The study, led by Putri Kusuma Astuti and colleagues at the University of Debrecen, focused on ten adult ewes of the Hortobágyi Racka breed, five black-coated and five white-coated, all of similar age and body weight, raised together under identical conditions and feeding management. Skin samples were collected from the posterior cervical region of each animal at an abattoir in northern Hungary in May 2023, preserved rapidly in RNAlater solution, and frozen at minus seventy degrees Celsius until analysis. By contrasting two coat colors within a single breed, the researchers deliberately minimized the confounding effects of breed-level genetic structure, allowing them to home in on expression differences genuinely associated with pigmentation rather than with broader ancestry.

The team used RNA sequencing, a technique that captures a snapshot of every gene being actively transcribed in a tissue at a given moment. Total RNA was extracted from the skin samples and converted into sequencing libraries using the QuantSeq 3′ mRNA-Seq kit, which focuses on the tail ends of messenger RNA molecules. The libraries were sequenced on an Illumina NovaSeq 6000 platform, generating more than fifty-four million reads across nine animals, as one black-coated sample failed to sequence. The reads were then aligned to the domestic sheep reference genome, the ARS-UI Ramb v3.0 assembly, using the STAR alignment tool, and gene-level counts were quantified with HTSeq. Sophisticated normalization and statistical modeling in the limma and edgeR frameworks, including the voom transformation that accounts for the peculiar mean-variance behavior of count data, allowed the researchers to identify genes whose expression differed significantly between the two color groups.

The results were strikingly one-sided. Of the 108 differentially expressed genes identified using a threshold of fold change greater than 1.5 and p less than 0.05, fully 83 were upregulated in the black-coated sheep, while only 25 were downregulated. The upregulated set was dominated by a tightly coordinated pigmentation network: melanogenesis enzymes such as TYR, TYRP1, and DCT; melanosome structural proteins including PMEL, MLANA, GPNMB, and TRPM1; melanosome transport genes like OCA2, SLC45A2, SLC24A5, and SLC24A4; and upstream melanocyte regulators including PAX3, IRF4, and RAB32. Nine of these genes, among them TYRP1, PMEL, TRPM1, MLANA, SLC24A4, SLC45A2, DCT, OCA2, and DDC, remained significant even after the most stringent multiple-testing correction. In other words, the skin of black Racka sheep is not merely darker; it is running an entire pigment-production program at far higher intensity.

Yet the story did not end with pigment. Functional enrichment analysis using Gene Ontology and KEGG pathway databases revealed that the differentially expressed genes clustered into pathways extending well beyond melanogenesis. The upregulated genes were significantly enriched in tyrosine metabolism, melanogenesis, metabolic pathways, thermogenesis, and oxidative phosphorylation, the latter being the mitochondrial process that generates most of a cell’s ATP. Gene-term networks and protein-protein interaction analysis built from the STRING database reinforced this picture, showing a coherent melanogenesis module centered on the TYR-DCT-MLANA axis, but also revealing separate modules devoted to mitochondrial and redox metabolism, involving genes such as NDUFS2, SLC25A4, QDPR, and AKR1B1, and to cell cycle and proteostasis signaling. Enriched molecular functions included calcium channel activity and calcium-potassium-sodium antiporter activity, reflecting the fact that melanin production inside melanosomes is tightly regulated by organellar ion homeostasis.

Why should coat color be entangled with cellular energy metabolism and stress biology? The authors point to a growing body of evidence that melanin synthesis is a biochemically demanding and redox-active process. Eumelanin, the pigment responsible for dark coloration, is a powerful antioxidant and an effective blocker of ultraviolet radiation, but producing it consumes cellular resources and can generate reactive oxygen species along the way. Pheomelanin, which produces lighter colors, is even more antioxidant-intensive, drawing on the cell’s stores of glutathione and cysteine. This means that the choice between dark and light pigmentation is not simply cosmetic; it carries real consequences for a skin cell’s oxidative balance, energy budget, and resilience under environmental stress such as intense sun and heat.

The study’s findings feed into an unresolved debate in livestock science about whether lighter or darker coats confer an advantage under a warming climate. Light-colored coats physically reflect more solar radiation and absorb less heat, which would seem advantageous in hot environments. Yet the evidence is mixed: some studies of grazing sheep in hot-arid conditions have found that black-coated animals maintained lower rectal temperatures than white-coated ones, while other research on goats under prolonged heat stress found white and mixed coats most adaptable. A recent gene-editing experiment in Holstein cattle, in which the PMEL gene was modified to dilute coat color, showed that lighter calves absorbed significantly less heat and light than their darker counterparts. The Racka data do not settle the question, but they suggest a mechanism by which dark pigmentation could carry its own compensatory benefits: the upregulation of genes like TRPM1, implicated in DNA repair and calcium signaling during heat stress, and TYRP1, reported to regulate oxidative stress and support cellular adaptation in extreme conditions.

There is also an intriguing immunological dimension. Melanocytes, the pigment-producing cells of the skin, are increasingly understood as active participants in immune signaling. They can present antigens to T lymphocytes, express immune-interaction molecules such as ICAM-1 and CD40, and release cytokines that shape inflammatory responses. Highly melanized melanocytes may suppress inflammation more effectively, partly through the immunosuppressive effects of L-DOPA and related compounds, whereas lightly pigmented skin may mount stronger cytokine-mediated immune signaling. The researchers also note that pigment regulation and the body’s stress-hormone system share biochemical machinery, notably the proopiomelanocortin molecule, which feeds into both pigmentation and cortisol synthesis. This overlap raises the possibility that coat color and stress physiology are coupled through shared hormonal pathways, a hypothesis the authors frame as speculative but testable.

The team validated their sequencing results using quantitative reverse-transcription PCR on ten selected genes spanning pigmentation, immune, and metabolic functions. Eight of the ten genes were confirmed to be significantly more highly expressed in black sheep skin, with TYRP1 showing the largest difference between the groups. The authors are careful, however, to spell out the limitations of their work. With only four black and five white animals, statistical power is modest, and the study included no direct measurements of heat tolerance, UV response, oxidative stress markers, or skin temperature. The links between pigmentation and adaptation remain, in their words, hypothesis-generating rather than causal, awaiting protein-level assays and controlled heat-challenge experiments in larger populations.

Even with those caveats, the study delivers a compelling molecular portrait of a famous breed and a provocative idea: that the color of a sheep’s fleece is the visible tip of a much larger biological iceberg. Beneath the black and white coats of the Hortobágyi Racka lie coordinated differences in ion transport, mitochondrial bioenergetics, redox buffering, and proteostasis, suggesting that pigmentation and environmental resilience may have evolved together. As climate change intensifies heat stress on livestock worldwide, understanding how coat color genes intertwine with cellular stress machinery could help breeders select animals not just for appearance, but for the hidden physiology that keeps them thriving in an increasingly hostile environment.

Subject of Research: Transcriptomic differences between black- and white-coated Hortobágyi Racka sheep skin

Article Title: Coat Color‐Associated Transcriptomic Differences of Black and White Hortobágyi Racka Sheep Using the RNA‐Seq Technique

Article References: Astuti, P. K., Bagi, Z., Bodrogi, L., & Kusza, S. (2026). Coat Color‐Associated Transcriptomic Differences of Black and White Hortobágyi Racka Sheep Using the RNA‐Seq Technique. Ecology and Evolution, 16(10), Article e74397. https://doi.org/10.1002/ece3.74397

Image Credits: AI Generated

DOI: 10.1002/ece3.74397

Keywords: Hortobágyi Racka sheep, coat color, RNA-seq, melanogenesis, transcriptomics, pigmentation, heat stress, oxidative stress, mitochondrial metabolism, TYRP1, melanocytes, livestock adaptation

The research team assembled an unusually powerful resource for dissecting the trait: a population of 932 second-generation offspring produced by intercrossing white Duroc boars with Erhualian sows. Because juvenile striping appears in this intercross population alongside solid-colored animals, the pedigree provided the statistical contrast needed to hunt for the trait’s genetic architecture. The researchers performed genome-wide association studies, or GWAS, using both chip-based genotyping and imputation-based approaches that leverage whole-genome sequence data to fill in millions of additional variants. This dual strategy allowed them to scan the pig genome for DNA markers that reliably track with the presence or absence of stripes across hundreds of animals, a standard technique in quantitative genetics that correlates inherited variation with observable traits.

The scan produced two suggestive association signals, one on pig chromosome 6 and another on chromosome 8. On chromosome 6, the strongest positional candidate was MC1R, the melanocortin 1 receptor gene that sits at the heart of the melanin switching system in mammals. MC1R acts as a molecular dial that determines whether pigment cells produce dark eumelanin or reddish-yellow pheomelanin, and mutations in this gene are responsible for some of the most famous coat color variants in dogs, horses, cattle, and pigs. On chromosome 8, the candidate gene was KIT, a receptor tyrosine kinase gene famous for its role in the migration and survival of pigment-producing cells during embryonic development. KIT disruptions classically produce white spotting patterns, because pigment cell precursors fail to reach their destinations in the skin.

Genotyping of the candidate mutations revealed a striking pattern. The striped pigs in the study did not carry the MC1R c.67_68insCC insertion mutation, and they also lacked the KIT duplication that produces characteristic white patterning in domestic pigs. This tells a coherent story: these two well-known pigment mutations, when present, appear to disrupt or erase the juvenile stripe pattern entirely. But the inverse did not hold. Some animals in the population that lacked both mutations still failed to show stripes, indicating that the presence of a striped coat cannot be explained by these two loci alone. Something else in the genome must be required to switch the stripe program on, prompting the team to dig deeper.

That deeper search took the form of a conditional GWAS, a statistical refinement in which the researchers re-ran the association analysis while accounting for the effects of the already-identified loci. By statistically removing the contribution of the MC1R and KIT regions, the analysis could expose additional genetic signals that had been masked by the stronger effects. This approach uncovered a further genomic locus, again on chromosome 8, and the gene nearest to the new association signal was CORIN. CORIN encodes a membrane-bound serine protease with a well-documented role in pigmentation biology; in other mammals, corin mutations are known to shift the balance of the melanocortin pathway and alter coat color patterning. Its emergence from the conditional analysis suggests that CORIN may act as a modifier that cooperates with the primary pigment genes to permit the striped pattern to develop.

Identifying the genes involved was only half the puzzle. Juvenile striping is fundamentally a spatial phenomenon: neighboring regions of the same dorsal skin follow different pigment programs, producing alternating dark and light bands. To understand how one patch of skin knows to go dark while its neighbor stays light, the team collected skin samples from the dark stripes and light stripes of three 2-day-old striped piglets and subjected them to parallel comparative transcriptome sequencing, or RNA-seq, which measures which genes are active in each tissue. The spatial comparison revealed that TYRP1, tyrosinase-related protein 1, was significantly upregulated in the dark-stripe skin. TYRP1 is an enzyme working directly in the melanin biosynthesis pathway, supporting the production of eumelanin, so its elevated activity in dark bands fits neatly with the visible difference in pigment deposition between the two stripe types.

The most technically ambitious component of the study was its use of ATAC-seq, the assay for transposase-accessible chromatin using sequencing. This method exploits a laboratory enzyme that preferentially inserts sequencing adapters into open, loosely packed stretches of chromatin, thereby creating a genome-wide map of regulatory regions where DNA is physically accessible to transcription factors. Because gene activity is largely controlled by which regulatory switches are open at any given moment, ATAC-seq reveals the layer of regulation that sits above the genome sequence itself. The team profiled accessible chromatin separately in dark-stripe and light-stripe skin, identifying peaks specific to each compartment and quantifying their quality using standard metrics such as the fraction of reads falling within peaks.

That chromatin map delivered a compelling lead. The researchers identified an accessible chromatin region located 1.8 kilobases downstream of the TYRP1 gene, a position consistent with a distal regulatory element such as an enhancer. Enhancers are DNA sequences that can lie far from the gene they control, binding transcription factor proteins and boosting gene expression in specific tissues or, in this case, specific skin territories. The analysis suggested that this downstream accessible region may contain binding sites for SOX10, the SRY-box transcription factor 10, a master regulator of the neural crest lineage from which pigment cells are born. If SOX10 or related factors bind preferentially to this open region in dark-stripe skin, it could explain how TYRP1 expression is boosted in the dark bands while remaining lower in the light ones, providing a plausible molecular mechanism for the spatial patterning of the stripes.

Taken together, the study sketches a layered genetic model of porcine juvenile striping. At the top level, the presence or absence of disruptive pigment mutations matters: the MC1R c.67_68insCC insertion and the KIT duplication appear capable of overriding the stripe program, which is why striped coats vanish from most domestic pig lineages where these mutations have been selected, whether deliberately or incidentally, during breeding. Below that, a conditional locus near CORIN likely contributes to whether the stripe program can be expressed at all in animals free of the disruptive mutations. And at the finest level, the pattern itself is executed through spatially restricted gene regulation, with an accessible chromatin element downstream of TYRP1, potentially controlled by SOX10-family transcription factors, driving higher melanogenic activity in dark-stripe skin than in adjacent light-stripe skin.

The work also demonstrates the power of integrating multiple genomic technologies on a single well-structured population. GWAS pinpointed where in the genome the relevant variation lies, RNA-seq revealed which genes behave differently between the patterned territories, and ATAC-seq illuminated the regulatory landscape that might explain that differential behavior. Each method alone would have left the picture incomplete; the association signals could not say how stripes are patterned, and the expression data could not say why TYRP1 differs between bands. For pig breeders and evolutionary biologists alike, the findings carry practical weight. Understanding which mutations erase juvenile striping clarifies how domestication reshaped coat appearance, and the identification of CORIN and the TYRP1 regulatory region provides molecular markers that could be used to track or manipulate the trait. More broadly, the striped piglet joins the growing list of mammalian color-pattern systems, from zebrafish stripes to big-cat rosettes, in which the interplay of pigment genes and chromatin-level regulation is being decoded base pair by base pair.

Subject of Research: The genetic and molecular mechanisms underlying juvenile stripe coat patterning in pigs

Article Title: Unraveling porcine juvenile striping: insights from the integration of genomics, transcriptomics and ATAC-seq

Article References: Xiong, S., Cui, D., Xiao, S., Li, L., & Guo, Y. (2026). Unraveling porcine juvenile striping: insights from the integration of genomics, transcriptomics and ATAC-seq. BMC Genomics. https://doi.org/10.1186/s12864-026-13377-4

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13377-4

Keywords: juvenile stripes, wild boar, pig genetics, GWAS, MC1R, KIT, CORIN, TYRP1, ATAC-seq, RNA-seq, SOX10, coat color