Two new structural mutations in the 5' region of the ASIP gene cause diluted feather color phenotypes in Japanese quail

Abstract

Background: In quail, two feather colour phenotypes i.e. fawn-2/beige and yellow are associated with the ASIP locus. The aim of our study was to characterize the structural modifications within this locus that explain the yellow mutation (large deletion) and the fawn-2/beige mutation (assumed to be caused by a different structural modification).

Results: For the yellow phenotype, we identified a complex mutation that involves a 141,162-bp long deletion. For the fawn-2/beige phenotype, we identified a 71-kb tandem duplication that comprises one unchanged copy of ASIP and one copy present in the ITCH-ASIP fusion gene, which leads to a transcript coding for a normal ASIP protein. Although this agrees with previous reports that reported an increased level of ASIP transcripts in the skin of mutant animals, we show that in the skin from fawn-2/beige embryos, this level is higher than expected with a simple duplication of the ASIP gene. Thus, we hypothesize that the 5' region of the ITCH-ASIP fusion gene leads to a higher transcription level than the 5' region of the ASIP gene.

Conclusions: We were able to conclude that the fawn-2 and beige phenotypes are caused by the same allele at the ASIP locus. Both of the associated mutations fawn-2/beige and yellow lead to the formation of a fusion gene, which encodes a transcript for the ASIP protein. In both cases, transcription of ASIP depends on the promoter of a different gene, which includes alternative up-regulating sequences. However, we cannot exclude the possibility that the loss of the 5' region of the ASIP gene itself has additional impacts, especially for the fawn-2/beige mutation. In addition, in several other species including mammals, the existence of other dominant gain-of-function structural modifications that are localized upstream of the ASIP coding sequences has been reported, which supports our hypothesis that repressors in the 5' region of ASIP are absent in the fawn-2/beige mutant.

Fig. 1

ASIP locus in quail. a Schematic representation of the ASIP locus in quail. Each gene is represented with a specific color: the coding exons are schematized by dark rectangles while the 5′ UTR exons are represented in a lighter shade. The 5′ UTR exons with potential transcription start sites are symbolized by a rectangle with an arrow. b Presentation of the ASIP gene and ASIP transcripts. We expect nine types of transcripts encoding the same ASIP protein. For the exons, we used the names proposed by Gluckman and Mundy [23]. The coding exons are Ce1, Ce2 and Ce3. We did not consider the ASIP transcript-6 as proposed by [23], because the sequence of the 5′ UTR exon relative to this transcript (novel6) was not included in this region of quail chromosome 20. The double pink arrow shows the 21.4-kb region highlighted in this study (see also Fig. 3)

Fig. 2

Characterization of the yellow and fawn-2/beige mutations. a Schematic characterization of the genomic region in quail carrying the yellow deletion. The breakpoint upstream from the deletion was named 5′BKPT-Yel (for 5′ breakpoint), the downstream breakpoint was named 3′BKPT-Yel, and the junction point was named JUNCT-Yel. The colored arrows represent the different primer pairs used to validate the variant with a deletion. The fragment JUNCT-Yel was sequenced and includes an insertion of 241 bp of unknown origin (represented in purple). b Schematic characterization of the genomic region in quail carrying the fawn-2/beige tandem duplication. The breakpoints upstream and downstream from the duplication were named 5′BKPT-fawn-2 and 3′BKPT-fawn-2, respectively, and the junction point was named JUNCT-fawn-2. The colored arrows represent the different primer pairs used to validate the variants with a deletion. The fragment JUNCT-fawn-2 was sequenced and is represented in brown

Fig. 3

ASIP locus in yellow and fawn-2/beige quail genomes. The deletion characterized in yellow and the tandem duplication identified in fawn-2/beige quails concern the same region. The gene organization on the reference genome is drawn in the middle. The two structural events both lead to the formation of a fusion gene. As on Fig. 1, 5′ UTR exons that could be the starting site of a transcript are symbolized by a rectangle with an arrow. In animals  heterozygous for the yellow allele, the fusion gene RALY-ASIP can produce a fusion transcript [15] coding for a normal ASIP protein. In animals carrying the fawn-2/beige allele, the fusion gene ITCH-ASIP can produce three possible transcripts coding for the ASIP protein from the three exons including a TSS

Fig. 4

Quantitative expression of the transcripts from the ASIP region in fawn-2/beige versus wild-type quails 15-days embryos (skin). Blue: wild-type embryos [n = 10, excepted for ASIP-tr2 (n = 6) and ASIP-tr3 (n = 8)]. Brown: embryos homozygous for the fawn-2/beige mutation (n = 9, except for ASIP-tr2 and ASIP-tr3 where n = 7). The abundance of transcripts is expressed in arbitrary units (AU), values are mean ± standard deviation. ***Significant difference with p value < 0.001. The different graphs represent the transcription from: a ITCH; b AHCY; c the ITCH-ASIP fused gene; d ASIP-tr2 and e ASIP-tr3 representing alternative transcripts from the ASIP and ITCH-ASIP fused genes; f overall amount of all ASIP-coding transcripts