| Pacific abalone(Haliotis discus hannai)is one of the important economic aquaculture shellfish in China.It has been expanded the culture range due to the southward migration of abalone in the north of China,and its aquaculture center has shifted from the north to Fujian province in the south,and the breeding scale has been unprecedentedly developed.Therefore,it is essential to explore the effects of a mass scale transfer of Pacific abalone to the south for culture and the exchange of juvenile abalones between different hatcheries in China on the genetic diversity and genetic structure of the pacific abalone populations in the past 30 years and to clarify the current status of germplasm resources of abalone in China,especially in the original area of Changshan archipelago.In this study,mitochondrial COI and Cytb genes were were selected for genetic variation analysis of six populations that were collected from Zhangzhou,Fujian(ZZ),Dalian,Liaoning(DL),Rongcheng Shandong(RC),and Changshan archipelago including Nanhuangcheng Island(NH),Daqin Island(DQ),and Tuoji Island(TJ),which supplemented the genetic information of Pacific abalone based on mitochondrial gene research.Then the development of EST-SSR genetic markers were carried out through the Pacific abalone transcriptome data,applicated in population genetic analyses,and the genetic variation and genetic structure of different populations of Pacific abalone in China were further analyzed.In addition,as the temperature in southern regions such as Fujian Province,the main producing area of Pacific abalone,is significantly higher than that in its natural habitat,the high summer mortality rate has become one of the main factors restricting the development of abalone culture.In order to explore the adaptability of Pacific abalone to high temperature,we took the northern Pacific abalone as the research object to selected the hemolymph tissue after high-temperature stress for 6 h,24 h,and 48 h for transcriptome sequencing.And some key differential genes were screened successfully.The result not only enriches the data of Pacific abalone germplasm resources in China but also further deepens people’s understanding of the molecular mechanism of Pacific abalone response to high-temperature stress.1.The genetic diversity and population genetic structure of six Pacific abalone populations were analyzed by using the mitochondrial cytochrome C oxidase subunit(COI)gene and cytochrome b(Cytb)gene.The results showed that 48 variation sites and 30 haplotypes were detected in the COI fragments of 730 bp.The haplotype diversity and nucleotide diversity of 6 populations were 0.586(NH)-0.897(TJ)and0.0056(DL)-0.0081(TJ),respectively.59 variation sites and 32 haplotypes were detected in the Cytb fragments of 730 bp.The haplotype diversity and nucleotide diversity of 6 populations were 0.605(RC)-0.909(TJ)and 0.0077(DL)-0.0120(TJ),respectively,showing the characteristics of high haplotype diversity and high nucleotide diversity.The results of Fst value and AMOVA between populations based on COI and Cytb genes showed that there was significant genetic differentiation among most populations(P<0.05),and the genetic variation mainly came from within populations.2.Eighteen polymorphic EST-SSR loci were successfully developed based on the hemolymph transcriptome data of Pacific abalone,and thirteen higher polymorphic EST-SSR loci among them were selected for genetic variation analysis of six populations The results showed that the average number of observed alleles was 8.0769(RC)-11.3848(DQ)in each population.The mean observed heterozygosities and expected heterozygosities were range 0.6818-0.9130 and 0.7581-0.8223,respectively.The number of observed alleles in the DQ,NH,and TJ populations was significantly higher than that in the RC population(H=14.755,df=5,P=0.011).If excluding NH and TJ from the cultured population,compared with the DQ wild population,the alleles of the cultured population decreased by 22.79%.The pairwise Fst values and analysis of molecular variance(AMOVA)revealed significant population differentiation among most populations,the population of RC and ZZ showed the largest population differentiation(Fst=0.1334).The UPGMA phylogenetic tree and structural analysis divided six populations into two groups(group1: NH,DQ,and ZZ;group2: DL,TJ,and RC),and there was no consistency between geographical distance and genetic distance.These results may reflect the mass scale transfer of Pacific abalone to the south for culture and the exchange of juveniles between hatcheries have caused gene drift,and different breeding conditions lead to a higher degree of genetic differentiation between RC and ZZ populations.In conclusion,this study provides valuable information for the genetic variation and genetic differentiation of Pacific abalone in China,it would contribute to the management and restoration of Pacific abalone resources,in particular,to the Pacific abalone origin area of the Changshan archipelago.3.Through the experiments on the feeding rate and oxygen consumption rate of Pacific abalone at different temperatures,we determined 28℃ as the temperature of heat stress.Then the hemolymph tissues of the heat stress group(28℃)and the control group(17℃)were sequenced after 6h,24 h,and 48 h.The results showed that 2213,2337,and 1420 differentially expressed genes(DEGs)were found after 4h,24 h,and48h of heat stress,respectively.The common DEGs of the three groups were 314.Thirteen DEGs were verified by the q RT-PCR experiment,and the expression trend was consistent with that of RNA-seq,which further verified the accuracy of transcriptome sequencing data.The results showed that DEGs were mainly involved in high-temperature adaptation-related pathways such as antigen processing and presentation and endoplasmic reticulum protein processing,including HSPs,GRP78,ERP29,CALR,and other genes to maintain cellular protein homeostasis,and immunerelated TLR6,MyD88,and NFIL3 genes were significantly up-regulated,which may be used to resist pathogen infection. |
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