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Team Teases Apart Genetics of Ancestral Camouflaging Horse Coat

Dun stallions.

NEW YORK (GenomeWeb) – The relatively pale coat and primitive markings of dun-colored horses appears to reflect an ancestral coat color pattern created by uneven distribution of a pigment-controlling transcription factor in the horse hair follicle, according to a new study in Nature Genetics.

An international team led by investigators in Sweden and the US used SNP genotyping, comparative genomics, and microscopy to show that dun patterning — considered a form of wild-type camouflage — is caused by asymmetric expression of a gene called TBX3 that codes for a pigment-squelching transcription factor.

"In growing hairs, TBX3 mirrors the distribution of melanocytes, the cells that produce pigment," co-first author Kelly McGowan, a researcher affiliated with the HudsonAlpha Institute for Biotechnology and Stanford University, said in a statement. "Our results suggest that TBX3 affects differentiation of specific cells in the hair, creating a microenvironment that inhibits melanocytes from living in the 'inner' half of the hair," McGowan explained.

Still, many domestic horses have acquired intense coloring due to mutations that alter TBX3. The researchers discovered that these non-dun phenotypes come in two flavors: a non-dun1 allele suspected of being present prior to horse domestication and a non-dun2 allele that arose more recently.

Diluted dun coloring appears to be wild type in horses, turning up in the wild Przewalski's horse as well as several wild ass species, the team noted.

Dun horses are known for pale coats punctuated by a dark stripe down the back and, in some cases, zebra-like stripes on their legs or shoulders.

"We were really curious to understand the underlying molecular mechanism why Dun pigment dilution did not affect all parts of the body," co-senior author Leif Andersson, an animal breeding and genetic researcher at Uppsala University, said in a statement.

With that in mind, McGowan, Andersson, and colleagues took a microscopic look at hair from the hindquarters of dun horses, demonstrating that just a portion of the hair is pigmented. They saw the same pattern in Przewalski's horse hairs, while non-dun horse hair and hair from the dun dorsal stripe had more uniform pigment dispersal.

After mapping the Dun coat locus to a chromosome 8 region containing the TBX3 gene, the team searched through existing genome sequences from a heterozygous dun horse and a non-dun horse, uncovering a structural variant downstream of the transcription factor gene. This stretch of sequence spanned more than 1,600 bases in dun horses but was missing from the horse reference genome.

By genotyping hundreds of horses from many different breeds, the researchers found that two-thirds of non-dun horses carry homozygous forms of the deletion. The remaining horses were heterozygous for the deletion or missing it entirely.

But not all of the non-dun horses had the same coat features: non-dun1 had some dun-related markings, while non-dun2 horses did not have the dorsal or leg stripes associated with dun coloring.

Quantitative RT-PCR and RNA sequencing experiments indicated that TBX3 levels are dramatically diminished in skin samples from non-dun1 and non-dun2 animals, leading to a jump in expression for several pigment-related genes.

In dun horses, meanwhile, the team saw uneven TBX3 expression across different parts of the hair follicle and body.

"The region of the body where TBX3 is expressed may account for the stripe pattern, whereas the region of the hair where TBX3 is expressed may account for color intensity," McGowan said.

Turning to sequence data for two-dozen modern domestic, modern Przewalksi's, or ancient horses, the researchers developed a 384-SNP panel to explore genetic differences between dun, non-dun1, and non-dun2 horses in more detail.

Together with genome resequencing data for domestic, Przewalski's, or hybrid horses, the SNP data pointed to two variants that — with or without the chromosome 8 deletion —distinguish between dun, non-dun1, or non-dun2 phenotypes.

Moreover, a non-dun1 allele turned up in ancient horse samples going back some 42,700-years, pointing to the presence of non-dun horses prior to domestication.