Genome-Wide Association Mapping and Identification of Candidate Genes for the Rumpless and Ear-tufted Traits of the Araucana Chicken

Araucana chickens are known for their rounded, tailless rumps and tufted ears. Inheritance studies have shown that the rumpless (Rp) and ear-tufted (Et) loci each act in an autosomal dominant fashion, segregate independently, and are associated with an increased rate of embryonic mortality. To find genomic regions associated with Rp and Et, we generated genome-wide SNP profiles for a diverse population of 60 Araucana chickens using the 60 K chicken SNP BeadChip. Genome-wide association studies using 40 rumpless and 11 tailed birds showed a strong association with rumpless on Gga 2 (P raw = 2.45×10−10, P genome = 0.00575), and analysis of genotypes revealed a 2.14 Mb haplotype shared by all rumpless birds. Within this haplotype, a 0.74 Mb critical interval containing two Iroquois homeobox genes, Irx1 and Irx2, was unique to rumpless Araucana chickens. Irx1 and Irx2 are central for developmental prepatterning, but neither gene is known to have a role in mechanisms leading to caudal development. A second genome-wide association analysis using 30 ear-tufted and 28 non-tufted birds revealed an association with tufted on Gga 15 (P raw = 6.61×10−7, P genome = 0.0981). We identified a 0.58 Mb haplotype common to tufted birds and harboring 7 genes. Because homozygosity for Et is nearly 100% lethal, we employed a heterozygosity mapping approach to prioritize candidate gene selection. A 60 kb region heterozygous in all Araucana chickens contains the complete coding sequence for TBX1 and partial sequence for GNB1L. TBX1 is an important transcriptional regulator of embryonic development and a key genetic determinant of human DiGeorge syndrome. Herein, we describe localization of Rp and Et and identification of positional candidate genes.


Introduction
There are hundreds of domestic chicken breeds worldwide [1]. Breeds were generally developed for meat and egg production, but morphological traits, plumage color, and other distinctive characteristics were also selected. The Araucana chicken, originally from Chile, is a multi-purpose breed initially established for its blue-shelled eggs [1,2]. Araucana chickens are also known for two other distinguishing traits: a rounded, tailless rump and protruding ear-tufts. Although these traits segregate in the population, the United States Araucana breed standard requires show birds to possess both phenotypes.
The rumpless phenotype is characterized by the absence of all free caudal vertebrae and the uropygial gland [3]. Without underlying skeletal support, birds with caudal truncation lack a fleshy rump and tail feathers [3]. An intermediate rumpless phenotype, wherein some caudal vertebrae are present but irregularly fused together, is thought to result from a modifier gene introduced through crosses with non-Araucana tailed chickens [3,4]. The rumpless phenotype arises from a defect in caudal patterning that is controlled by a dominant gene (Rp) [3]. Rumpless Araucana chickens may be heterozygous or homozygous for this locus. In test matings, all rumpless intermediates were determined to be heterozygous (Rp/rp + ) [3]. Homozygosity is underrepresented among chicks from rumpless to rumpless matings, indicating that the Rp/Rp genotype has reduced viability [3,5]. Birds having at least one copy of Rp have increased mortality in the embryo stage, with death occurring at 17 to 21 days of incubation [3]. Rumpless birds also have reduced fecundity as adults [3].
Ear-tufts are feather-covered, epidermal protrusions originating near the ear canal ( Figure 1). The mass of tissue forming the protrusion, or peduncle, is believed to develop as a result of the incomplete fusion of the hyomandibular arches, and it can vary in position and length (from 2 mm to 2 cm) [6,7]. Tufted chickens may also have structural rearrangement of the ears [6]. Abnormalities include irregularly shaped external ear openings and shortened or absent external auditory canals [6].
Inheritance studies indicate that tufted is governed by a dominant locus, Et [6,8]. Test matings show that all tufted birds are heterozygous (Et/et + ) and that homozygosity for Et is lethal at about 17-19 days of incubation [6,8]. Lethality among a portion of heterozygous birds is also reported, appearing to occur at 20-21 days of incubation [8]. Post-hatch mortality is significantly higher among tufted chickens [6,8].
Because tufts can occur unilaterally or bilaterally and may differ in size from one side to the other, Et is proposed to have variable expressivity [6]. In addition, a paucity of tufted progeny from mating studies in 1978 suggests reduced penetrance of the tufted locus [6]. In 1981, Somes and Pabilonia identified a tufted male that produced excessive tufted progeny when crossed with an et + / et + White Leghorn (86%), and they speculated that Et/Et birds may occasionally reach maturity [8]. The non-tufted chicks from the Et/Et male produced tufted progeny when crossed with an et + / et + White Leghorn, indicating that their predicted genotype does not match their phenotype, providing further evidence for variable penetrance.
The aim of our investigation was to localize the genetic bases for the rumpless and tufted phenotypes of the Araucana chicken. To this end, we generated genome-wide SNP profiles for 60 Araucana chickens using the 60 K chicken SNP BeadChip [9]. Using a genome-wide association approach, we elucidate the chromosomal regions harboring Rp and Et and identify strong candidate genes for each trait.

Results
Case/control analyses were carried out using 40 rumpless and 11 tailed Araucana chickens ( Figure 2a). Seven birds described as having partial tails by their breeders were excluded from the rumpless association analysis because of uncertainty concerning their phenotype. A total of 191 SNPs were associated with the rumpless phenotype (P raw #0.0001), 72 of which were located on Gga 2 ( Figure 2b). The most significant result obtained was for SNP Gga_rs13637596, located on chromosome 2 at position 88.95 Mb (P raw = 2.45610 210 , P genome = 0.00575). The next two most significant results were for proximal SNPs located at 89.17 Mb (P raw = 1.20610 29 , P genome = 0.0119) and 89.19 Mb (P raw = 1.20610 29 , P genome = 0.0119).
Analysis of genotypes in the Gga 2 region revealed a 2.14 Mb haplotype (87.99-90.13 Mb) predicted to contain five genes ( Figure 3). All 40 rumpless birds had at least one copy of the haplotype: 18 were homozygous and 22 were heterozygous. Partial tailed birds were heterozygous. The haplotype was absent in its entirety from the 11 tailed birds. Three tailed birds were heterozygous for partial blocks of the haplotype and further delimit the critical interval to 0.74 Mb (88.77-89.51 Mb). This region contains two candidate genes: Irx1 and Irx2.
Analyses for association with the tufted phenotype, using 30 cases and 28 controls, resulted in 31 significant SNPs, 11 of which map to Gga 15 ( Figure 2c). The most significant results were for SNPs Gga_rs10730189 (P raw = 6.61610 27 , P genome = 0.0981) and Gga_rs15762547 (P raw = 9.19610 27 , P genome = 0.118), located at positions 1.33 Mb and 1.30 Mb on chromosome 15, respectively. Four other proximal SNPs also reached significance (Figure 2d).
Analysis of genotypes reveals that 29 of 30 tufted birds shared a haplotype extending from the telomere of Gga 15 to position 1.75 Mb. These birds were heterozygous for the complete haplotype. Two of 28 non-tufted birds were also heterozygous for the haplotype in its entirety. A single tufted bird shared only part of the 1.75 Mb haplotype, defining a 0.58 Mb (0.90-1.48 Mb) critical interval that is heterozygous in all 30 tufted birds and contains 7 genes. Because tufted is nearly always recessive lethal, blocks of homozygosity for the tufted haplotype were identified to  reduce the number of candidate genes. Homozygosity blocks in three birds flank a 60 kb interval harboring two genes: TBX1 and GNB1L (Figure 4).

Discussion
In this study, we used genome-wide SNP profiles to localize genes causative for two breed-defining phenotypes of Araucana chickens, rumpless and ear-tufts. We took advantage of the fact that both traits segregate independently in the population by using a single data set to carry out an association analysis for each trait. Haplotype analyses based on inheritance patterns were used to identify positional candidate genes for both traits.
We identified a rumpless haplotype spanning 2.14 Mb and five genes on chromosome 2. The haplotype is present in the heterozygous or homozygous state in rumpless birds. All 7 birds with partial tails are heterozygous for the rumpless haplotype and likely represent the intermediate phenotype described by Dunn and Landauer [3]. Because rumpless is dominant and fully penetrant, we further delimited the critical interval by identifying regions of the haplotype shared by tailed birds. A 0.74 Mb region common to all rumpless birds, and absent from 11 tailed birds, harbors Rp.
These data reveal that Rp maps to a region of Gga 2 that is distinct from the predicted location of genes previously associated with caudal truncation [10][11][12][13][14]. The 0.74 Mb critical interval contains the Iroquois homeobox genes, Irx1 and Irx2. The Iroquois genes encode transcription factors that function in patterning and regionalization of tissues early in development [15]. Irx1 and Irx2 are prepattern and proneural genes first identified in Drosophila and Xenopus [16,17]. Studies of gene function suggest that Irx genes have redundant yet distinct roles in development [18,19]. Irx genes have been knocked out in mice and zebrafish with little effect on tail development [19][20][21][22][23]. However, the rumpless phenotype is dominant, suggesting that misexpression of Irx1 or Irx2 may underlie the trait, rather than loss of function.
We identified SNPs on Gga 15 that are strongly associated with the tufted phenotype and define a 0.58 Mb haplotype for which all tufted birds in our cohort are heterozygous. No birds are homozygous for the complete tufted haplotype. These data support conclusions from previous inheritance studies that suggest nearly 100% of tufted birds are heterozygous, and that Et/Et is lethal [6,8].
Two non-tufted Araucana chickens are heterozygous for the tufted haplotype. These birds may signify reduced penetrance. Penetrance of the tufted allele is estimated to range from 86% to 96% [6,8]. Based on the assigned phenotypes and the associated haplotype, we observed 94% penetrance in our cohort. Alternatively, these birds may have been incorrectly phenotyped by their breeders due to short peduncles or missing protruding feathers.
The 0.58 Mb haplotype harbors 7 protein-coding genes. Unlike rumpless, identification of the tufted haplotype in non-tufted birds could not be used to narrow the critical interval because of reduced penetrance. However, because homozygosity for Et is nearly always lethal, we were able to prioritize candidate gene selection using heterozygosity mapping. Tufted birds with blocks of homozygosity extending into the 0.58 Mb common haplotype were identified, and these regions were deemed less likely to  harbor the Et locus. These data indicate that Et is located in a region containing partial coding sequence for GNB1L, which encodes a protein implicated in neuropsychiatric disorders [24,25], and complete coding sequence for TBX1 [26], an important transcriptional regulator of embryonic development.
Haploinsufficiency for TBX1 is considered to be the key genetic determinant of human DiGeorge syndrome (DGS), which is caused by a heterozygous chromosomal deletion of 22q11.2 [27]. While the clinical phenotype is highly variable, DGS is characterized by craniofacial and cardiovascular abnormalities. Malformations in DGS are attributed to disturbed segmentation and patterning of the pharyngeal structures [28]. Auricular defects common in DGS include narrow or absent external ear canal and protruding ears [29]. Homozygosity for null mutations of TBX1 in mice and zebrafish causes a range of phenotypic effects similar to DGS, including abnormal ear development [30,31]. Based on phenotypic similarities between the malformations causing ear tufts and DGS, TBX1 is a highly plausible candidate gene and the primary focus of ongoing work to identify the genetic basis for eartufts in Araucana chickens.
In conclusion, we used genome-wide association and haplotype analyses to localize Rp and Et to chicken chromosomes 2 and 15, respectively. In addition, we identified candidate genes that are immediate targets for future work.

Ethics Statement
This study was approved by the Clemson University IACUC protocol number 2011-041 and IBC protocol number 2010-041.

Study Cohort
Whole blood for DNA was collected from 6 different flocks of Araucana chickens from the United States. Phenotypic information and photographs, when available, were provided by owners. Birds with tufts of any size and on either side of the head were classified as tufted. Because both traits segregate in the Araucana population, birds were selected to ensure that the phenotypes were balanced. Our study cohort comprised 60 Araucana chickens: 21 rumpless/tufted birds, 20 rumpless/non-tufted birds, 7 tailed/nontufted birds, 5 tailed/tufted birds, 5 partial/tufted birds, and 2 partial/non-tufted birds. Genomic DNA was isolated using the DNeasy blood and tissue kit (QIAGEN, Valencia, USA) and adjusted to a concentration of 50 ng/uL.

Genome-wide Association Mapping
SNP genotypes were generated using the Illumina 60 K chicken SNP BeadChip, which has 57,636 SNPs across chromosomes 1 through 28, Z, W, and two unmapped linkage groups [9]. BeadChips were processed by DNA Landmarks (Quebec, Canada), according to manufacturer's protocols. Raw data files were analyzed using GenomeStudio's Genotyping Module to generate SNP calls. The PLINK Input Report Plug-in v2.1.1 was used to format the data. For analysis, Gga 27, Gga 28, Gga Z, Gga W, and microchromosomes were all identified as chromosome zero. Case/control analyses using 56,685 SNPs were performed using PLINK [32]. Two birds with excessive missing data were excluded from all analyses. By convention, P raw values #0.0001 were considered significant. Permutation testing, using 100,000 iterations, was carried out using PLINK.