Expression Analysis of Candidate Genes in White and Black Spots of Skin Tissue in Lori Bakhtiari Sheep

Sheep are the main source of wool and its fiber characteristics, such as diameter, length, and color, which are determined by genetics and environmental factors, are key features in the economic value of sheep wool. In sheep, white wool has the highest economic value due to its dyeability, thus, the identification of mechanisms responsible for coating color determination is very important from an economic point of view. In general, the coat color is determined based on the amounts and types of melanin produced and released by the melanocytes in the skin tissue (Ito et al., 2000). The genetic basis and genes involved in coat color are well understood in rodents, although many of these genes are incorporated in coat color regulation in other species; including Sheep also have a common role. In Iran, the Lori Bakhtiari sheep is one of the most important breeds of sheep in terms of the use of its wool in the textile and carpet industries. In his breed, the dominant coat of the wool is white, although sometimes a percentage of dark brown and pale brown is also observed (Saadat Nouri, M. & Siah Mansour, 1368). However, in this breed, some animals have black spots on their coat, which leads to a decrease in wool quality. Since MC1R, ASIP, KLF4 and MITF genes play an important role in controlling and determining coat color in mammals, the purpose of this study was to investigate the expression of these genes in two phenotypes of white and black spots in the skin tissue of Lori Bakhtiari sheep. Skin samples were obtained from both white and dark parts of 14 white-coated sheep with black spots and total RNA was extracted. The quality and quantity of extracted RNAs were evaluated by agarose gel electrophoresis and spectrophotometer. Extracted RNA samples were exposed to DNase1 enzyme digestion to remove the possible contamination of genomic DNA. Also, the quality of synthesized cDNA was evaluated using 1% agarose gel. In this research, in order to amplify a fragment of the studied genes, using the mRNA sequence of these genes in the GenBank database, appropriate primers were designed by Primer3plus software. To evaluate the relative expression of the target genes, β-actin and GAPDH genes were used as reference genes to normalize the data. Finally, BestKeeper and REST 2009 V2.0.13 software were used for the analysis of gene expression data. Based on the descriptive results of Ct values, MC1R and MITF genes revealed minimum and maximum expression stability among the target genes in skin samples with standard deviations of 1.34 and 3.62, respectively. In addition, the reference genes (β-actin and GAPDH) showed the highest stability among all the studied genes. No significant differences were observed in mRNA levels of MC1R, ASIP, KLF4, and MITF genes in the spotted skin tissue compared to the white part of the skin (p>0.05). However, the expression of the ASIP gene was more than 2 times in the spotted part compared to the white skin, but this difference was not significant (p=0.21). In addition, the MC1R gene showed minimum expression differences in black spots and white parts of the skin tissue. In addition, the MITF and MC1R genes showed the highest and lowest levels of expression in skin samples of Lori Bakhtiari breed sheep with average Ct of 25.86 and 30.42, respectively. However, among all the studied genes, the lowest mRNA level was observed for the GAPDH gene with an average Ct of 35.96. Mammalian coat color results from various factors such as the degree and distribution of melanin pigment and the interaction between genotype and environment (B. Li et al., 2018). In addition, melanogenesis is a complex process that includes melanocyte growth, melanosome formation, melanin synthesis, melanin transport, and melanosome release (Ito & Wakamatsu, 2011). According to the conducted studies, a large number of genes are involved in the mechanism of coat color determination, but two genes, MC1R and ASIP, play an essential role in the regulation and control of coat color (Searle, 1968). In our study, no significant difference was observed in the expression of MC1R, ASIP, KFL4, and MITF genes in the spotted compared to the white part of the skin tissue in Lori Bakhtiari sheep. These results showed that the development of skin spots is not under the control of the studied genes in Lori Bakhtiari sheep, and genes or other factors can play roles in the creation of dark spots in this breed.