Chicken Egg White Spreading Area's Genetic Analysis And OC-17's Functional Mechanism On Calcium Carbonate Mineralization

Improvement of important economic traits in livestock is the major task of animal breeding.Egg white and eggshell quality are important economic traits in poultry.In this study,we analyzed the genetic variant of egg white spreading area and the role of ovocleidin-17(OC-17)on calcium carbonate precipitation to provide new understanding for egg white and egg shell quality improvement.We measured the total egg content spreading area(TECA),outer thin albumen spreading area(OTAA),inner thick albumen spreading area(ITAA)and yolk spreading area(YA)for 2,395 newly laid eggs from 786 White Leghorn pure line layers in two generations.The internal and external quality traits of eggs from the 2nd generation hens were also measured.Then,we estimated the genetic parameters of egg content spreading areas and analyzed the phenotypic and genetic correlations between egg content spreading area and egg quality traits.The differences in the properties of eggs with similar egg weight but markedly different TECA were also analyzed.The heritability estimates for TECA,OTAA,ITAA,and YA were moderate,with values of 0.25,0.23,0.42 and 0.25,respectively.Highly negative phenotypic and genetic correlations of ITAA with albumen height and Haugh unit were found;while the phenotypic correlations of OTAA with albumen height and Haugh unit were low and the genetic correlations of OTAA with albumen height and Haugh unit were moderate.Higher outer thin albumen(OTA)weight and lower inner thick albumen(ITA)weight were found in eggs with larger TECA.Therefore,the TECA may be significantly affected by the relative amount of OTA and ITA.The process of "plumping" in the shell gland may had a markedly influence on the ratio of OTA to ITA.A total of 191 White Leghorn pure line layers were genotyped using the chicken Afymetrix 600K single nucleotide polymorphism(SNP)genotyping array.Univariate genome-wide association studies(GWAS)were performed for TECA,OTAA and ITAA using GEMMA software.We identified 2,1 and 0 genome-wise significant SNPs(P<4.09E-6),and 23,23 and 34 genome-wise suggestively significant SNPs(P<8.17E-5)associated with TECA,OTAA and ITAA,respectively.All associated SNPs were located in 4,4 and 3 genome regions for TECA,OTAA and ITAA,respectively.Eleven candidate genes(TGM4,GALNT1,MUC6,LRRC8B,RARB,EPRS,ARL5B,PRKCA,RAB11B,AKAP7 and WDR19)associated with egg white spreading areas were found,which were mainly involved in the process of gonad development,cell capacity homeostasis regulation and translation,post-translation modification,and extracellular secretion for proteins.Ovocleidin-17(OC-17)is a major protein involved in the eggshell mineralization.But the specific role of OC-17 and its binding sites on calcium carbonate deposits are still ambiguous.Previously,specific amino acid residues of OC-17 were predicted to interact with calcium carbonate surface,thus we targeted these residues to deletion or replacement and constructed 4 OC-17 mutants,Ser61 deletion mutant type and 3 alanine substitution mutants Ml-M3(Ml:Ser27/Arg28/Ser31/Arg34/Arg35,M2:Ser85/Arg86/Arg89 and M3:Arg46/Ser47/Arg52),to explore the differences of mineralization function of wild and mutant OC-17s.Recombinant OC-17s were expressed in cultured human embryonic kidney(HEK)293T cells and purified as the soluble secreted proteins.In vitro calcium carbonate precipitation was performed using the sitting drop method,and the precipitates were characterized by X-ray diffraction(XRD),fourier-transformed infrared(FTIR)and scanning electron microscopy(SEM).We report that(1)OC-17 wild type stabilized or promoted the transient formation of ACC,and induced a high nucleation density resulting in the formation of smaller calcite crystals.Therefore,OC-17 is a strong candidates to regulate the initiation of eggshell mineralization that strongly influences the eggshell ultimate biomechanical properties;(2)Serine61 deletion mutant type had a lower efficacy on reducing crystal size and promoting the formation of calcite compared with wild type;(3)Alanine substitution mutant types M1-M3 catalyzed the growth of calcite,which increased the crystal size and reduced the crystal number simultaneously.Therefore,four important domains of OC-17 binding to the surface of calcium carbonate deposits were confirmed by experimental method in our study.