Using EQTL Mapping To Reveal Expression Regulation Mechanism Of Temperature-and Salinity-adaptive Critical Genes In Oyster

Global climate change has been intensifying under human industrial activities since 1950 s.It leads to the continuous rising of ocean temperature and the larger difference in ocean salinity.Ocean with high salinity is becoming saltier,while ocean with low salinity is becoming lighter.Coastal ecosystems,such as intertidal and estuary zone,are more susceptible to human activities and climate change,where the temperature and salinity fluctuate wildly.It is one of the harshest ecosystems on the earth.Marine organisms living there are facing greater selective pressure.As the representative species of intertidal and estuary zone,oyster has important both economic and ecological value.However,worldwide wild germplasm resources have declined sharply in recent years.Aquaculture was also troubled by summer large-scale mortality for many years.Therefore,breeding highly adaptive oysters is the urgent demand for germplasm conservation and aquaculture.Phenotypic plasticity and genetic variation are two significant strategies to cope with environmental changes for sessile marine organisms.As the bridge linking genetic variation and phenotypic plasticity,gene expression plays an important role in regulating the formation of adaptive phenotypes.The effect of genetic variation regulating gene expression(regulatory variation)on phenotypic plasticity is usually dependent on environment.However,the genetic regulatory mechanism of genotype-environment interaction(G × E)was poorly understood in marine organisms.Therefore,we study the genetic basis of G × E by studying the genetic regulation mechanism of gene expression.For identifying vital regulatory variations in adaptive response and predicting adaptive potential of oyster under global climate change,we carried out the following studies:1.Linkage mapping of high-temperature responsive critical genes based on a genetic mapCrassostrea gigas and Crassostrea angulate,distributed in the northern and southern regions of China,are sister species with differentiation in temperature adaptation.They are ideal material systems to study the mechanism of temperature adaptation.The study was conducted in a pseudo-backcross family(ZF2-3)of C.gigas and C.angulate.The expression levels of 21 high-temperature responsive genes in 106 progeny were detected under heat shock(35 ℃,3 h).Expression quantitative trait locus(eQTL)mapping was conducted using a high-density genetic linkage map.A total of56 significant eQTL were mapped for 19 genes,including 164 distant-eSNP(distanteQTL SNP).The associations between vital eSNP and the corresponding target genes were verified in an independent family(ZF2).The results further confirmed the significant association between the three vital distant-eSNP and the corresponding target genes.It is suggested that they regulate the expression level of target genes in a trans-acting manner.In other words,they influence the transcriptional activity of target genes by regulating the expression of eGenes(genes with an eQTL).Specifically,Marker13973 for HSPA9 was located in approximately 2 Kb upstream of Ribosomal protein L10a(RPL10A),which may interact with HSPA9.Marker14346-48 and Marker14346-85 for ATG7 were located in approximately 3 Kb downstream of nuclear respiratory factor 1(NRF1),which can bind to the promoter region of ATG7 for regulation.The distant-eSNP may regulate the expression level of target genes(HSPA9and ATG7)by influencing the transcription of eGene(RPL10A and NRF1).The distanteSNP identified in this study are potential regulatory variants for high-temperature adaptation and can be applied to genetic improvement of heat-tolerant oysters.2.Genome-wide association analysis of temperature-and salinity-adaptive critical genes based on liquid chip(1)The development of liquid chipCrassostrea ariakensis is widely distributed along northern and southern estuary regions of China,where temperature and salinity have differentiation.The northern and southern C.ariakensis populations occur adaptive differentiation.Population genetics and transcriptome analysis identified some selective genomic regions and temperatureand salinity-adaptive genes.In this study,we developed a 30 K single nucleotide polymorphism(SNP)targeted genotyping liquid chip for C.ariakensis,which covered selective genomic regions in adaptive divergence of northern and southern oysters and the gene body and upstream/downstream regions of candidate temperature-and salinity-adaptive genes.The target genomic regions were enriched by hybridization capture using biotin-labeled double-stranded DNA(dsDNA)probes,and sequenced with high depth.Target SNPs were extracted and genotyped.A total of 24,541(81.8%)high-quality SNPs were successfully genotyped in 655 samples.Most of the 30 K SNP were located in intergenic and regulatory regions,whereas 7% were located in coding region.These high-quality SNP were located in 953 genes,including 100 heat-and 348high-salinity-responsive genes.Compared to the genotypes obtained from previous genome-wide resequencing,74.2% of theSNPs had a consistency rate of 100%,with an average consistency rate for each sample of 97.8%.Population structure analysis showed three divergent populations(northern,southern,and middle)on the coast of China,consistent with the results using whole-genome resequencing data.The chip provided favorable supports for the quantitative genetics and population genetics in C.ariakensis.(2)Genome-wide association analysisBased on the above genotyping results of 256 southern estuarine oysters(TSG3)that were domesticated in the north for two generations,we conducted genome-wide mapping of eQTL for 23 temperature-and salinity-adaptive genes to reveal the regulatory mechanism of gene expression.We identified 1,194 eQTL eSNP in eight genes,including 433 local-eSNP and 722 distant-eSNP.The expression variation explanation(EVE)of local-eSNP(9.95%)was higher than that of distant-eSNP(9.15%).Clustered local-and distant-eSNP with high linkage disequilibrium(LD)were identified for TRAF7,SLC6A5,GGT,and DAP3.Among which,we identified a localregulatory LD block containing 68 local-eSNP and a distant-regulatory LD block including 20 distant-eSNP in TRAF7.Some vital eSNP were further validated in an independent population(NSS),and several reliable regulatory variants were identified.There was a local-eSNP located in about 1 Kb upstream region of TRAF7 that may regulate TRAF7 transcription by influencing promoter activity.The two distant-eSNP of TRAF7 were located 4 Kb downstream of the FBXL4.They may influence the expression level of TRAF7 by affecting FBXL4 in a trans-acting manner.3.G × E analysis for critical eSNPIn this study,we performed genome-wide mapping of eQTL for 89 1-year-old oysters(TSG3)culture in northern environment and made comparisons with the results of southern environment.Eleven shared local-eSNP in BAAT and SLC6A5 were obtained but no distant-eSNP,which implied that local-eSNP were highly conserved,whereas distant-eSNP were environment-specific.In addition,we analyzed G × E for the 38 vital eSNP and two shared tag local-eSNP identified above.A high proportion(85%)of the eSNP exhibited significant G × E effects.By constructing mix-linear model of G,E and G × E and drawing gene expression reaction norms,two patterns of G × E were identified including 21 crossing and 19 nonparallel patterns.This study provides insights into adaptive evolutionary mechanisms and phenotypic response prediction of marine invertebrates to variable environments.4.Studies on the function and regulatory mechanism of CaTRAF7TRAF7 is the last member of the tumor necrosis factor(TNF)receptor-related factor family.Previous studies have confirmed that CaTRAF7 is strongly selected in northern and southern environmental adaptation.We conducted high-temperature(41℃)and high-salinity(60‰)shocks in southern population,respectively.The results showed that CaTRAF7 was significantly up-regulated after heat shock for 8 h and reached the highest expression level at 24 h.But there was no significant change under high-salinity stress.To further study the function and regulation mechanism,we firstly cloned the CDS sequence and 2 Kb upstream regulatory region of CaTRAF7.The total length of CDS sequence is 2064 bp,encoding 687 amino acids.CaTRAF7 of C.ariakensis lacks the zinc finger domain compared with human,but has an extra serinerich low complexity region(LCR)at the N-terminal.The transcriptional activity of NF-κB was decreased after CaTRAF7 was overexpressed in HEK293 T cells.In addition,we knocked down CaTRAF7 using si RNA.The results showed that expression levels of apoptosis-related genes were significantly down-regulated,which suggesting that CaTRAF7 involved in caspase-mediated apoptosis pathway in estuarine oyster.Furthermore,we detected promoter activity of upstream regulatory regions of CaTRAF7 for southern and northern oysters using dual-luciferase reporter system.The results showed that the promoter activity of southern estuarine oyster was significantly higher than that of northern oyster.We also identified a local-regulatory regions with high LD,including 15 significant SNP and 7 INDEL,four of which were close to the eSNP obtained by single-locus association analysis.In conclusion,we firstly investigated the regulation mechanism of temperatureand salinity-adaptive genes,such as HSPs and TRAF7,at the whole-genome level.We initiatory revealed the genetic regulation basis of G × E and identified vital local-and distant-regulatory variants.In addition,the function and regulatory mechanism of CaTRAF7 were further studied.The results in the study can provide a theoretical basis for the assessment and prediction of adaptive potential of marine organisms under global climate change,the restoration of oyster resources and the breeding of highly adaptive populations.