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

Chicken blue-greenish coloration (BGC) was known as a classic Mendel trait caused by a retrovirus (EAV-HP) insertion in the SLCO1B3 gene. Lueyang black-boned chicken (LBC) BGC is light and varies continuously, implying that LBC BGC may be controlled by a new molecular mechanism. The aim of this study was to provide an insight into the molecular basis of LBC BGC. The EAV-HP was detected in the BGC (n = 105) and non-BGC LBC (n = 474) using a PCR-based method. The association of SLCO1B3 expression in shell glands and sequence variants in a 1.6-kb region upstream from the transcription start site of SLCO1B3 with eggshell color and biliverdin (pigment for BGC) concentration was studied. Promoter activities of haplotypes in the 1.6-kb region were analyzed by luciferase reporter assay. This study did not found the EAV-HP in BGC and Non-BGC LBC, but detected a strong positive correlation between levels of SLCO1B3 expression in shell glands and biliverdin concentrations. A total of 31 SNP were found in the 1.6-kb region. Twenty-two of 31 SNP formed 42 types of haplotypes in the re-sequenced samples (n = 94). Haplotype 4 was present in higher frequency in the BGC (52%) than Non-BGC (3%). Haplotype 13 was significantly associated with Non-BGC (Non-BGC vs. BGC = 26% vs. 6%). In line with the above associations, Haplotype 4 showed higher (P < 0.05) levels of SLCO1B3 expression in shell glands, biliverdin concentration, and promoter activity than Haplotype 13. This study confirms that LBC BGC is not caused by the EAV-HP, but remains to be associated with the change of SLCO1B3 expression. Haplotype 4 accounts to some extents for the molecular basis of LBC BGC. The new molecular mechanism supports LBC BGC independently evolved.