Narrowing the wingless-2 mutation to a 227 kb candidate region on chicken chromosome 12

Abstract

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders.

Two Proximally Close Priority Candidate Genes for diplopodia-1, an Autosomal Inherited Craniofacial-Limb Syndrome in the Chicken: MRE11 and GPR83

“…Full details of the array setup and methods can be found in Robb and Delany (2012b). (Note that the same array was utilized to sequence 2 other UCD-congenic developmental mutations, coloboma [co.003] and wingless-2 [wg-2.331]; Robb and Delany 2012b; Robb et al 2013; Webb et al 2018.) The other 2 congenic lines, which mapped to different chromosomes (GGA Z and 12), were utilized as controls for GGA 1 comparisons (Robb et al 2011; Robb 2012; Robb and Delany 2012b).…”

“…The discrimination of non-causative polymorphic elements (naturally occurring in the introgressed region) from the potentially causative polymorphic elements was accomplished by a comparative approach. Polymorphisms were identified by comparing the Dp-1.003 causative (sequenced) region to variants identified in other chicken genetic lines including: the other inbred congenic lines ( wg-2 , Webb et al 2018, and co , Robb et al 2013) utilized on the capture array, the chicken reference genome (UCD-001), or any other previously reported polymorphism (e.g., within NCBI [ex. dbSNP] and the UCSC genome browser).…”

A Premature Stop Codon in RAF1 Is the Priority Candidate Causative Mutation of the Inherited Chicken Wingless-2 Developmental Syndrome

“…The wg-2 mutation is autosomal recessive and embryonic lethal [3]; thus, phenotypically normal heterozygous carriers are viable. The wg-2 congenic inbred line (Wingless-2.331) was created by repeatedly crossing wg-2 carriers into the highly-inbred UCD (University of California, Davis) 331 line, selecting carrier parents each generation (confirmed by test mating) before the resulting birds were crossed inter se and the new line was closed [4,5]. The uniform genetic background conferred by UCD 331 (>99% inbred) [6] facilitated the use of genetic and genomic techniques such as SNP arrays, linkage mapping, and the capture array described herein to study the wg-2 introgressed candidate region.…”

“…The DNA retaining the causative element (also known as the causative region) was first mapped to a 2-Mb span of chromosome 12 (GGA 12) by employing a 3K SNP array; the region’s size was successively narrowed over several generations by genotyping birds at linked SNPs [5]. These techniques ultimately mapped the causative element to a 227-kb region on GGA 12 [5].…”

“…The DNA retaining the causative element (also known as the causative region) was first mapped to a 2-Mb span of chromosome 12 (GGA 12) by employing a 3K SNP array; the region’s size was successively narrowed over several generations by genotyping birds at linked SNPs [5]. These techniques ultimately mapped the causative element to a 227-kb region on GGA 12 [5]. A prior study testing the capture array technique in chicken used two pooled mutant individuals and identified a large amount of sequence variation in the causative region in the wg-2 line [8].…”