Mechanism Of Heterosis In Hybrid Tilapia, Oreochromis Niloticus ♀ × O.aureus ♂

Heterosis has been widely applied in improving growth rate,aquaculture production and fish flesh quality for domestic aquaculture breeders.However,the molecular mechanism of heterosis is unclear yet.As a hybrid fish species,hybrid tilapia "Auni"(Oreochromis niloticus × O.aureus)has many advantages,such as fast growth performance,high male ratio,strong tolerance to low oxygen,temperature and bacterial disease.So it is popular with aquaculture companies and farmers and has become an important tilapia variety in aquaculture.Hybrid tilapia "Auni" has several relative advantages compared with its parents,such as increased growth rate,stronger stress resistance and disease resistance,making it a valuable object for analysing fish heterosis.Currently,studies on the mechanism of heterosis in hybrid tilapia are limited to a few regulation gene expressions.There is a lack of overall and in-depth research on the mechanism.Therefore,this study was carried out from four aspects:(1)Analysis of multi-traits heterosis of hybrid tilapia.(2)Transcriptome basic on the heterosis of hybrid tilapia.(3)Molecular basis of miRNA on the heterosis of hybrid tilapia.(4)Role of specific expression of mapk14 a allele in the heterosis of hybrid tilapia.This paper mainly elaborates on the molecular basis of heterosis in growth,stress resistance and disease resistance of hybrid tilapia "Auni",which will be beneficial for fish breeding and the genetic analysis of animal heterosis.The results and main conclusions are presented as follows:1.Analysis of multi-traits heterosis of hybrid tilapia.Groups of Oreochromis niloticus(NL),O.aureus(AR)and hybrid tilapia "Auni"(HY)of the same age were established through artificial oxytocin and simultaneous incubation of fertilised eggs.The three groups were cultured in the same pond after seedlings cultivation and PIT(passive integrated transponder)electronic labelling injection.Comparative analyses of growth,metabolic,antioxidant and non-specific immune characters among the three groups were conducted to elaborate on the traits differences among NL,AR and HY.It was revealed that HY showed significantly improved phenotypic characteristics compared with NL and AR,in absolute body-weight gain rate,specific growth rate,body-length growth rate and body-height growth rate during the 90 days culturing period(p<0.05).The metabolic characteristics were significantly increased in serum glucose concentration in HY compared with NL and AR(p<0.05).Meanwhile,it was close to NL but considerably higher than AR in cholesterol concentration(p<0.05).In intestinal tissues,HY showed significantly increasing protease,lipase,amylase activities and cholecystokinin concentration than NL and AR(p<0.05);all four characters showed a trend of HY>NL>AR among the three groups.Moreover,HY showed improved characters beyond NL and AR,or close to the dominant expression of AR in antioxidant indices and non-specific immune parameters,such as catalase,superoxide dismutase,total antioxidant capacity,glutathione,glutathione peroxidase,acid phosphatase,alkaline phosphatase,lysozyme,immunoglobulin Ig M and Ig D.These results support a comprehensive description of heterosis in growth,stress resistance and disease resistance in HY.2.Transcriptome basic on the heterosis of hybrid tilapia.The RNA-seq technology was used to conduct a comparative transcriptome analysis of liver and intestinal tissues among NL,AR and HY groups to understand the gene expression differences.A total of 35175 expressed genes were detected by comparison and assembly,of which 32734 were annotated known genes,and 2441 were newly discovered unknown genes,accounting for 93.1% and 6.9%,respectively.Furthermore,3097 differentially expressed genes(DEGs)were detected in the livers of NL,AR and HY,in which 2561,1050 and 822 DEGs were detected in the NL-AR,HY-AR and HYNL combinations,respectively.In HY-AR and HY-NL combinations,65.4% and 67.8%of DEGs overlapped with those in the NL-AR combination,respectively.4980 DEGs were detected in intestinal tissues,in which 3392,2470 and 1261 DEGs were found in the NL-AR,HY-AR and HY-NL combinations.55.2% and 54.7% of DEGs overlapped with those in the HY-AR and HY-NL combinations.It was shown that the gene expression differences between HY and parents were mainly due to the differences between the male and female parents.The expression classification analysis of DEGs was performed among HY,NL and AR,which showed that 48.4%,47.9% and 3.7% of DEGs were classified into expression additivity,expression level dominance(ELD)and expression level over-dominance(ELOD)patterns in the liver tissues,respectively.DEGs accounted for 31.9%,63.7%and 4.4%of additivity,ELD and ELOD patterns in the intestine tissues,respectively.The above result indicated that ELD and ELOD expression patterns accounted for a high proportion of DEGs among HY,NL and AR.In addition,61.3%and 51.2%of the ELD pattern genes were up-regulated in the liver and intestine of HY,and 66.4%and 52.3%of the ELOD pattern genes were up-regulated in the liver and intestine,respectively.It was revealed that the gene expression advantages of parents were integrated into the regulation of the non-additive expression genes in hybrid tilapia.Moreover,these non-additively expressed genes(ELD and ELOD)were significantly enriched in pathways related to cell proliferation,RNA transcription and translation,digestion and absorption,growth and development,substance metabolism(amino acids,lipids and sugars)and developmental hormone synthesis.These enriched pathways indicated that hybrids differ from parent average gene expressions in growth,metabolism and immunity capacity.The results promoted our understanding of the basis of multi-traits heterosis in hybrid tilapia.3.Molecular basis of miRNA for heterosis in hybrid tilapia.To understand the differences in post-transcriptional regulation,sRNA sequencing technology was used to compare miRNA expression in NL,AR and HY liver tissues.After comparison and assembly,242,246,and 260 known miRNAs were annotated,and89,77 and 99 unknown miRNAs were newly discovered in NL,AR and HY,respectively.One hundred two different expressing miRNAs(DEMs)were detected in the three groups,including 88 DEMs in NL-AR combinations,48 DEMs in HY-AR combinations,and 27 DEMs in HY-NL combinations,respectively.Meanwhile,79.2% of DEMs in NL-AR and 82.1% of DEMs in HY-AR overlapped with DEMs in NL-AR,indicating that the DEMs between HY and parents were mainly derived from differences between NL and AR in post-transcriptional regulation.The expression classification analysis of DEMs was performed among HY,NL and AR,which showed that 39.2%,59.8%,and1.0% of the DEMs showed additivity,ELD,and ELOD patterns,respectively.It was indicated that miRNAs with ELD pattern played an important role in post-transcriptional regulation of the genes of HY.Furthermore,264 putative miRNA-mRNA interaction pairs were predicted,including 53 DEMs and 158 DEGs.It showed that 70.8% and 29.2%of the putative miRNA-mRNA interaction pairs were divided into ELD-NL and ELDAR patterns,respectively.It was concluded that miRNAs in HY were more similar to NL in the post-transcriptional regulation process.The GO and KEGG enrichment analyses were performed on DEGs of the putative miRNA-mRNA interaction pairs,which showed that these DEGs were mainly enriched in cell proliferation,steroid hormone synthesis,lipid and protein metabolites and innate immune response,etc.The result suggested that these miRNAs,which were mainly involved in regulating the expression of genes in above pathways,played an important role in improving growth,development,fat and protein metabolism,and immune and stress resistance in HY.Our results demonstrated that miRNAs were closely related to the regulation of non-additive expression of genes involved in the growth,development,and non-specific immunity of hybrid tilapia and deepened our understanding of the molecular basis of miRNAs for heterosis in hybrid tilapia.4.Role of specific expression of mapk14 a allele in the heterosis of hybrid tilapia.The previous chapter found that mitogen-activated protein kinase(mapk14a)is nonadditive expressed in HY,which played a key role in several pathways,including the endocrine system,immunity response and oxidative metabolism.Therefore,the coding region and 5’ flanker region sequences of mapk14 a were analysed to find specific SNP sites between NL and AR groups,and allele-specific expression mode was studied in HY.The regulatory mode of cis-acting and trans-acting elements from NL and AR subgenomes in mapk14 a allele expression in HY was analysed,which involved the mapk14 a expression changes.A total of 7 SNPs sites were found in the three groups,all of which were synonymous mutations.The SNPs in NL,AR and HY groups were 5,3and 7,respectively.There were two specific SNPs sites in the NL-AR combination,namely S4(G.841 A>G)and S7(G.1357 G>A),and S7 was taken as a marker in the allele-specific expression pattern in HY.It was found that the expression of the mapk14 a allele showed some deviation in the transcription,and the ratio of the mRNA transcripts number of alleles(G)from NL to that of alleles(A)from AR was 77: 23.The result indicated that NL subgenomic alleles dominated the expression of mapk14 a in HY.Meanwhile,the quantitative analysis of mapk14 a expression levels showed a trend of HY > NL > AR among the three groups(p < 0.05),in which the expression levels of HY and NL were 1.90-and 1.48-times AR,respectively.A compensatory interaction of cis+ trans acting elements were found in mapk14 a transcription of HY by calculation.Moreover,there were 8 specific SNPs sites between NL and AR groups in the comparative sequences analysis of the mapk14 5 ’flanking region.There were two putative transcription factor binding site variations in the promoter region of mapk14 a,which may result in a gene expression efficiency difference between NL and AR alleles.The results described the regulation modes of mapk14 a allele transcription in the effect of the cis-acting and trans-acting elements of NL and AR subgenomes,which are beneficial to explain the mechanism for non-additive expression regulation of plenty of functional genes in HY related to growth and immunity response.It is important to elaborate further the mechanism of heterosis in growth,disease resistance and stress resistance of hybrid tilapia and to guide the breeding of more superior traits hybrid tilapias in future.