Genetic Parameters Estimate And Candidate Gene Identification Of Eggshell Color In The Lueyang Black-Boned Chicken

Lueyang black-boned chicken is a local breed from Lueyang County,Shaanxi Province.Due to the lack of systematic breeding before,the eggshell color of the Lueyang black-boned chicken has great variability,which has a very adverse impact on the sales of eggs and the creation of brands.In this study,on the basis of quantifying eggshell color,the heritability of chromaticity indexes was estimated,and the relationship between the synthesis genes CPOX and ALAS1 of protoporphyrin(responsible for reddish-brown eggshell coloration)and the transport gene SLCO1B3gene of biliverdin(responsible for blue-green eggshell coloration)and eggshell color and eggshell pigment content were studied.The results will promote our understanding of the genetic basis of the color of the eggshell,screen out the key regulatory genes,and lay a foundation for the establishment of the color selection scheme and the development of molecular-assisted breeding methods.841 hens were selected,3 eggs were collected from each chicken,eggshell color was quantified by L*a*b chromaticity system,and animal models were used to estimate the heritability of chromaticity indexes and the genetic correlation between the indexes.Brown shell(n=8),light brown shell(n=8)and blue shell(n=8)hens were selected,and 3 eggs were collected from each chicken,and the content of protoporphyrin and biliverdin in eggshell were determined spectrophotometrically.The expression of SLCO1B3,ALAS1 and CPOX genes in the eggshell gland was detected by q PCR.Blue shell(n=105)and non-blue shell(n=474)samples were selected and the distribution of EAV-HP in the population of Lueyang black-boned chicken was detected by PCR.The 1.6 kb region upstream of SLCO1B3 gene was resequenced,and haplotypes were constructed with PHASE v2.1.1,and the relationship between haplotypes and gene expression,eggshell pigment content and color was analyzed.Haplotype 4(Hap4)and haplotype 13(Hap13)were cloned upstream of the luciferase reporter,and the effect of haplotype on promoter activity in the promoter region of SLCO1B3 gene was analyzed by diluciferase reporter system.Among the three chromaticity indexes,the distribution of L value and b value was more concentrated,and the L and b values of 50%of the samples were concentrated between 73.3～79.1 and 13.1～18.2.However,the distribution of A value is relatively scattered,and 50%of the samples are distributed between 1.4～7.1.The coefficient of variation of 88.2%for the a value was higher than 5.7%for the L value and 24.0%for the B value.The heritability of a value(h~2=0.77)was significantly higher than that of L value(h~2=0.46)and b value(h~2=0.37).Between eggshell pigment and eggshell color,protoporphyrin concentration was strongly correlated with the three chromaticity indexes(L:r=-0.86,a:r=0.73,b:r=0.88),but the concentration of biliverdin was only strongly negatively correlated with the a value(r=-0.73).The expression level of CPOX in brown shell chicken was 1.5 times of that in light brown shell(P<0.05),but there was no significant difference in the expression of ALAS1(P>0.05).In this study,no EAV-HP distribution was detected in the population of Lueyang black-boned chicken,but a strong positive correlation between SLCO1B3 expression level and biliverdin concentration was found(r=0.86).In the promoter region of SLCO1B3 gene,hap4 appeared at a higher frequency(52%vs.3%)in blue shell samples,while hap13 appeared at a higher frequency(26%vs.6%)in non-blue shells.The expression level,biliverdin content and promoter activity of SLCO1B3 in Hap4 were significantly higher than those of hap13(P<0.05).These results show that(1)a value is an optimal eggshell color quantification index that is sensitive to eggshell color changes and is mainly regulated by genetic effects;(2)protoporphyrin is the key pigment that affects the eggshell color of Lueyang black-boned chicken,CPOX is a candidate gene significantly associated with the color depth of the brown shell of Lueyang black-boned chicken;(3)The blue shell of Lueyang black-boned chicken is controlled by a new molecular mechanism,and hap4is a functional haplotype that affects the expression of SLCO1B3 and the color of the blue shell of Lueyang black-boned chicken.