Mammalian coat color exhibits a wide range of shades and is dictated by melanin production in melanocytes (melanogenesis). Melanogenesis involves a complex molecular regulation . In order to understand the molecular mechanisms of coat color formation, previous studies have reported the generation of ESTs from both sheep and alpaca skin through traditional Sanger sequencing [16, 17]. A previous study examined differences in gene expression associated with black spots in fleece of Corriedale sheep using microarray technology . To further investigate genes that may play important roles in sheep skin, particularly in fiber/coat pigmentation, over 100 million transcriptome sequence reads were generated from white and black sheep skin using the Illumina technology. From these reads there were 37,768 known unigenes identified as expressed in sheep skin, among which 2,235 were differentially expressed in black versus white sheep skin. It is acknowledged that study design was not optimal due to limited biological replication because single pooled samples (n = 3 per coat color) were used in transcriptome sequencing analysis and the same three samples from white sheep skin and from black sheep skin were used individually for quantitative real time PCR validation of the sequencing results. Despite such limitations, results have significantly enhanced understanding of sheep skin transcriptome composition and potential differences in gene expression associated with coat color that are foundational to further study in the future.
Genes encoding for ribosomal proteins, keratin family members and keratin associated proteins were among the most highly expressed genes detected in sheep skin. The ribosome is a central player in the translation system and its function is to decode the nucleotide sequence carried by the mRNA and convert it into an amino acid primary structure . Abundant presence of ribosomal proteins in sheep skin suggests the importance of high rates of protein translation in sheep skin. In channel catfish skin, the expression of ribosomal proteins was high presumably due to higher levels of translational activities [20, 21]. Of the top 30 highly expressed genes in sheep skin, all 9 keratin family members and keratin associated proteins displayed down regulation in black sheep skin, which was the same as observed in piebald Merino sheep skin . Collectively, results support Garcia’s view that no single keratin gene alone appears to be responsible for the coat color trait . Hair keratins contain a much higher content of cysteine residues in their non-helical domains and thus form tougher and more durable structures via intermolecular disulfide bond formation . Therefore, high expression of keratins is likely crucial for fleece strength. Genes encoding for important oxidative and dehydrolytic enzymes such as NADH5 and COX1 were also highly expressed in sheep skin. The coenzyme NAD (nicotinamide adenine dinucleotide) is a key electron-carrier which mediates hundreds of reactions. The redox state of the NAD–NADH couple plays a central role in energy metabolism , signal transduction , and transcriptional regulation , which is consistent with the need for mitochondrial biogenesis, energy and other proteins during the strong metabolism characteristic of adult sheep skin development .
The human hair follicle (HF) has a variable response to potent androgens, such as testosterone (T) and dihydrotestosterone (DHT). The pilosebaceous unit (including HF and sebaceous gland) enzymatically converts weak androgens, such as dehydroepiandrosterone (DHEA) and androstenedione (AD), to more potent androgens, such as T and DHT. In HF of scalp, androgens shorten the anagen growth phase of the hair cycle, causing the HF to regress and recede. The conversion of androgens is dependent on oxidized-reduced pyridine cofactors, NAD, NADH, and NADPH . So, the high level of expression of NADH likely improves the conversion of androgens in certain body regions, influencing terminal hair growth.
Transcription factors (TFs) perform important regulatory functions by controlling a variety of cellular processes . In the mouse genome, 1,445 genes were identified to encode for TFs and 983 were expressed in the brain . In the current studies expression of 527 TF genes was detected in sheep skin, including general TFs such as endothelial differentiation-related factor 1 isoform alpha, DLX3, JUN, ATF4 and GATA3. The high level of expression of these genes detected in skin reflects their importance in regulation of general transcriptional pathways in sheep skin.
Several novel genes were also identified in sheep skin, and a portion of such genes were differentially expressed. Two of the novel genes detected lacked ORF in sequence reads detected, and were highly abundant and exclusively expressed in black sheep skin. BLAST analysis of these 2 novel genes did not find any similar sequences in NCBI database (including EST), suggesting that they could be specific to sheep skin. The differentiated phenotype of melanocytes must be due, at least in part, to differential transcription of melanocyte-specific genes . Thus, these two novel genes may play an important role in promoting pigmentation and dark coat colors.
The GO and KEGG pathway analyses of differentially expressed genes revealed that most were associated with the function of cell and cell part ontology categories. Of particular interest in our dataset were pathways related to pigmentation and melanogenesis. Of the differentially expressed genes, the genes in the category related to ‘the components of melanosomes and their precursor’ and ‘Eumelanin and pheomelanin’ were up-regulated in skin from sheep with black coat color. The function of genes in “the components of melanosomes and their precursor” and ‘Eumelanin and pheomelanin’ are melanin synthesis and the switch between eumelanin and pheomelanin . The darker pigmentation of skin, and possibly of hair, is associated with a higher numbers of melanosomes, although the number of melanocytes remains constant [7, 31]. Melanocytes in black hair follicles contain the greatest number of melanosomes (which are eumelanosomes), while the melanosomes in brown hair bulbs are smaller and those in blonde hair are very poorly melanised. The relationship of less melanin with lighter skin/hair phenotype has been reported in several species, including humans , alpaca , llama  and horse . In both domestic sheep and Soay sheep, light coat color is known to be due to a decrease in the ratio of eumelanin to pheomelanin, relative to black coat color .
Genes in the ‘Melanosome construction/protein routing (HPS-related)’ ontology category, such as HPS5, Lysosomal trafficking regulator (Lyst) and Pallidin, were all down-regulated in skin of sheep with black coat color. The functions of genes in the ‘Melanosome construction/protein routing (HPS-related)’ categories are related to organelle biogenesis . The key to melanin production is the organelle that is the site of melanogenesis, the melanosome, whose architecture, intracellular distribution and enzyme catalog are critical . HPS5 protein is a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2) and its deficiency can result in Hermansky–Pudlak syndrome (HPS-5) . HPS is a disorder of lysosome-related organelle (melanosome) biogenesis, resulting in oculocutaneous albinism [37, 38]. It has been reported that HPS5 melanocytes have an approximately normal contingent of the melanogenic protein, TYR. Elucidation of the relationship between lower level of expression of HPS5 and other genes in this ontology category with black coat color phenotype will require further investigation.
Among the differentially expressed coat color genes, TYRP1 showed the greatest level of differential expression in black versus white sheep skin. TYRP1, one of the members of the tyrosinase family, is a I type membrane bound protein that is expressed in both melanocytes and the retinal epithelium. TYRP1 is involved in the distal eumelanic pathway and plays a role in stabilizing TYR, which is the critical and rate-determining enzyme in melanogenesis . There existed a significant association between coat color and TYRP1 in Soay sheep . In the free-living Soay sheep, coat color is either dark brown or light tawny color. The light phenotype is determined by homozygosity of a single recessive amino acid change (G → T transversion) at coding position 869 in the TYRP1 gene . This is consistent with studies in domestic sheep, where light coat color is known to be due to a decrease in the ratio of eumelanin to pheomelanin, relative to black coat color .