Molecular characterization of tyrosinase gene (exon 1) in camels of Saudi Arabia

Arabian camels belong to the genus Camelus (the old world camels), which includes only two species, the one-humped (Camelus dromdarius) and two-humped camels (Camelus bacterianus). Camels are unique animals in many aspects and cannot be compared with other farm animals in their physiological responses or in their adaptation to arid environment (Sweet, 1965; Schwartz, 1992). They are used in transportation, trade, agricultural work, tourism, race and beauty contests and as a source of economically important products, such as meat, milk and wool (Groeneveld et al., 2010).
        
Mehaia et al., (1995) reported that indigenous camels in Saudi Arabia can be classified into different ecotypes: Majaheim or Malha, Wadah, Hamra, Safrah and Omani (Wardeh, 1989; Wardeh and Al-Mustafa, 1990; Elamin and Wilcox, 1992). The Majaheim ecotype is restricted to central Arabia, with some spread in the east as well. It is large, blackish-brown in color and the breeders consider it the best local variety available for milk production. The Wadah ecotype is the camel of the western part of Arabia and is also found in the central and northern part of the country. It is small, white in color and produces small amounts of milk. The Hamra ecotype, fawn in color, is found in small numbers in the central and northern parts of the country. The Safrah ecotype is restricted to the northern part of the country. It is brownish-yellow with a small head and a large abdomen. The Omani ecotype is light in weight and is used mainly for riding and racing.
        
In the last decades, the population of dromedary camel have declined at a very high rate because of the continuous development of modern transportation, the improvements in agricultural mechanization, and the development of camel products (Ming et al., 2016). To avoid a decline in the population of dromedary camel, its genetic resources should be preserved for sustainable utilization and conservation through assessment of genetic variability using genetic markers.
        
Single nucleotide polymorphism (SNP) in tyrosinase gene (TYR) is now widely used as a genetic marker for analyzing genetic variations and mammalian coat color phenotypes (Everts et al., 2000). TYR was identified and characterized by Giebel et al., (1991) who showed that TYR consists of five exons and four introns. Exon1 of TYR is of interest in the investigation of genetic variation and in the analysis of coat phenotypes in C. dromedarius. The TYR gene is expressed into its product tyrosinase, which is located in melanocytes. The cells of melanocytes are specialized in producing melanin, a pigment that confers color to skin, hair and eyes (Shah et al., 2005). Mutations in TYR were responsible for the albino phenotype in mammals and chicken and influence quantitative traits in mice (Schmidtz et al., 2001).
        
Shah et al., (2005) designed a primer pair based on the partial sequences of mouse and human that can be used for the amplification of an 820-bp fragment in exon 1 of the camel tyrosinase gene. By sequencing the exon 1 of TYR from different Pakistani camel breeds, they detected a single nucleotide polymorphism (C>T) at coding position 200 after the ATG, causing an amino acid substitution (Pro>Leu). Shah et al., (2008) investigated the genetic variation between the breeds of C. dromedarius in Pakistan by genotyping of TYR using PCR-RFLP, recording significant differences in the genotype frequency between the breeds. The allelic variants of TYR in Sudanese camel breeds were studied using PCR-RFLP analysis (Ishag et al., 2013) revealing that there are no significant differences in the allele frequency between the breeds and there is no significant association between the camel coat color and TYR genotypes (TT, CC, TC). In Egyptian populations, genotyping of TYR was done using PCR-RFLP technique (Alam et al., 2015), and only one SNP (C/T) was detected in exon1 among the five tested camel breeds and this nucleotide substitution was recommended as a marker for assessing the genetic biodiversity among camel breeds reared in Egypt.
        
Previous studies on genetic variability among C. dromedarius populations based on polymorphisms in sequences of exon 1 of TYR showed considerable discrepancies in the genetic variation data in C. dromedarius populations from different countries. No strong evidence on the relation between polymorphisms in the TYR exon 1 and coat color was reported as in other mammals and limited data are available on genetic variation in Saudi Arabian populations. The objective of this study, therefore, was to evaluate the genetic variability in C. dromedarius populations of Saudi Arabia, and to determine the relationship between the polymorphism in TYR exon 1 and coat color phenotypes.

Animal Resources
 
Hair samples were collected from 166 camel individuals representing six indigenous breeds (28 Magaheem, 29 Maghateer, 18 Shaul, 28 Hamra, 19 Sofr, 24 Sawahli) and one exotic breed (20 Alsumalia). The collected samples were kept at -20°C for further use. The samples taken from Alsumalia population were collected from individuals immediately released from quarantine.
 
DNA Extraction
 
Genomic DNA was extracted from hair roots using the QIAgen DNeasy blood and tissue kit (Hildane, Germany). The concentration and quality of DNA was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA). All the extracted DNA samples were stored immediately at -80°C for further use.
 
Amplification of Exon 1
 
The exon 1 of TYR was amplified from camel DNA samples using two PCR primers, forward 52-AGC CTG TGC CTC CTC CAA GAA-32 and reverse 52-TGC ATC CAT ACA AAG AAG TCA TAA-3', which yielded a 474-bp product. Polymerase chain reaction (PCR) amplifications were carried out in a 25-µL reaction volume containing 100 ng of template DNA and 2 µL of each of the 10 µM primers. To reduce the possibility of cross contamination and variation in the amplification reactions, master mixes containing all PCR reagents including the Kapa Taq polymerase enzyme (KAPA Biosystems, Boston, MA, USA), except DNA template and primers, were used. The amplification was performed using Gene Amp PCR system 9700 thermocycler (Applied Biosystems, Warrington, UK). The amplification protocol included denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 40 s and extension at 72°C for 1 min, and final extension at 72°C for 5 min. PCR products were electrophoresed on 1% agarose gel, which was subsequently stained with ethidium bromide and the amplified bands were detected under UV light using a gel documentation system (Amersham Biosciences, Uppsala, Sweden).
 
DNA sequencing and sequence analysis
 
PCR products were extracted from the gel and sequenced at the Advanced Genetic Technologies Center (http://www.uky. edu /Centers/ AGTC/). The DNA sequences were edited and aligned using BioEdit software (Hall; http://www.mbio. ncsu edu Bioedit/bioedit.html). The BLAST algorithm was used to search the NCBI GenBank database (http www.ncbi.nlm.nih.gov) for homologous sequences. The BioEdit software was also used to detect SNPs and indel mutations.
 
Data analysis
 
A dendrogram was constructed based on Nei’s genetic distance employing UPGMA (Nei, 1978) using SYSTAT 13.1 software.

Tyrosinase has a role in determining the coat color and significantly effects some phenotypic characteristics in farm animals (Oetting and King, 1999; Aigner et al., 2000; Beermann et al., 2004; Schmutz et al., 2004; Ishag et al., 2013). This gene codes for the TYR enzyme that is a key enzyme in the melanogenic metabolic pathway leading to coat color pigmentation in mammals. Mutations in TYR are responsible for the albino phenotype in these organisms. The albinism is due to the loss of expression of TYR, which prevents melanin synthesis.
        
In this study, a 474-bp fragment from exon 1 of TYR was amplified and sequenced from camels representing seven populations (Majaheem, Maghateer, Shaul, Hamra, Sofr, Sawahli and Alsumalia) raised in Saudi Arabia (Fig 1). Two nonsynonymous SNPs were detected. The first one (C>T) was at coding position 200, which led to a Leu>Pro substitution at amino acid position 67 of the TYR protein (Fig 1) and the second (C>T) was at coding position 523, which led to a Leu>Phe substitution at amino acid position 175.
 

        
There were significant differences in the genotypic frequencies among the seven populations (Table 1). The genotypic frequencies at position 200 were 0.48, 0.084 and 0.43 for CC, TT and CT genotypes, respectively. All the populations possessed the three detected genotypes in different frequencies with the exception of the HA population that lacked the TT genotype and SU that lacked the CC and TT genotypes and only had the CT genotype (Table 2). The detection of the CT genotype only in Alsumalia population contradicted the findings of Alam et al., (2015) who reported the three genotypes in Alsumalia breed. This discrepancy can be interpreted on the basis that samples in their study might have been taken from individuals introduced to Egypt and there was an exchange of genes with other Egyptian breeds through breeding. The highest CC, TT and CT genotype frequencies were found in SO (0.79), SH (0.28), and SU (1.00) camels, respectively (Table 1). The highest genotype frequency was for CC followed by those for CT and TT and the frequency of allele C was dominant over that of allele T (Table 1). The dominance of allele C over allele T was also reported by Shah et al., (2008, 2012) and Alam et al., (2015) in their study on Pakistani and Egyptians camel breeds. The similar frequency of C allele in MG and MJ breeds could be due to the fact that these two breeds are often raised together in mixed herds (Ishag et al., 2013). The low frequency of TT genotypes was also reported by Shah et al., (2005, 2008) and Alam et al., (2015) in their studies on Pakistani camel breeds. They attributed their results to the active selection against TT genotype resulting from local preference. However, because active selection against TT genotype was not restricted to Pakistani camel breeds but was extended to Egyptian and Saudi Arabian camel breeds, it can be suggested that the active selection against TT genotypes could be due to global environmental preferences.
 

 
The frequency of genotypes CC, CT and TT matched their frequencies in the study of Ishag et al., (2013) and Alam et al., (2015) on Sudanese and Egyptian camel breeds, respectively. Although, Ishag et al., (2013) reported that there was a significant effect of SNPs in TYR on shoulder height and there was no significant influence on other phenotypic measurements or on the coat color in Sudanese camel; our results suggest a possible association between TYR allele C and the darkness of camel coat color, where the highest frequencies of allele C were present in MG (0.54), SO (0.79) and SL (0.54) and the camels of these breeds possess dark colors ranging from black to red (Table 2). In addition, MG, which has black color, was found to have genotype CC at position 523 where the other breeds had TT. The association of C allele with coat color contradicted the suggestion of Alam et al., (2015) and Shah et al., (2008 and 2012) that the coat color is association with T allele. The active selections of Pakistani, Egyptian and Saud Arabian camel breeds against the TT genotype and the lack of TT genotype in HA breed supports our suggestion.
        
The cluster analysis of the data for genotypic frequencies showed the presence of two clusters, one included the MG population and the other contained the other populations (Fig 2). The second cluster was divided into two sub-clusters: one included the SU population and the other contained SH, MJ, SL, HA and SO populations. Based on these data, it can be suggested that the SH, MJ, SL, HA and SO breeds were derived from SU breeds. Moreover, the genetic distance between MG and other breeds led us to conclude that the Saudi camel populations were derived from MG. In addition, the genetic distance between MG and other populations and the presence of polymorphism at position 523 in both MG and wild Bactrian camel (the combination of mutations in TYR deposited in GenBank under the accession number 010973375.1) suggests that wild Bactrian camel might be the immediate ancestor of Saudi populations.
 

It is concluded that there is (1) a moderate but significant difference in the allele frequencies of TYR exon 1 among the Saudi Arabian camel breeds, (2) a significant association between coat color and CC genotypes, (3) a new mutation at position 523 in exon 1 of TYR in MG breed (genotype CC) and (4) active selection against the TT genotype. It is also concluded that wild Bactrian camel might be the immediate ancestor of Saudi Arabian breeds. Further studies with larger number of camel breeds, collected from a wide geographical area, are required to investigate and verify the associations between TYR exon1 and coat color.

We extend our appreciation to King Abdulaziz City for Science and Technology (KACST) for funding this work through project number MS-36-60. The authors thank the Deanship of Scientific Research and RSSU at King Saud University for their technical support.