Exploring The Genetic Basis Of Hypoxia Tolerance Trait In Tilapia

Tilapia(Oreochromis niloticus)is native to Africa,shaped like a crucian carp,so it is also called Africa crucian carp.Tilapia is cultured as one of the key freshwater fish of the world aquaculture,and it is regarded as one of the main source of future animal protein.Tilapia has strong fertility and environment adaptability.Low dissolved oxygen(hypoxia)can cause serious damages to aquatic organisms and even the entire aquatic ecosystem,and it is one of the most serious challenges to aquaculture.However,the molecular mechanism of fish hypoxia adaptation is still not very clear.Tilapia solved well to hypoxia environment,and it is considered as a perfect material for hypoxia research.We used tilapia to briefly analyze hypoxia trait genetic basis from three aspects.First,for some hypoxia conserved candidate genes,HIF1? and its inhibitors HIF1?n,we cloned the whole CDS sequences and analyzed their structures and functions.Then,we explored transcriptome-wide differentially expressed genes and differentially expressed exons.And we also identified and analyzed genome-wide hypoxia trait QTLs in a Nile tilapia family.The main results are as follows:1.Clone,structure and function analysis of hypoxia relevant candidate genes.We cloned and sequenced the whole length of HIF1? and HIF1?n CDS sequences,and analyzed the conserved structures of HIF1? and HIF1?n gene and protein sequences.Then we identified 21 and 2 amino acid sites significant associated with aquatic and terrestrial phenotype by SigniSite 2.1 Server on HIF1? and HIF1?n protein sequences,respectively.Positive selection analysis of HIF1? and HIF1?n with PAML found these two genes do not exist strong positive selection effect,but some sites on HIF1? sequence truly suffered selection pressures.Spatial and temporal expressions of HIF1? and HIF1?n mRNA revealed that,HIF1? gene has different expression patterns in different tissues and different hypoxia exposure time.While the expression patterns of HIF1?n gene in hypoxia were generally increased.2.Transcriptome-wide differential expressed genes and exons identification and function analysis.Hypoxia treatment for 12 h heart and gill tissues of tilapia were transcriptome sequenced.Cuffdiff was used to identify differential expressed genes and exons between hypoxia treatment group and control group.We identified 284 differentially expressed genes and 103 alternative spliced genes in heart tissue and 239 differentially expressed genes and 34 alternative spliced genes in gill tissue.We successfully validated the differential expressions of almost all selected genes and exons with fluorescence quantitative PCR experiment.GO annotation and Pathway analysis significantly associated these genes with the categories related to energy conservation,protein synthesis and immune response.Interestingly,we found different enrichment categories(GO,pathway and protein class)within and between the differential expressed genes and alternative spliced genes categories,which shows the different roles of these two kind of genes in hypoxia.3.Tilapia hypoxia trait genome QTL analysis.Using two high throughput sequencing methods,QTLseq and ddRADseq,we analyzed the tilapia hypoxia trait genome QTL.These two methods both identified QTLs in LG3 and LG14 associated with hypoxia significantly.To verify the accuracy of the identified QTLs,we developed four microsatellite markers in the QTL regions of LG3 and LG14.Each of the two QTLs of the LG3 and LG14 has one marker verified the significant differences of phenotype and genotype.We focused on the LG3 and LG14 QTL regions,and found a hypoxia-related gene GPR132 in the QTL region of LG3 and another hypoxia related gene ABCG4 in the QTL region of LG14.Structure and function analysis of these two genes showed that,some SNPs in the exons of the two genes significantly associated with hypoxia trait.And fluorescence quantitative PCR experiments also found the expressions of the two genes were associated with hypoxia.The results showed that,the two genes were very likely to associate with hypoxia.Through the adoption of various methods,we analyzed the genetic basis of hypoxia trait in tilapia.We identified tilapia hypoxia trait important genome QTL regions and candidate function genes and obtained molecular markers closely linked to hypoxia trait.This research laid a foundation for using molecular markers(or genes)assisted breeding technology to breed hypoxia tolerant new fish lines.