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. 2016 Jun 2;12(6):e1006071.
doi: 10.1371/journal.pgen.1006071. eCollection 2016 Jun.

A Complex Structural Variation on Chromosome 27 Leads to the Ectopic Expression of HOXB8 and the Muffs and Beard Phenotype in Chickens

Affiliations

A Complex Structural Variation on Chromosome 27 Leads to the Ectopic Expression of HOXB8 and the Muffs and Beard Phenotype in Chickens

Ying Guo et al. PLoS Genet. .

Abstract

Muffs and beard (Mb) is a phenotype in chickens where groups of elongated feathers gather from both sides of the face (muffs) and below the beak (beard). It is an autosomal, incomplete dominant phenotype encoded by the Muffs and beard (Mb) locus. Here we use genome-wide association (GWA) analysis, linkage analysis, Identity-by-Descent (IBD) mapping, array-CGH, genome re-sequencing and expression analysis to show that the Mb allele causing the Mb phenotype is a derived allele where a complex structural variation (SV) on GGA27 leads to an altered expression of the gene HOXB8. This Mb allele was shown to be completely associated with the Mb phenotype in nine other independent Mb chicken breeds. The Mb allele differs from the wild-type mb allele by three duplications, one in tandem and two that are translocated to that of the tandem repeat around 1.70 Mb on GGA27. The duplications contain total seven annotated genes and their expression was tested during distinct stages of Mb morphogenesis. A continuous high ectopic expression of HOXB8 was found in the facial skin of Mb chickens, strongly suggesting that HOXB8 directs this regional feather-development. In conclusion, our results provide an interesting example of how genomic structural rearrangements alter the regulation of genes leading to novel phenotypes. Further, it again illustrates the value of utilizing derived phenotypes in domestic animals to dissect the genetic basis of developmental traits, herein providing novel insights into the likely role of HOXB8 in feather development and differentiation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Muffs and beard phenotype in Chinese domestic chickens.
(A) Huiyang Bearded (HB) chicken. (B) Silky-feather chicken with Muffs and beard. (C) Newly hatched Mb chicks from the HB broiler breed. (D) Newly hatched wild-type chicks from White Leghorn breed. Male (E) and female (F) Huiyang Bearded chickens that were founders of the HB & HQLA family. Male (G) and female (H) birds from the High quality chicken Line A were also founders of HB & HQLA family.
Fig 2
Fig 2. Results from the Genome-Wide Association, linkage and shared IBD analyses on the Muffs and beard (Mb) trait in HB × HQLA population.
(A) The Manhattan plot from the Genome-wide association analysis for the Mb phenotype at 10 weeks of age. X-axis shows the physical positions in Mb for each marker along the chromosomes, and the y-axis shows -log10 p values for the association tests. (B) A scatter plot illustrating all SNPs tested on GGA27 for the Mb trait at 10 weeks of age. The peak SNP (rs13620154 on GGA27 at 1,959,687 bp) is marked with a filled triangle, while other SNPs are marked with dots. The colors of the dots indicate their LD (r2) with the peak SNP. (C) The whole-genome linkage analysis for the Mb trait at 10 weeks of age. The x-axis shows the genetic positions in centiMorgan (cM) along chromosomes, and the y-axis shows the F values for each position. Vertical dashed lines are used to distinguish the chromosomes. (D) Shared IBD analysis is shown in schematic form in this plot. Each bar represents the Mb locus identified in linkage analysis for one F2 bird. Bars in red refers to chromosomal segments originated from line HB, bars in blue refers to segments originated from line HQLA. Four breakpoints of recombination were indicated by the corresponding SNP names on the x-axis. The dashed lines indicated the boundary defined by corresponding recombinant individuals. The arrows pointed out the location of the final fine-mapped 48-kb interval between two SNPs (rs14301514 and rs16205185).
Fig 3
Fig 3. Illustration of the read depth analysis that confirmed the copy number variations on GGA27 and the following fine-mapping of their structural rearrangements.
(A) The log2 fold-change values from whole genome re-sequencing data illustrating the read depth differences between the HB and HQLA breeds. This analysis validates the presence of the CNVs on GGA27 in chickens with the Muffs and beard phenotype that were previously identified using a CGH array experiment. (B) Schematic illustration of the CNV rearrangements in the Mb locus on GGA27. (C) Genes located within the three duplicated CNV regions include the 3’ sequence of PSMC5 and entire SMARCD2 in CNV1 (green shadow), entire HOXB8 and HOXB7 in CNV2 (blue shadow), 5’ sequence of CCR7, 3’ sequence of KRT222, and entire SMARCE1 in CNV3 (pink shadow).
Fig 4
Fig 4. Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern.
The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the CNVs were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified PCR product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.
Fig 5
Fig 5. Differential expression of genes in the CNV regions in adult tissues.
(A) The ectopic HOXB8 expression in the facial skin was discovered by reverse-transcription PCR analysis. The negative control was non-template PCR reaction. The relative mRNA expression of (B) PSMC5, (C) SMARCD2, (D) HOXB8, (E) HOXB7, (F) CCR7, (G) SMARCE1, and (H) KRT222 in the dorsal skin, liver and facial skin from adults were analyzed by qPCR. Barely detectable expression of HOXB8 was also identified. All tissue samples used came from Mb/Mb, mb/mb, and Mb/mb adults. Mb/Mb, mb/mb, and Mb/mb represent Mb homozygous, wild-type homozygous, and heterozygous genotypes for the Mb locus regulating the Mb trait. ***p<0.001, **p<0.01, *p<0.05.
Fig 6
Fig 6. Relative gene expression during the critical stages of feather development.
Gene expression analyses were performed in dorsal and facial skin tissue from embryos and chicks. Samples from Mb/Mb and mb/mb chickens were collected at embryonic (E) days 7.5, 8.5, 9.5, 10.5 and two weeks after hatching. The expression of (A) PSMC5, (B) SMARCD2, (C) HOXB8, (D) HOXB7, (E) CCR7, (F) SMARCE1, and (G) KRT222 in the dorsal and facial skin were normalized to GAPDH. Ectopic expression of HOXB8 was observed in the Mb chickens during the whole period of feather development.
Fig 7
Fig 7. In situ hybridization scan of HOXB8 expression in chicken embryos with different genotypes at the Mb locus.
Skin samples in (A)—(D) were hybridized with HOXB8 antisense probes, and in (E)—(H) were hybridized with sense probes as the negative control. The ectopic expression of HOXB8 can be observed in the Mb/Mb chickens at the age of E10.5 and E12.

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