| Pinctada fucata martensii is one of the main species for marine pearls cultivated in China.It belongs to warm water shellfish and holds the ability for tolerance to low temperature.Natural groups are mainly distributed in the south sea area of Shenzhen.There have been many winter cold waves that have led to large-scale deaths of the pearl oyster,and have limited the breeding areas of the pearl oyster.Therefore,the cultivation of low-temperature-resistant population is the prerequisite for the development of the northward migration of pearl oyster.Our lab has bred a low temperature resistant line(R)of pearl oyster.Compared with the base population,the low-temperature resistant ability of the selected line is significantly improved.In this study,genome resequencing technology was used to compare the genome of the F3 generation of low-temperature resistant lines and Beibu Gulf wild population(W)and to establish a genomic variation information database for screening positive selection signature between the breeding line and wild population.This study is significant in exploring the molecular mechanism of the breeding progress of low-temperature-tolerant selection line,and to accumulate basic data and theoretical basis for the development of molecular marker-assisted breeding to shorten the breeding progress.The specific results of the study are as follows:1.A total of 1317.5G of effective data were obtained from genome resequencing.The proportion of bases with a mass value greater than 20 in the total reads length was greater than 94.5%.The comparison rate of all samples was between 96.96% and 98.28%,and the effective sequencing depth was between 12.30 x and 21.24 x.A total of 40,599,399 SNPs were obtained,of which 13,646,863(33.61%)SNPs were located in m RNA,1,843,887(4.54%)SNPs were located in CDs region,18,310(0.045%)SNPs may affect gene or protein function.A total of 12418755 indels were detected,of which 3,643,503(0.29%)were located in m RNA and 56600(0.46%)in CDS.A total of 9,767,962 SV were detected in 60 individuals,of which 29,906(0.31%)SVS were located in m RNA and 9,468,356(96.93%)SVS in CDS.2.Comparative analysis of the two populations showed that the R population showed a positive significance.There are 636 genes in the selected region,and these genes may improve the low temperature tolerance of the R population by improving cell membrane fluidity,regulating energy metabolism,regulating the gene transcription and translation which intends affect the degradation of proteins and apoptosis.In combination with literature to further explore the molecular processes and related functions of these genes,we found 26 key genes related to the low temperature tolerance of P.f.martensii,which are ABCC1,ABCC6,DGAT1,GPAT,ITGB,ROCK2,dynein,insulysin,MDH,V-ATPase-d,JAK2,CBP,PIAS2,SETD8,AFF4,TBP,AP1,POLR2 A,e IF3,UBE2G2,TRIM71,MID1,Cullin1,Cullin4,BAX and BIRC3.3.A total of 13896 SNPs were obtained from the 26 key gene SNP loci screened out,of which 1517(10.91%)and 1367((9.84%))SNPs were located in the promoter and exon respectively.7 Genes have no SNP sites in the exon region.The genetic polymorphism and linkage disequilibrium analysis of SNP loci located in the exon region of the gene showed that the 26 gene SNP loci belonged to moderate polymorphism and low polymorphism,no high polymorphism.Hardy-Weinberg equilibrium analysis results show that most of the SNP sites in the gene are consistent with Ha-temperature equilibrium(P> 0.05)and linkage disequilibrium analysis showed that there is no random association between SNP.4.To analyze the SNP loci with significant differences between the two populations of the above 26 gene exon regions(p <0.05),we performed haplotype analysis on these loci.The results show that Pm-ABCC1 gene block2 genotype GTG,block4 genotype ATC,block5 genotype GTGT and GCGT,block6 genotype TAGTT,block7 genotype AG,block8 genotype TGGTGATA,block9 genotype CC;Pm-ABCC6 genotype TT;Pm-dynein gene genotype CAA;Pm-insulysin gene genotype AA;Pm-V-ATPase-d gene block 1 genotype CTG,block 2 genotype TG and block 3 genotype AG;Pm-CBP gene block1 genotype TC,block2 genotype TAAGCCT and CGAAATC,block3 genotype AT,block4 genotype CGAA and GAGG;Pm-PIAS2 gene genotype GA;Pm-UBE2G2 gene block1 genotype GGA,block2 genotype TA,block3 genotype GAT;Pm-TRIM71 gene small genotype GC;Pm-cullin1 gene block 1 genotype TT and block 2 genotype ATGGCAC;Pm-IAP1 gene block1 genotype AGTTGCCTATCTT,block2 genotype AA,block3 genotype GTC,GCC,CCT were excellent type for low temperature tolerance.5.Based on the data of expression library of P.f.martensii under low temperature stress,the expression levels of 6 genes(Pm-CBP,Pm-AP1,Pm-PIAS2,Pm-TRIM71,Pm-cullin1 and Pm-V-ATPase-d)among the 26 key genes were significantly different between the temperature-AP1 propriate group and the low-temperature group,respectively.The full-length of genes(Pm-AP1,Pm-PIAS2,Pm-cullin1 and Pm-V-ATPase-d)were obtained by rapid amplification of c DNA ends(RACE).q RT-PCR results showed that all 4 genes were expressed in the detected tissues,and the difference was significant(P<0.05).The time-series expression results in gill under temperature stress showed that 4 cold-tolerant genes showed higher expression under low temperature stress(P <0.05). |
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