Screening Of Low Temperature Tolerance Markers And Transcriptome Analysis Of Takifugu Rubripes

Takifugu rubripes(Takifugu rubripes)is an important mariculture fish,belonging to the order Telraodontiformes,family Tetradontidae and genus Takifugu.It is a warm-temperature and broad-salt benthic carnivorous fish.It is mainly distributed in the coast of Japan,in the Korean Peninsula,Russia and China’s Yellow Sea,Bohai Sea and East China Sea.It is an important fish in North China’s mariculture and export to earn foreign exchange.Low temperature tolerance is an important trait and a target for genetic improvement of Takifugu rubripes.Molecular marker technology is one of the main tools for genetic improvement.This paper completed the screening of microsatellite molecular markers for low temperature tolerance of Takifugu rubripes.Based on a large number of genetic populations,the genetic linkage map of high density Takifugu rubripes was constructed,and the QTLs for low temperature tolerance traits were mapped.The transcriptome of Takifugu rubripes in normal temperature group and cold stress group was sequenced,the related pathways were analyzed,and some genes were detected by fluorescence quantitative PCR.This study provided a theoretical basis for the breeding of low temperature tolerance traits and the study of molecular mechanism of Takifugu rubripes.Specific research contents are as follows:1.SSR combined with BSA technology was used to screen low temperature tolerance related microsatellite markers for Takifugu rubripes population.Firstly,300 juvenile rubripes were treated with low temperature,and 34 individuals with low temperature tolerance(group S)and no low temperature tolerance(group D)were obtained,respectively.Genomic DNA was extracted from 15 animals randomly selected from the two groups,and mixed pools of cold and cold resistant DNA were constructed.Then 148 pairs of microsatellite primers were used to scan them.The results showed that four markers(fms45,fms82,fms100 and fms182)amplified different bands in the mixed pool of cold and cold tolerant DNA.The results showed that the frequency of individuals amplified by fms100 with 116 bp bands in group S and group D was 53% and 18%,respectively,and the frequency of 132 bp bands in group S and group D was 59%(group S)and 24%(group D),respectively.The frequency of the 125 bp individuals amplified by fms182 in group S and group D was12% and 35%,respectively.The P values of the chi-square test were all less than 0.05,indicating a significant difference.2.A high density linkage map of Takifugu rubripes was successfully constructed by collecting 138 pedigree samples,with a length of 3164.81 c M and a coverage rate of 99.46%.The male map contained a total of 2788 Binmarkers,with a total length of2526.551 cm,an average length of the linkage group of 114.85 cm,and an average marker interval of 0.95 cm for the male map.The female map contained a total of1807 Binmarkers,the length of which was 1931.653 c M,the average length of linkage group was 87.80 c M,and the average marker interval of the female map was 1.12 c M.Except for LG1 markers,other linkage groups were relatively uniform.In this study,1,339,627 SNP loci were detected,and a total of 6 QTL regions were found to be associated with low temperature tolerance traits.They were located at LG7,LG10,LG15,LG16,and LG22,respectively,among which LG10 had the largest LOD value(5.87),and 16 SNP loci related to low temperature tolerance were screened.3.The culture of Takifugu rubripes is easily affected by temperature,and the study of the key regulatory genes of low temperature stress is of great significance for the breeding of Takifugu rubripes with low temperature tolerance.RNA-seq was performed by Illumina sequencing technology to screen out differentially expressed genes(DEGs)from kidney tissues and heart tissues of puffer rubripes at 23℃ and5℃,respectively.The results showed that a total of 1679 DEGs were screened in the kidney tissue,among which 635 DEGs were up-regulated and 1044 DEGs were down-regulated by low temperature relative to optimal temperature.The GO functional classification and enrichment analysis of 1679 DEGs showed that a total of692 DEGs were annotated into 20 subclasses of the three GO functional classifications,mainly involving biological regulation,stress regulation,and catalytic activity.In the KEGG functional enrichment analysis,a total of 1206 genes were involved.It is mainly enriched in the metabolic pathways of glycine,serine and threonine,primary bile acid biosynthesis,arginine and proline metabolism,proteoglycan pathway and circadian rhythm pathway.A total of 919 DEGs were screened in heart tissue,of which 341 DEGs were up-regulated and 578 DEGs were down-regulated by low temperature relative to optimal temperature.The GO functional classification and enrichment analysis of 919 DEGs showed that a total of692 DEGs were annotated into 20 subclasses of the three GO functional classifications,mainly involving biological regulation,cellular processes,and catalytic activity.In the KEGG functional enrichment analysis,a total of 800 genes were involved.It is mainly enriched in glycosome biosynthesis related pathways,circadian rhythm pathways,RNA transport pathways and antigen-related pathways.The expression of 15 significantly enriched DEGs was analyzed by q RT-PCR,and 3genes involved in the antigen-associated pathway of heart were found to be up-regulated(P <0.05,heat shock 70 k Da protein 1,hspa4 a,hspa44b).Two genes involved in arginine and proline related pathways in kidney were significantly up-regulated(P <0.05,odc1,nos1).Two genes involved in kidney circadian rhythm were significantly upregulated(P <0.05,gsk3 bb,per3).