shrimp from 40 families were infected with Vp _AHPND.Resistance to Vp AHPND was measured as survival time,and phenotypic records were collected for a total of 762 shrimp.Of those shrimp,447 shrimp was genotyped by liquid chip“Yellow Sea Chip No.1”.The variance components of AHPND resistance of the reference population were predicted with methods of p BLUP based on a numerator relationship matrix（Matrix A）and ss GBLUP based on a combined pedigree and genomic relationship matrix（Matrix H）and Bayes.GEBV（EBV）of resistance were predicted by 8 methods including p BLUP,ss GBLUP,Bayes A,Bayes B,Bayes C,ss BR-A,ss BR-B and ss BR-C,and accuracies of GEBV（EBV）were calculated by five fold cross validation.The result showed that the estimated heritability of AHPND resistance used p BLUP and ss GBLUP methods were 0.17±0.063 and 0.25±0.077,respectively.The accuracies of prediction used by p BLUP,ss GBLUP,Bayes A and ss BR-C were 0.15,0.18,0.20 and 0.27,respectively,and the highest prediction accuracy was obtained by ss BR-C,which was 80% higher than that of p BLUP.To evaluate the accuracy of genomic prediction in close practical situations,2 individuals were randomly selected from each family as the validation population,and then the rest of the individuals of same family were used as the reference population.The prediction accuracy results show that the prediction accuracy obtained by p BLUP,ss GBLUP Bayes A and ss BR-C is 0.15,0.18,0.20,0.27 respectively,in which ss BR-C is 80%higher than that of p BLUP.In sum,these results suggest that genomic selection has the great potential to accelerate the AHPND resistance of Litopenaeus vannamei.Genomic wide association study（GWAS）has been used to detect the markers associated with AHPND resistance.When P<0.0001 was used as the genomic significance standard,9 SNP markers associated with AHPND resistance were screened,which explained 38.36%phenotypic variation,of which 3 loci were mapped to 34 linkage groups.When P<0.00001 was used as the genomic significance standard,4 SNP markers associated with AHPND resistance were screened,which explained 19.57%phenotypic variation,of which 2 loci were mapped to 21 linkage groups.（3）Analysis of genomic selection effect of AHPND resistance in candidate Litopenaeus vannamei.In 24 sibs families of Litopenaeus vannamei,5 meal and 5 female shrimp were selected to construct a candidate population.Genomic information of 238 shrimp was genotyped using the40K chip.GEBVs for AHPND resistance of candidate were predicted by ss BR-A method with genomic information and phenotypic information of reference population.To design and optimize breeding programs for producing families,high,medium and low GEBV broodstock were selected,and then 7 high resistance families,12 intermediate resistance families and 5 low resistance families were produced,and finally,offsprings of 24 families were infested.The result of infestation experiment showed that when over survival rate was 50%average family survival rates of high,medium and low groups were 69%,61%and 31%,respectively.When all individuals died,average survival times of individuals in high,middle and low resistance groups were 568.59h,496.94h and 256.47h,respectively.Thus,Using GEBV to guide breeding can produce families with high AHPND resistance and further increase the genetic gain of AHPND resistance in core breeding population.