Genetic Linkage Map Construction And QTL Mapping And Cloning And Expression Of Heat Shock Protein 22 Gene In The Pearl Oyster Pinctada Fucata

The pearl oyster,Pinctada fucata,is the main shellfish producing seawater pearls in the world.It's widely distributed in the main tropical and temperate oceans of the Pacific Ocean,Indian Ocean and Atlantic Ocean.It is also an important mariculture species in China.Its cultivated pearls are known as "South Sea" pearls and have extremely high economic value.The selective breeding research of P.fucata has undergone a period of more than 10 years,and has achieved fruitful results.With the continuous development of modern biotechnology,genetic breeding of various aquatic animals,including P.fucata,has ushered in new perspectives and development opportunities.Compared with traditional family breeding,which requires multiple generations for more than 10 years,modern biotechnology can greatly accelerate the efficiency of breeding,and can also respond to the current situation of germplasm decline,aquaculture environment deterioration,etc.,and provide multiple levels of more accurate proposals for genetic breeding solutions.The genome-wide SNP genotyping technology can bring technological advancements and upgrades to the genetics and breeding work of P.fucata,which has broad research and application prospects.Therefore,this study took the pearl oyster P.fucata as the research object,and the research included(1)the development,annotation and individual genotyping of large scale SNP markers based on simplified genome technology Genotyping by Sequencing.(2)using the developed SNP markers to construct high density genetic linkage map and combining the collected phenotypic information of the family to carry out QTL localization and analysis.(3)cloning the heat shock protein gene HSP22 of P.fucata,and studying the effect of carotenoids on expression of HSP22 gene under high temperature stress.The main results are as follows:(1)Sequencing libraries of 978 individuals from the parents and corresponding progenies of the constructed 10 P.fucata family lineages were constructed based on GBS simplified genome technology and sequenced using the Illumina high-throughput sequencing platform HiSeq X Ten.The statistical results showed that the average number of tags obtained for all samples was 312,008,and the average sequencing depth is 13�,and the coverage of the genome ranged from 0.84% to 12.50%.The sequencing data of all the samples of P.fucata was genotyped and a total of 129,584 polymorphic SNPs were obtained.The annotation results showed that the overall variation rate was 1 variant every 4,875 bases;(2)The genetic linkage map of A7 family was constructed.The number of SNP mapped markers was 2,485.A total of 14 genetic linkage groups were generated.The total map length was 1,697.01 cM.The average marker interval of all linkage groups was 0.69 cM;QTLs were mapped to 9 traits,and thirteen major QTLs related to 7 traits were mapped.The LOD value of each QTL ranged from 3.57 to 5.78,which explained that the range of phenotypic variation ranged from 17.0% to 26.1%.(3)The cDNA sequence of heat shock protein 22 gene(GenBank accession number:MG013985,named PfHSP22)from the pearl oyster P.fucata was obtained.The full length of PfHSP22 cDNA was 2,187 bp,including a 699 bp ORF(open reading frame)which encoded 232 amino acid residues.The qRT-PCR(quantitative real-time PCR)analysis revealed that the PfHSP22 gene was expressed in various tissues of orange adductor muscle individuals with high carotenoids content and white adductor muscle individuals with low carotenoids content,the highest expression of PfHSP22 was observed in foot,and the expression of PfHSP22 in orange group were significantly higher than that in white group at normal temperature(P<0.05).During the high temperature stress process,the expression of PfHSP22 in each tissue generally showed a downward trend in the first 3 hours,and then showed a complex change of increase of decrease.There were several tissues whose time for PfHSP22 gene expression to rise significantly is later in the orange group than in the white group,which may be caused by differences in carotenoid content.This indicated that the level of carotenoids may affect the basic expression of PfHSP22 gene.These results provide a basis for a further study on the interaction between carotenoids and HSP22 gene under high temperature stress.This study is an application of GBS simplified genome sequencing technology to the development of large-scale SNP markers in P.fucata,and it is the first genetic linkage map of P.fucata based on GBS.The 13 main QTLs we targeted can be used for subsequent screening of key genes,which has important guiding significance for the breeding work of P.fucata.This study obtained the full sequence of HSP22 gene from P.fucata,which lays the foundation for the study of HSP22 protein function;and explored the effect of carotenoids on the expression of HSP22 gene,which can provide a theoretical basis for the breeding of orange-colored adductor muscle family in P.fucata.