Figure 1.
The eggshell color and the expression of SLCO1B3 in the uterus of blue-shelled and non-blue-shelled chickens.
(A) Eggshell colors of homozygous blue-shelled (O*LC/O*LC), heterozygous blue-shelled (O*LC/O*N) and brown-shelled (O*N/O*N) of Dongxiang chickens. (B) The expression analysis of the four genes which are located in the refined region between markers L4 and L5 in the uterus of Dongxiang blue-shelled hens (n = 16) and Dongxiang non-blue-shelled hens (n = 16) by RT-PCRs. DX-BS: blue-shelled Dongxiang, DX-NBS: non-blue-shelled Dongxiang. (C) Analysis of SLCO1B3 expression in the uterus. Expression data were presented as fold relative to heterozygous blue-shelled Dongxiang chickens (O*LC/O*N) by the comparative Ct method (2−ΔΔCt). SLCO1B3 is exclusively expressed in blue-shelled chickens, and the amount of SLCO1B3 transcripts in homozygous blue-shelled chicken (O*LC/O*LC) is approximately two to three folds of that in heterozygous individuals (O*LC/O*N). DX-BS: blue-shelled Dongxiang, LS-BS: blue-shelled Lushi, DX-NBS: non-blue-shelled Dongxiang, LS-NBS: non-blue-shelled Lushi. (D) Micrographs of cDNA in situ hybridization for SLCO1B3 mRNA in the uterus from blue-shelled and non-blue-shelled chickens. (E) Differential expression of SLCO1B3 transcript in the uterus from blue-shelled heterozygotes using genomic DNA (gDNA) as control. The polymorphic position g. 67334934 G>T was used to monitor differential expression using pyrosequencing. T and G at this position correspond to blue-shell and non-blue-shell alleles, respectively. Due to two Ts next to the SNP at 3′end, the peaks of T in the schema contain three Ts including one T from blue-shell allele and two Ts from non-blue-shell allele. The percent expression on the peaks for T and G are the T or G at g. 67334934 G>T. (F) Summary of the detection of differential expression in uterus and liver from six heterozygotous blue-shelled (O*LC/O*N) birds.
Figure 2.
Refined localization of O on chicken chromosome 1.
The black bar represents the short arm of chicken chromosome 1. Numbers in middle column are recombination fractions between markers and O, the corresponding LOD scores are shown in the last column. Here, O is assigned to the interval of L4 and L5 at Chr1: 67296991–67419904. Four candidate genes in the interval, EAV-HP insertion with recombination fraction and LOD in the parentheses, SRY (sex determining region Y)-box 5 (SOX5) for pea comb phenotype [17], ev1 marker [16] are indicated at the left of the black bar.
Table 1.
Distribution of allelic frequencies of SNPs and EAV-HP in SLCO1B3 in several blue-shelled and non-blue-shelled breeds.
Figure 3.
The EAV-HP insertion in blue-shelled chickens.
(A) Schematic diagram of the relationship of a complete avian leukosis virus and the EAV-HP retrovirus. Some putative cis-regulatory elements in long terminal repeat (LTR) are underlined. The diagram was redrawn by referencing Sacco et al. [22]. (B) Arrangement of EAV-HP retrovirus and SLCO1B3 in chicken genome. EAV-HP integrates into the 5′ end of SLCO1B3 in inverted orientation. When SLCO1B3 is transcribed, an extra 24-bp sequence from the EAV-HP is also compiled into SLCO1B3 transcript. The asterisk indicates the site of 24-bp sequence from EAV-HP, and arrows indicate the direction of transcription. (C) EAV-HP junction sites in blue-shelled chickens of Dongxiang, Lushi and Araucana breeds. The arrows show the EAV-HP inserted sites and the underlined sequences are the EAV-HP integration specific sequences. The sequences in red denote the inserted EAV-HP.
Table 2.
EAV-HP insertion distributions in various populations.