Two cis-regulatory SNPs upstream of ABCG2 synergistically cause the blue eggshell phenotype in the duck

Abstract

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.

A new molecular mechanism supports that blue-greenish egg color evolved independently across chicken breeds

“…The identical types and deposition mode of eggshell pigments support homology of eggshell color between dinosaur and modern bird which can be traced back to a single evolutionary origin in eumaniraptorans ( Wiemann et al, 2018 ). Not only was BGC early present in oviraptors ( Wiemann et al, 2017 ), but it is commonly distributed in modern bird species ( Siefferman et al, 2006 ; Wang et al, 2013 ; Chen et al, 2020 ; Hamchand et al, 2020 ). Different molecular mechanisms for chicken and duck BGC question the single evolutionary origin of BGC, and support that BGC should independently evolved among different avian species laying BGC eggs ( Wang et al, 2013 ; Chen et al, 2020 ).…”

“…Blue-greenish coloration ( BGC ) is an ancient and common eggshell color. It appears not only in nonavian dinosaurs ( Wiemann et al, 2017 ), but in some modern avian species ( Siefferman et al, 2006 ; Wang et al, 2013 ; Chen et al, 2020 ; Hamchand et al, 2020 ).…”

“…BGC is mainly caused by biliverdin IX, a product from oxidative degradation of heme ( Kennedy and Vevers, 1976 ; Ryter and Tyrrel, 2000 ). With the change of biliverdin contents and the involvement of protoporphyrin IX, another eggshell pigment responsible for red-brown color ( Kennedy and Vevers, 1976 ), BGC eggs show a wide spectrum of chroma from blue-greenish eggs laid by chicken and duck ( Wang et al, 2013 ; Chen et al, 2020 ), deep blue eggs laid by eastern bluebird ( Siefferman et al, 2006 ), olive-green eggs laid by elegant crested tinamou to blackish-green eggs laid by emu ( Hamchand et al, 2020 ). BGC was hypothesized to signal female quality to male which allocates more parental care for their offspring depending on antioxidant properties of biliverdin ( Stocker et al, 1987 ; Mereno and Osorno, 2003 ).…”

“…BGC of Dongxiang chickens was caused by a retrovirus (EAV-HP) insertion in 5’ flanking region of SLCO1B3 gene ( Wang et al, 2013 ). The causative gene controlling duck BGC was ABCG2 ( Chen et al, 2020 ). Lueyang black-boned chicken ( LBC ) is one of indigenous breeds laying BGC eggs in China.…”

The study of candidate genes in the improvement of egg production in ducks – a review

“…GWAS and selective sweep analysis reveal genetic loci responsible for feed conversion ratio ( FCR ) of egg production performances in ducks ( Liu et al, 2021b ). The identified SNPs at the cis-regulatory region of ABCG2 gene are related to egg color in Mallard ( Liu et al, 2021a ) and the 2 cis-regulatory SNPs upstream of ABCG2 can cause blue eggshell phenotype in duck ( Chen et al, 2020 ). The summarize findings from several studies using QTL, GWAS and selection signature, and cis-regulatory are presented in Table 2 .…”