Genetic Characteristics,optimal Dietary Protein Level And Molecular Mechanism For Heterosis Of Hybrid Strains Between Megalobrama Amblycephala And Culter Alburnus

(1)The topmouth culter(Culter alburnus,CA)is a typical carnivorous fish,whose meat is delicious,but growth rate is slow.The blunt snout bream(Megalobrama amblycephala,MA)is a typical herbivorous fish,which grows faster,but its meat quality is inferior to topmouth culter.By intergeneric crossing between blunt snout bream and topmouth culter and sequential backcrossing,five hybrid groups of MC(MA?×CA?),BC-1(MA?×MC?),BC-2(MC?×MA?),BC-3(CA?×MC?)and BC-4(MC?×CA?)with different genetic backgrounds were obtained through artificial insemination in this study.All these hybrids showed a high fertilization rate,hatching rate and survival rate.Five hybrid groups were diploid and they had the similar DNA content and same karyotype(2n=48=18m+26sm+4st)with their parents.The morphological characters of five hybrids were intermediate between their parents,while two backcross groups of BA-1 and BC-2 were more similar to their famale parent MA,BA-3 and BC-4 were more similar to their male parent CA,respectively.The female and male of the five hybrids had normal gonadal development.Moreover,protein content of muscle for five hybrids was significantly(P < 0.05)higher and carbohydrates content was significantly(P < 0.05)lower than their parents.In addition,IB morphology was classified into 7 shape categories in all four groups,A total of 125 IBs in backcross BC-1 appeared to be intermediate between the number of IBs of the female parent MA(124)and male parent CA(137),IBs of BC-1 were significantly lower than that of its parent culter(137)and BC-4(132)backcross bream culter)and the IB morphological complexity of the backcross BC-1 was intermediate between its parents.In conclusion,the backcross BC-1 had advantageous characteristics,including meat quality,excellent morphology and fewer and simpler IBs,making it a hybrid of great commercial value.(2)The present study was to investigate the effect of dietary protein levels on growth performance,feed utilization,intestinal morphology and digestive enzyme gene expression of Megalobrama amblycephala,Culter alburnus and their hybrids.Using fish meal and soybean meal as main protein source,5 kinds of feed with protein level of 25%,30%,35%,40% and 45% were prepared for feeding.90 day culture test showed that the feed protein level had significant influence on the weight gain(WG),feed conversion ratio(FCR),specific growth rate(SGR)and shape index of 7 kinds of fish(P < 0.05).The protein levels of 7 kinds of fish at the maximum weight gain rate were 30.14%,57.97%,36.97%,33.48%,33.85%,39.55% and 41.42%,respectively.Meanwhile,the protein level was 29.84%,46.67%,37.56%,32.60%,33.35%,40% and 40.45% at the minimum feed conversion ratio.Taking the WG rate and FCR as the comprehensive consideration,the optimal protein levels for 7 kinds of fish were 29.99%,52.32%,37.26%,33.04%,33.60%,39.77% and 40.93%,respectively.The histological sections of small intestine showed that the level of feed protein had a significant effect on the morphology of the small intestine.The backcross BC-1 showed the best state at 35% protein level.Similarly,expression of digestive enzyme genes(TRY,CTRL1,ELA1 and CPA2)reached a high level at 35% protein level.(3)The backcross BC showed heterosis on growth performance compared to its parents.But the molecular mechanisms of its heterosis still remain poorly understood.Based on RNA sequencing and gene expression profiling,we conducted comparative transcriptome analyses of liver between the backcross BC and its parents.Three hundred twety-five and 872 differentially expressed genes(DEGs)were identified between BC and MA and between BC and CA in the liver,respectively.GO enrichment analysis of all DEGs showed that metabolic process,cell part and binding were the most three enriched GO terms.Further analysis revealed the expressions of digestive enzyme genes such as TRY,ELA1,CTRL1,CPA2 and BAL,and GH/IGF axis related genes such as IGF1,IGF2 a and IGFBP2 b in the liver of the backcross BC were up-regulated significantly,which is in accordance with the growth superiority of BC.Meanwhile,expressions of genes related to the protein and fatty acid synthesis pathway,such as PI3 KR,RAPTOR,EIF4 E,CS,MDH,FASN,ELOVL1,ELOVL5 and ELOVL6 were significantly up-regulated,these changes may contribute to increased protein and fatty acid synthesis in the backcross BC.The results suggested that the activity of the upstream GH/IGF system,along with the up-regulated digestive enzyme genes as well as protein and fatty acid synthesis pathway in liver,led to heterosis of the backcross BC.(4)According to the standard of ratio(B/A)> 2.0 or ratio(B/A)< 0.5,fifty seven differently expressed proteins(DEPs)were identified,including 42 up-regulated and 15 down-regulated DEPs in‘BC vs.MA'.And 434 DEPs were identified,including 42 up-regulated and 15 down-regulated DEPs in ‘BC vs.CA'.In total,there were 491 DEPs that were differentially expressed in the BC compared to its parents,and among those genes,27 overlap DEPs were found in ‘BC vs.MA' and ‘BC vs.CA'.GO enrichment analysis of all DEPs showed that metabolic process,cell part and binding were the most three enriched GO terms.Further analysis revealed that protein digestion and absorption pathway in BC was more active than their parents,in which the expression of chymotrypsin and ribosomal protein were up-regulated significantly.Meanwhile,the digestion,absorption and synthesis pathways of fatty acids are more active,including the bile secretion pathway,fat digestion and absorption pathway and the fatty acid biosynthesis pathway and fatty acid elongation pathway.In addition,the vitamin digestion and absorption pathway and mineral absorption pathway were also significantly enhanced.These pathways of the differential protein are most consistent with the pathway of the differentially expressed genes in the transcriptional group,which proves that these pathways are the key pathways of heterosis.(5)Elongases of very long chain fatty acids(ELOVL),which were involved in the biosynthesis of highly unsaturated fatty acids(HUFA).ELOVL1 and ELOVL6 are two members of the ELOVL family and are mainly responsible for 1 carbon-carbon chain extension.Here,we characterizedthe expression patterns of elovl1 and elovl6 in backcross species,which are retained in many fish species.Both elovl1 and elovl6 encode 324-and 267-aa peptides,respectively,which share a low sequence identity of 30%.During embryogenesis,both elovl1 and elovl6 m RNAs were lowly detected at zygotes but gradually increased and then constantly expressed after 24 hpf and 32 hpf,respectively.Whole-mount in situ hybridization demonstrated that elovl1 m RNA was transcribed at the eyes and anterior notochord at 12 hpf,while the elovl6 m RNA was only detected at the anterior notochord at the same period.At 24 hpf embryos,elovl1 m RNA was transcribed at the eyes,skin and tailbud,while the elovl6 m RNA was only detected at the skin and posterior somites at the same period.At 55 hpf embryos,elovl1 m RNA was detected in the eyes,midbrain,hindbrain and tailbud,elovl6 m RNA was detected in the midbrain,skin and posterior somites.In adult fish,both elovl1 and elovl6 m RNAs was strongly expressed in the brain,liver,kidney,eye and skin,but examined relatively low in the intestine,muscle and gill,besides a relative low expression for the elovl6.During starvation,both elovl1 and elovl6 m RNAs were mainly significantly down-regulated in the brain,kidney and liver,but up-regulated in the brain of elovl6.Our results suggest that elovl1 and elovl6 genes play important roles in regulating growth and development in backcross species.