Study On Eggshell Color Traits And Genetic Effects Of LOC101800257 Gene In Leizhou Black Duck

Eggshell color,a qualitative character that reflects the external appearance of an egg and performance of egg quality,it plays a great economic importance to duck industry.In this study,the egg quality and nutrient component of Leizhou black duck green and white eggshell at 300 days were tested.Meanwhile,the eggshell pigment contents were determined to analyze the correlation with eggshell color.The relative expression of green eggshell candidate gene LOC101800257 in uterus,oviduct and liver from different growth stages Leizhou black duck were determined by using real-time quantitative PCR?RT-PCR?.Finally the single nucleotide polymorphisms of LOC101800257 gene were detected to analyze the correlation between the polymorphisms of SNP locus and green eggshell trait by sequencing and PCR-SSCP methods.Consequently,all these tests above aims to reveal the regulation of LOC101800257 gene on the formation of green eggshell and provide the molecular breeding materials for green eggshell breeding of Leizhou black ducks.The results showed that:?1?There was a significant difference of eggshell color between the white ones?62.02?and green ones?51.58??P<0.05?.The egg yolk color and cholesterol content in green shell egg were significantly higher than that of white shell egg,while the other egg quality indexes and nutrient content showed no significant difference between white and green eggshell.At the same time,there was a significant correlation between eggshell color and egg yolk color,eggshell thickness and eggshell strength.?2?The biliverdin content in Leizhou black duck green eggshell was 0.0253mg/g,which was significantly higher than that of white eggshell?0.0083mg/g?and much higher than protoporphyrin content in Leizhou black duck green eggshell?0.0069mg/g?.There was a significant correlation between eggshell color and total eggshell pigments and biliverdin contents?P<0.05?.?3?RT-PCR results showed that:LOC101800257 gene was mainly expressed in liver while its relative expression in uterus and oviduct from Leizhou black ducks were little.The relative expression in liver were significantly difference between Leizhou black duck who lay green eggshell and white eggshell.In different growth stages of Leizhou black ducks,the expression of LOC101800257 gene in liver increased as the time decreases initially and increases afterwards.The highest relative expression is 0.385 in 27 weeks old,while the lowest expression is 0.022 in 43 weeks old.?4?Two new single nucleotide polymorphisms?SNP?were found in 13815 bp and15325bp of LOC101800257 gene respectively via designing ten specific primers to cloned the partial exons and introns in LOC101800257 gene of Leizhou black ducks.The former SNP locus located in the 9^thexon,which was named c.1406A>G on the level of cDNA.It belongs to the missense mutation owing to base A to G mutation led to the original encoding amino acid isoleucine?ILe?mutation into valine?Val?.The latter SNP locus located in the 11^thintrons,which was named c.1642+16A>G owing to the A to G mutation in non coding region.The A and G allele gene frequency of c.1406A>G locus were 0.554 and 0.446 respectively,and heterozygous genotype AG was the dominant genotype.The A and G allele gene frequency of c.1642+16A>G locus were 0.683 and 0.317 respectively,and wild type AA was the dominant genotype.In the statistic population of Leizhou black ducks,two SNP locus were in the Hardy-Weinberg non-equilibrium state?P<0.01?.The expected heterozygosity,effective number of alleles and PIC of c.1406A>G locus were0.494,1.977 and 0.372 respectively.Meanwhile,the expected heterozygosity,effective number of alleles and PIC of c.1642+16A>G locus were 0.433,1.763 and 0.339 respectively.All these two SNP locus showed abundant genetic polymorphism.The genotype combinations GGGG from c.1406A>G and c.1642+16A>G locus showed significant correlation with green eggshell traits,which can be used as a molecular marker for green eggshell breeding.