Identification Of Genes Related To Eggshell Blueness In Chinese Dongxiang Blue-Shelled Chickens

Eggshell blueness is an interesting object for chicken genetic studies and blue-shelled chicken industry,especially after the discovery of the causative mutation of chicken blue eggshell.In China,Dongxiang chickens have been selected for blue eggshell and the population was systematically fixed on the dominant homozygous genotype of SLCO1B3 gene.Despite the fact that all of the hens lay blue-shelled eggs,the color intensity of the eggs varies from dark to light blue.Thus,in the present study,we conducted the basic analysis of standard biliverdin and protoporphyrin pigments of blue-shelled eggs to obtain the phenotype traits;quantity of biliverdin(QB),quantity of protoporphyrin(QP),and quantity of total pigments(QT).Thereafter,we tested the mRNA expression levels for three potential regulatory genes(i.e.SLCO1B3,HOI,and BLVRA)involved in the heme pathway to assess the correlation of these candidate genes with eggshell color intensity trait.Our results demonstrated no significant association for these genes with the trait.The follow-up replication studies were performed in two populations of Chinese Dongxiang blue-shelled chicken underlying three traits of blue eggshell pigments.In order to further verify and confirm a set of identified SNP loci that anchored previously by genome-wide association study(GWAS).A total of 146 individuals of the second generation derived from the former GWAS population,as well as 280 individuals from an alternative independent population,were employed for genotyping by MALDI-TOF MS in a genotype-phenotype association study.Eighteen SNPs evenly distributed on the GGA21 significant region were successfully genotyped in the two populations.Our results showed that four SNPs(rs313867043,rs16177219,rs16177126,and rs16177212)and(rs315586328,rs16177219,rs313199923,and rs16177126)were significantly associated with QB and QT in the N146 and N280 populations,respectively(P<0.05).In the N280 population,two SNPs(rs315477097 and rs13602462)were associated with the three phenotype traits,while SNP rs16177212 was related only to QP.Further,the SNPs were narrowed down to a region of-653.918 Kb on GGA21 that harbors five candidate genes:AJAP1,TNFRSF9,C10RF174,CAMTAI,and CEP104.Interestingly,two mutual SNP loci(rs16177219 and rs)6177126)associated with CEP104 and AJAP1 genes were discovered between the first and the latter population.The genotypic effects of the SNPs in the two populations were further divided into additive and dominant effects.In the N146 population,three SNPs(rs313867043 rs16177219,and rs177212)showed significant dominant effects on QB and QT(P<0.05),while for the N280 population,SNP rs315477097 showed significance in QB and QT for the additive effect while its dominant effect related only to QP(P<0.05).The additive genotype effect of three different SNPs(rs315586328,rs15180009,and rs313199923)was significant for QB(P<0.05),whereas the additive effect of SNP rs16177212 was significant for QP(P<0.05).Three haplotype blocks were detected in the two populations,of which the haplotype combinations in Block 2 and 3 were noticed significantly associated with QP(P<0.05)in the N280 population.Shell gland,liver,and spleen tissues of dark and light blue-shelled chicken were chosen from the previous two populations for the detection of mRNA expression of the five candidate genes.The results showed differential expression levels of these genes in the three tissues of dark and light blue egg shelled chicken.The identified genes in this study were detected in human.However,the specific function of these genes has not yet been defined clearly in chickens and further in-depth studies are required to explore the new functional role in chicken eggshell blueness.It is well known that eggshell color is controlled by multiple genes,but the mechanism to identify the variation appeared on the eggshell has not been clearly understood or determined until now.In this study,a transcriptome profile using high-throughput sequencing has been effectively used for screening the differentially expressed genes(DEGs).RNA sequences(RNA-Seq)from the shell gland of dark and light blue-shelled chicken(N280 population)were examined using HiSeq 2000 to identify and compare the differential expression of functional genes related to eggshell color intensity traits.We detected that 46,338 transcripts were expressed between the two groups of dark blue(DB)and light blue(LB)eggshell chickens.Of the 46,338 transcripts,917 were significantly different(P<0.05)of which 412 were up-regulated in(DB)and 505 were down-regulated in(LB).When the filter criteria of Fold change>2 was applied,883 transcripts were obtained,some of which are involved in,erythrocyte differentiation and development,mitochondrial iron transportation,and integral component of membrane.Interestingly,in this experiment,AJAP1 gene was also observed differently expressed between DB and LB.Beside,FAM210B that is further considered a significant candidate gene for chicken eggshell blueness.In summary,we emphasize the important role of AJAP1 that showed through our results.In addition to,the other candidate genes that might interact and function effectively on chicken eggshell blueness.We believe that identification of genes associated with the variations of blue eggshell color may provide a new insight into the genetic basis of these traits and benefit for designing an efficient selection strategy in laying hens breeding programs to produce eggs with uniform or desired eggshell colors.