| IMB(Intermuscular bone)is one of the most important factors influencing the flesh quality of common carp.If IMB could be reduced or erased,the flesh quality of common carp would be greatly improved.However,few literatures about IMB are reported,and the scientific community had different opinions about whether it’s possible to carry out selective breeding program for reducing or erasing IMBs for common carp.To investigate the genetic mechanism of IMB in common carp(Cyprinus carpio),in this study,studies on genetic parameters,QTL(Quantitative Trait Loci),GWAS(Genome Wide Association Study)and transcriptome analyses of IMB tissue were performed.Through this study,we obtained heritability and genetic correlation of IMB,and characterized candidate genes related to development of IMBs.Furthermore,we established two knock-out zebrafish lines(ngs-/-and cilp-/-)and preliminarily investigated its function.The main results are followed.1.Thirty full-sib families including 871 individuals were used to calculate the heritability and genetic correlation,the results showed that the heritability of the total IMB count,Y-shaped(YS)IMB count and I-shaped(IS)IMB count were moderate and significant(P<0.001),with the values of 0.15 ± 0.08,0.44± 0.12 and 0.23 ± 0.08,respectively.Furthermore,QTL mapping was performed with 8 families including 522 individuals,and 8 QTLs were found for IMBs,YS IMBs and IS IMBs,which the phenotypic variance explained by QTLs ranged from 16.20%～56.26%.Within the QTL regions,29 genes associated with osteogenesis,osteoclast and chondrocyte development and differentiation,and fin or limb development were identified as candidate genes for IMB development.2.Based on the 250 K SNP(Single Nucleotide Polymorphism)array,genome-wide association analyses of IMB,BW(Body weight),SL(Body length)and TH(Body thickness)were performed,and found that 28 SNPs with genome-wide significance(FDR<0.05)(Fals Discovery Rate)which could explain the phenotypic variation rate of 11.47%±9.1%,and 25 candidate genes were obtained for IMB;76 SNPs with genome-wide significance(FDR<0.05)for BW and 60 candidate genes were obtained,which could explain the phenotypic variation rate of 54.29%±8.3%;97 SNP markers with genome-wide significance(FDR≤0.05)for SL and 73 candidate genes were obtained,which could explain the phenotypic variation rate of 48.75%±11.18%;25 SNP markers with genome-wide significance(FDR≤0.05)for TH and 19 candidate genes were obtained,which could explain the phenotypic variation rate of 48.75%±11.18%.Gene-based analysis showed that 9 significant genes were obtained for IMB(FDR≤0.05);17 significant genes were obtained for BW;2 significant genes were obtained for SL;and one significant gene was obtained for TH.Furthermore,based on gene-set analysis,6 GO terms(Gene Ontology term)were significantly enriched for IMB,5 GO terms were significantly enriched for BW,10 GO terms were significantly enriched for SL,and 5 significantly enriched GO terms were obtained for TH.3.The transcriptome expression profiles of IMB,septum,muscle and vertebra were analyzed in mirror carp based on RNA-Seq,and the results showed that 38.9-53.6 M clean reads were obtained from each sample,The expression profile of IMB was similar to that of septum and muscle,and the correlation coefficient was up to 0.99.However,the expression profile was significantly different from that of vertebra,the correlation coefficient was only 0.37.The osteoblastic genes runx2a,sp7,and Osteocalcin were expressed in the 4 tissues,which indicated that the muscle and septum contained osteoblasts and osteoblastic progenitor cells that promote IMB formation.The differential expression analysis showed that 1575 differentially expressed genes were detected for IMB,septum and vertebra.The pathway enrichment analysis showed that the up-regulated genes of IMB compared to muscle and myoseptal were mainly enriched in PPAR(dre03320),Focal adhension(dre04510),ECM(dre04512),metallocarboxypeptiase activity(GO:0004181),bone development(GO:0060348),fin development(GO:0033334),appendage development(GO:0048736)and so on,these genes and pathways may influence the development and counts of IMB.4.The cilp and ngs zebrafish knock-out lines were established using CRISPR/Cas9 technology,and the functions to bone development were analyzed preliminarily investigated.Bones of cilp-/-and ngs-/-.and wild-type zebrafish were stained using alcian blue and alizarin red,and showed that some medullary spines and vein spines disappeared in cilp-/-and ngs-/-zebrafish,and enlargement of vertebra and fusion of joints were also observed.These results suggested that ngs and cilp might play a role in bone development.5.Using cilp-/-mutants,we investigated the expression levels of 10 genes related to bone development at 6 developmental stages(Blastula stage,Gastrula stage,Segmentation stage,Pharyngeal stage,Caudal bud stage,Hatching stage)as well as in 6 tissues(brain,liver,intestine,gonad,muscle in caudal,and bone)of adult.The results showed that the expression trends of bmp2a,dmpk,coll ala and cilp genes during the embryo development of cilp-/-and wild-type zebrafish were similar,while the expression of runx2a,bmp6,smad5,smad4a,sp7,sost and bglap genes showed opposite trends.The expression levels of cilp and bglap in the cilp-/-bone were lower than that of wild-type zebrafish,while the expression of the remaining 9 genes was higher than that of the wild-type.The expression level of smad5 was significantly different from that of the wild-type(P<0.05),and the other 9 genes showed no significant difference.6.Using zebrafish ngs-/-mutants as the materials,the expressed levels of 10 genes related to bone development were analyzed and compared to that of wild-type zebrafish.The results showed that the expression levels of smad5,smad4a,sost and bglap in the embryos of wild-type zebrafish were decreased,but the expression levels of bmp2a,coll ala and ngs increased.The expression levels of runx2a,bmp6 and sp7 increased and then decreased,while the expression levels of sost and dmpk decreased and then increased.With the embryonic development of ngs-/-mutant,the expression levels of runx2a,smad4a and bglap decreased,while the expression levels of bmp2a,smad5 and col1ala increased;the expression levels of sost,bmp6,dmpk and ngs decreased and then increased,while the expression levels of bmp6 and dmpk decreased after increasing.The expression levels of 10 genes in bone of ngs-/-mutation were not significantly different from that of wild-type(P>0.05).The expression of ngs in brain,intestine,gonad,muscle and bone of ngs-/-mutants were lower than that of wild-type,while only the expression level in liver was higher than that of wild-type. |
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