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.

禽蛋壳颜色是一个有趣的遗传特征。在这里，我们报道家鸭的蓝色蛋壳颜色是由ABCG2上游的两个顺式调控G向A过渡引起的，ABCG2编码外排转运蛋白。并列的蓝色蛋壳等位基因AA比白色蛋壳等位基因GG具有更高的启动子活性和更强的核蛋白结合能力。转录因子分析表明，CTCF在蓝色蛋壳和白色蛋壳等位基因之间的结合能力不同。击倒CTCF表达明显降低了蓝色蛋壳的启动子活性，但没有降低白色蛋壳的等位基因。DNA甲基化分析显示，在蓝蛋壳鸭和白蛋壳鸭中，CTCF结合位点上游区域的甲基化程度相似。然而，与白蛋鸭相比，蓝蛋鸭的结合位点下游的DNA甲基化水平降低了35％，而降低了35％。与致病位点的体外调控模式一致，ABCG2在蓝蛋壳鸭的子宫中表现出较高的表达，并且在其子宫内膜上皮细胞中也呈极化分布，分布在子宫内膜上皮细胞的顶端表面，并且朝向子宫腔，蛋壳上有色素的地方。

总之，我们的结果表明，ABCG2上游的两个顺式调控SNPs是鸭子蓝色蛋壳的致病突变。蓝色蛋壳变体通过募集CTCF结合上调了ABCG2的表达，其可能充当屏障元件，以保护下游区域免受上游出现的高甲基化水平的影响。ABCG2被确定为蓝色蛋壳的唯一候选致病基因。它可能充当胆囊泌素向子宫腔的外向转运蛋白。