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Avian eggshell color is an interesting genetic trait. Here, we report that the blue eggshell color of the domestic duck is caused by two cis-regulatory G to A transitions upstream of ABCG2, which encodes an efflux transporter. The juxtaposed blue eggshell allele A-A exhibited higher promoter activity and stronger nuclear protein binding capacity than the white eggshell allele G-G. Transcription factor analysis suggested differential binding capability of CTCF between blue eggshell and white eggshell alleles. Knockdown of CTCF expression significantly decreased the promoter activity of the blue eggshell but not the white eggshell allele. DNA methylation analysis revealed similar high methylation of the region upstream of the CTCF binding sites in both blue-eggshelled and white-eggshelled ducks. However, DNA methylation levels downstream of the binding sites were decreased and 35% lower in blue-eggshelled ducks than in white-eggshelled ducks. Consistent with the in vitro regulatory pattern of causative sites, ABCG2 exhibited higher expression in uteruses of blue-eggshelled ducks and also showed polarized distribution in their endometrial epithelial cells, distributing at the apical surface of endometrial epithelial cells and with orientation toward the uterine cavity, where the eggshell is pigmented. In conclusion, our results suggest that two cis-regulatory SNPs upstream of ABCG2 are the causative mutations for blue eggshells in ducks. The blue eggshell variant up-regulated ABCG2 expression through recruiting CTCF binding, which may function as a barrier element to shield the downstream region from high methylation levels present upstream. ABCG2 was identified as the only candidate causative gene for blue eggshells; it may function as an efflux transporter of biliverdin to the uterine cavity. Author summary: Avian eggshell color is an interesting genetic trait that has been related to numerous interesting biological functions, such as crypsis, mimicry, and protection from ultraviolet radiation. In ducks, blue eggshells are a dominant Mendelian trait. The color is preferred by customers and has become one of the main breeding targets in laying ducks in China. In this study, we identified that duck blue eggshells are likely caused by two cis-regulatory variations that synergistically up-regulate ABCG2 expression in the uterus. ABCG2 was identified as the only candidate causative gene for blue eggshell; it may function as an efflux transporter of biliverdin to the uterine cavity, where the eggshell is pigmented. Our study provides useful molecular markers for breeding of blue-eggshelled ducks. [ABSTRACT FROM AUTHOR]

Duck follicles enter different reproductive phases throughout life, and follicle gene expression patterns differ according to these phases. In particular, differentially expressed genes and related to development of follicle (mRNAs) play an important role to explore the key genes in this process; however, the expression profiles of these genes remain unclear. In this study, transcriptome sequencing was used to investigate the expression levels of duck ovarian genes, and comparative transcriptional analysis was carried out to identify differential genes, and cluster them into groups and function identification. The results showed differential expression of 593 coding genes between young and laying ducks, and of 518 coding genes between laying and old ducks. In further GO analysis, 35 genes from the comparison between old ducks and laying ducks have significant been changed involved in hormones related to follicle development. They include up-regulated genes StAR, CYP17, EPOX, 3β-HSD, CYP1B1 CYP19A1 and down-regulated genes SR-B1 in laying ducks hormone synthesis than old ducks. Among which EPOX is a key gene for time special highly expression during egg laying stage, and other key regulatory genes’ highly expression showed in young and laying stage, and lower expression showing with follicular development stopping. Therefore, EPOX is a key regulator for duck follicle development in laying period, its expression level increase 100 times higher than in youth and decrease 98% than stop laying period in duck life cycle. [ABSTRACT FROM AUTHOR]