Analysis Of Eggshell Pigmentation In The Chinese Indigenous Blue-Shelled Chickens

Eggshell color is of important aspects of egg quality and consumers tend to purchase eggs with particular colors. An EAV-HP insertion of SLCO1B3gene has been found to be the causative mutation for blue eggshell color in chicken in the former study. However there still remains a lot of interesting questions worth studying.One is the population diversity of blue eggshell chickens. Blue eggshell chicken populations have been reported in China, Europe and America. Especially in China, people found many "original" blue eggshell chicken populations distributing in different regions. In this study, we first evaluated phylogenetic relationships among six Chinese native blue-shelled chicken populations including Yimeng Blue, Wulong Blue, Lindian Blue, Dongxiang Blue, Lushi Blue and Jingmen Blue, along with two brown-shelled chicken subpopulations including Dongxiang Brown and Lushi Brown, by using21SNP markers from whole genomic region of SLCO1B3gene and EAV-HP insertion sequence. As an out-group breed, White Leghorn was included in the study. A high proportion of SNP polymorphism in SLCO1B3gene was observed in the Chinese chickens. The UPGMA tree clearly clustered the populations according to their eggshell color type and historical origins, which was further supported by the principal coordinates and STRUCTURE analyses. The six blue-shelled chicken populations are divided into two branches of the phylogenic tree. The low genetic diversity of SLCO1B3gene observed among the Chinese blue-shelled chicken populations demonstrates a gene flow in the chicken populations due to the geographical proximity and human migration. Aligning EAV-HP insertion sequences showed no difference among the blue-shelled chickens.The other interesting object is the color variation in the blue eggshell chickens with the same homozygous oocyan genotype of the EAV-HP insertion mutation in SLCO1B3gene. Through artificial selection, some of the population such as Dongxiang blue has been fixed on dominant homozygous genotype in SLCO1B3gene. However, although all of the hens lay blue-shelled eggs, the color density of the eggs is very different from dark to light blue. A genome-wide association study was performed using a high-density600K SNP array on a Chinese native blue-shelled chicken i.e. Dongxiang, to identify genes and chromosome regions associated with blue eggshell color density. Quantity of biliverdin (QB), protoporphyrin (QP), total quantity of the two pigments (QT) and color density of eggshell surface (CD) were measured as representatives for blue eggshell phenotypes. In total,49 SNP effects were detected on two significance levels including three SNP effects on genome-wide significance level (P<5.130E-07) associated with QT and QB, and46SNP effects on suggestive significance or chromosome-wide significance level (P<1.027E-05) associated with QT, QB, QP and CD. Two most significant SNPs were associated with QB and QT, located near or in the intron region of A JAP1and Clorf174genes on chicken chromosome21. Most suggested significant SNPs were also located near or in the intron or exon regions of known genes on this chromosome. These genes or markers might be applied to select the color density in blue eggshell chicken population. The functions of these genes have not been fully understood in chickens and call for further studies and might be required to confirm the functional implication for these newly identified SNP effects on blue-shelled chickens.Finally, it has been discovered that the EAV-HP insertion of SLCO1B3gene enhances the expression level in eggshell gland, while it has an opposite expression effect on liver. So, we were interested in the expression profile of the related candidate genes to investigate the potential functions in other tissues of chicken. We evaluated mRNA expression of SLCO1B3, SLCO1C1, SLCO1A2, LOC418189, PDE3A and HMOX1genes in11organs of a Chinese native blue-and non-blue-shelled female chicken, Yimeng, and expression of SLCO1B3in the same organs of females and12organs of males from three genotypes i.e. dominant, heterozygote and recessive. RNA was purified from heart, liver, spleen, lung, kidney, duodenum, jejunum, ileum, caecum, colon, rectum, ovary, shell gland and testes. The SLCO1B3was the most important gene with significantly higher expression in the reproductive system organs i.e. shell gland and testes of dominant and heterozygote in both male and female chickens, demonstrating an opposite expression level in liver. The lower expression of SLCO1B3in liver of dominant and heterozygote female and male chickens indicates alternative function of SLCO1B3in liver. Although a significant overexpression of SLCO1C1, SLCO1A2and HMOX1was detected in some organs, there was no significant higher expression of LOC418189and PDE3A in the studied organs of blue-shelled compare to non-blue-shelled hens. Moreover, higher and lower expression level of these candidate genes in some organs of blue-shelled compared with non-blue-shelled phenotypes may be due to their up-regulation and down-regulation role in biliverdin pathway and eggshell pigmentation mechanism, which is worthy of further investigation.