Identification And Validation Of Key Genes For Knob Trait In Geese

There was a protuberance at the base of the upper bill in geese domesticated from Anser cygnoides,called “knob”,which consisted of skin and bony protuberance,while the geese domesticated from Anser Anser were devoid of knob.The knob had morphological and size differences in different Chinese domestic geese breeds(except Yili goose),and the formation time was late.There were also differences between sexes,and the knob was larger and more obvious in male.However,there were few reports on the regulatory mechanism of knob formation.In this study,therefore,adult Lion head geese and Sichuan white geese with knob as well as adult Landes geese devoid of knob were used to reveal the histological differences of knob in different geese breeds through histomorphology,explore the molecular regulation mechanisms of the histological differences of knob skin and bone in different geese breeds through transcriptome,identify the candidate genes regulating knob trait by integrated analysis of transcriptome and whole genome resequencing,and verify in the paternal population of Tianfu meat geese,in order to provide a theoretical basis for the breeding of goose knob triat.The main results were as follows:1.The results of histomorphology showed that the goose knob was located on the frontal region.The skin consisted of epidermis,dermis and subcutaneous connective tissue and the bone consisted of bony matrix and osteocytes.The thicknesses of papillary dermis and reticular dermis of knob skin in Lion head goose were significantly thicker than that in other two geese breeds(P < 0.05),and the content of collagen in dermis was also higher(P< 0.05).The density of subcutaneous adipocytes was significantly higher in knob skin in Sichuan white goose than that in other two geese breeds(P < 0.05).Compared with Landes goose,the thicknesses of stratum corneum and epidermis of knob skin in Lion head goose and Sichuan white goose were significantly thicker,the height of papillary layer in dermis was significantly higher(P < 0.05),and numerous smaller locular adipocytes were scattered in the large locular adipocytes,as well as the ratio of short to long diameter of adipocytes was also higher(P < 0.05).However,the bony density of protuberance significantly decreased in Lion head geese and Sichuan white geese than that in Landes geese,and the thickness of the knob bone was also significantly thinner.In addition,the connective tissue and cartilage were also observed close to the bony matrix in Landes geese.2.The results of transcriptome showed that 2145,981,4862 differentially expressed genes(DEGs)were identified in knob skin,and 778,1711,2535 DEGs were identified in knob bone in Sichuan white goose vs Lion head goose(W vs S),Sichuan white goose vs Landes goose(W vs L),Lion head goose vs Landes goose(S vs L),respectively.Functional enrichment analysis showed that the DEGs in skin were significantly enriched in lipid synthesis and metabolism,cell proliferation,cell adhesion and extrallcelluar matrix structure,the DEGs in bone were in calcium signaling pathway,extracellular matrix structure and basic metabolism,and the DEGs in both skin and bone were in ECM signaling pathway.Notably,the DEGs of skin in W vs S were significantly enriched in cell proliferation and cell adhesion pathways.Between knob geese(S and W)and non-knob goose(L),592 and 952 DEGs were identified in skin and bone,respectively,through intersecting the W vs L and S vs L.Functional enrichment analysis showed that the DEGs in skin were significantly enriched in metabolic process,cell adhesion and lipid metabolism pathways,DEGs in bone were in calcium signaling pathway,metabolic process,and DEGs in both skin and bone were in extracellular matrix structure,cell adhesion,cytokine-cytokine receptor interaction pathways.Combined with the results of histomorphology,FABL7,LPL,FABP3,UBB,ACSBG1 enriched in PPAR signaling pathway were involved in regulating the knob skin thickening,CD38,CACNA1 H,NOS2,ATP2B2,CAMK1 G,CACNA1I,NOS1,RYR2,HTR4,AVPR1 A,PTAFR,HTR2 C,HRH1,P2RX1,GRIN1,ADCY3,GRM1 enriched in calcium signaling pathway were involved in regulating the knob bone protuberating,and HMMR and VTN enriched in ECM-receptor interaction were involved in both knob skin thickening and bone protuberating,which indicated a common regulatory mechanism between skin and bone.3.To further reduce the crucial candidate genes regulating skin and bone,351 selected genes were screened through whole genome resequencing.Integrated analysis of transcriptome and whole genome resequencing identified 17 and 21 genes in skin and bone,respectively,which were differentially expressed in transcriptomic level and were selected in genome level.Among these genes,KIAA2022,DIO2,ADAMTS3,TRPC6 were differentially expressed and were selected in both knob skin and bone at the same time.However,these genes were not on the PPAR,calcium signaling pathways and ECM-receptor interaction,indicating that these genes might be upstream genes regulating these signal pathways.Further analysis showed that KIAA2022 and DIO2 had the highest population differentiation index(Fst),variation detection showed that there were synonymous mutations in the coding region of KIAA2022,while there was a nonsynonymous mutation in the coding region of DIO2.This mutation didn’t change in Landes goose,while there was a G>A mutation in Lion head goose and Sichuan white goose which changed the protein secondary structure of DIO2,indicating that DIO2 gene might play an important role in regulating the protuberance of goose knob.4.Alignment of DIO2 coding sequence between Sichuan white goose and Landes goose showed that four polymorphic loci,g.419 A>G,g.533 A>G,g.725 C>A,g.799 C>T,were identified in Sichuan white geese compared with Landes geese.In the paternal population of Tianfu meat geese,the head dimensions of individuals with knob were significantly larger than that of individuals with non-knob(P <0.05),and the AA genotypes at the g.419 A>G and g.533 A>G loci or CC genotypes at the g.725A>C and g.799T>C loci in the DIO2 gene as well as haplotype AACC,diplotype AACC-AACC had wider beak and cranial width as well as longer nostril length in the population of non-knob geese(P < 0.05).These results indicated that DIO2 was considered as a key gene to regulate the presence or absence of goose knob,and its four polymorphic loci could be as the molecular markers for head size selection of goose without knob.Taken together,the present study analyzed the histomorphological differences and molecular regulatory mechanisma of knob(skin and bone)in different geese breeds as well as knob and non-knob geese,and DIO2 might be the crucial gene to regulate the knob trait.