Refine localizations of functional variants affecting eggshell color of Lueyang black-boned chicken in the SLCO1B3

Table eggs with color-uniformity shell are visually attractive for consumers. Lueyang black-boned chicken (LBC) lays colorful eggs, which is unde-sirable for sale of table eggs, but provides a segregating population for mapping functional variants affecting eggshell color. SLCO1B3 was identified as the causative gene for blue eggs in the Dongxiang , Araucana chick-ens. The aim of this study is to map functional variants associated with chicken eggshell color in the SLCO1B3. Eggshell color of LBC (n = 383) was measured using the L*a*b color space. SLCO1B3 was resequencing using a subset (n = 30) of 383 samples. Linkage disequilibrium among 139 SNP was analyzed. Association of 16 SNP in the SLCO1B3 and 8 in CPOX, ALAS1, ABCG2 genes with L*a*b were tested by a polygenic model (LMM) and a polygenic/oligogenic mixed model (BSLMM). Chromatin state annotations were retrieved from the UCSC database. Effect of SLCO1B3 variants distributed in mapping and upstream 1.6-kb regions on promoter activities were analyzed using dual-luciferase reporter assay. One hundred and thirty-nine variants maintained low linkage disequilibrium with80% of r2 less than 0.226. Fifteen SLCO1B3 variants were significantly associated with a*, of which 1B3_SNP108 was showed the strongest association , the largest effect on a*. In the BSLMM, 1B3_SNP108 alone appeared in the Markov chain Monte Carlo as major variants in 100% of posterior inclusion probabil-ity. None of variants in CPOX, ALAS1, ABCG2 were significantly associated with color indexes except that 2 ALAS1 variants were associated with L*. 1B3_SNP108 distributes in the Intron4 where 6 active enhancers and 1 ATAC island were enriched. However, 1B3_SNP108-containing constructs showed negligible activities in the reporter assay. No significant differences of activities between haplotypes were found for five 50- deleted promoter constructs. The data recognizes 1B3_SNP108 as a valuable marker for breeding of egg-shell color. Functional variants are localized in the region adjacent to the 1B3_SNP108 due to low linkage disequilibrium in the LBC. Our findings extend the role of SLCO1B3 from a causative gene for blue eggs to a major regulator driving continuous variation of LBC eggshell color.

Automated summary

The study investigates the functional variants associated with chicken eggshell color in the SLCO1B3 gene by measuring the eggshell color of Lueyang black-boned chickens and analyzing the genetic data. It identifies 15 variants in SLCO1B3 that are significantly associated with eggshell color. However, the functional analysis shows that one specific variant, 1B3_SNP108, does not have a significant effect on promoter activity. This study highlights the importance of SLCO1B3 and its variants in breeding eggshell color.