All experiments performed in this study were approved by the International Animal Care and Use Committee of the Yunnan Agricultural University. The care and use of animals fully complied with local animal welfare laws, guidelines and policies.

Three groups of sheep (Ovis aries) were used for the study. Two groups of sheep more than 1 year of age were identified in a flock of indigenous sheep in Nanping County, Yunnan Province, PRC. A group of 101 were classified as Nanping black-boned (NPBB) sheep and a second group of 106 were classified as Nanping normal (NPN) sheep. A third group of 82 mature Romney Marsh (RM) sheep were kept at the Yunnan Malong Sheep Stud Farm, Malong County, Yunnan Province, PRC. All sheep grazed on pasture consisting of natural grasses and herbs during the day and were penned at night.

A blood sample was collected from each sheep in the morning prior to releasing the sheep to pasture. The blood was collected from the external jugular vein by direct venipuncture, and each sample was distributed to six aliquots into sterile tubes. Sodium citrate was added to six of the aliquots prior to centrifugation to obtain plasma. All three tubes of blood or plasma were then snap frozen in liquid nitrogen before storing at −80^∘ C pending analyses.

At the time of collection of blood the wool color of each sheep was recorded as either black, white mixed (black, brown and white) or brown.

Whole blood samples from five sheep chosen randomly from each of the NPBB, NPN and RM groups were used for the identification of polymorphisms. Genomic DNA was extracted from each blood sample using a TIA Namp Blood Kit (Tiangen Biotech Co. Ltd., Beijing, China). The reference genomic sequence was obtained from the sheep (EF102110) TYRP1 gene complete coding region, which has been posted on COW BLAT Search (http://genome.ucsc.edu/cgi-bin/hgBlat).

Primers were designed in inferential introns using primer Premium 5.0 software to provide optimal polymerase chain reaction (PCR) products. Eight primers were designed to amplify each exon and its partial flanking region of the TYRP1 gene (Table 1). The PCR mixture contained 2 µL (20–50 ng) of genomic DNA, 1.5 µL of 25 mM Mg Cl₂, 1.0 µL of 10 pmol µL⁻¹ forward primer, 1.0 µL of 10 pmol µL⁻¹ reverse primer, 0.5 µL of 10 pmol µL⁻¹ dNTP (deoxy-ribonucleoside triphosphate) mixture, 0.4 µL of 5 U Taq polymerase and 2.5 µL of 10 × PCR buffer at a final volume of 25 µL. Each PCR reaction was performed in a Gene Thermal Cycle (Hangzou, China) using the protocol shown in Table 1. The PCR program started with denaturation at 94 ^∘C for 3 min, followed by 38 cycles of 94 ^∘C for 45 s, Ta (annealing temperature) for 45 s, 72 ^∘C for 60 s then an extended 72 ^∘C cycle for 5 min and finally 4 ^∘C to terminate the reaction. The PCR products were sequenced bi-directionally using an ABI 3730 DNA analyzer (Applied Biosystems Inc.) at the Sun Biotechnology Company (Beijing, China).

All collected samples except those sequenced previously were genotyped as follows.

Due to the lack of a suitable restriction nuclease for the single nucleotide polymorphism (SNP) g.203C > T, a bi-directional PCR allele-specific amplification (bi-PASA) system was designed to quickly and conveniently detect the genotype of SNP g.203C > T. The Bi-PASA method, based on a one-tube reaction, permits a distinction to be made between the different genotypes by using two outside primers and two inner (allele-specific) primers at the mutation point (Liu et al., 1997).

Table 1Primers and PCR conditions.

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The bi-PASA PCR reactions were performed in a mixture (25 µL) containing 2 µL of genomic DNA, 1 µL of 25 mM Mg Cl₂, 0.25 µL of 10 mM outside primer, 0.5 µL of 10 mM inner primer (Table 1), 0.5 µL of 20 mM dNTP mixture, 0.2 µL of 5 U Taq polymerase, 3 µL of 10 × buffer and 16.8 µL of water. The thermal cycling PCR conditions were 94 ^∘C for 3 min, 40 cycles at 94 ^∘C each for 45 s, 60 ^∘C for 45 s and 72 ^∘C for 45 s with a final extension at 72 ^∘C lasting for 5 min. Following the PCR an aliquot of 20 µL of the PCR bi-PASA product was separated on agarose gel (1.5 %, w : v), which was finally stained with ethidium bromide to permit visualization.

The single-strand conformation polymorphism (SSCP) method was used to identify SNP g.1202T > C by using another primer (SSCP-exon 6); see Table 1. The PCR mixtures contained 0.75 µL of 10 pmol µL⁻¹ forward primer and 0.75 µL of 10 pmol µL⁻¹ reverse primer with the remainder of the mixture as outlined above for the bi-PASA reactions. Aliquots of 2 µL of the PCR products were mixed with 5 µL of denaturing solution containing 98 % (w : v), deionized formamide, 0.01 mM EDTA, 0.25 % (w : v) xylene cyanol, 0.25 % (w : v) bromphenol blue and 2 % (w : v) glycerol; these were heated for 10 min at 98 ^∘C and then chilled immediately on ice. Denatured DNA was subjected to electrophoresis on polyacrylamide gel (14 %, w : v) in 1 × TBE buffer at a constant voltage of 200 V overnight at 4 ^∘C. Thereafter the gels were stained with 0.36 % (w : v) silver nitrate.

The method described by Pomerantz (1963) was used to measure the activity of tyrosinase in plasma. Briefly, L-dopamine was used as a substrate to measure the activity of the enzyme with a spectrophotometer set at a wavelength of 475 nm. One unit of enzyme was defined as the amount causing the uptake of 1 µL of O₂ per minute in a total volume of 3 mL including 1 mg of dopa, 0.1 M sodium phosphate buffer, a pH of 6.8 and a constant temperature of 37 ^∘C.

The concentration of plasma phaeomelanin (SpPM) was measured using a spectrophotometric assay (Ozeki et al., 1995, 1996a). An aliquot of 200 µL of each plasma sample was added to 800 µL of phosphate buffer with pH 10.5. The mixture was agitated vigorously for 10 min at room temperature then the mixture was centrifuged at 10 000 rpm for 10 min before 1 mL of chloroform was added to the supernatant to remove fatty impurities. The pale yellow aqueous layer containing phaeomelanin was cleared by centrifugation at 10 000 rpm for 10 min and the absorbance was measured at 400 nm (A₄₀₀). Plasma concentrations were expressed as the absorbance value A₄₀₀.

Plasma alkali-soluble melanin (SpASM, comprising phaeomelanin and eumelanin) was measured by the above spectrophotometric analysis, but samples were solubilized using 8M urea–1M sodium hydroxide instead of phosphate buffer. Plasma concentrations were expressed as the value for A₄₀₀.

Plasma total melanin (SpTM) also was measured by spectrophotometric analysis (Ozeki et al., 1995, 1996a). An aliquot of 100 µL of plasma was mixed with 900 µL of soluene 350 (Packard, Meriden, CT, USA) and then the mixture was heated in a boiling water bath for 45 min. After cooling, the absorbance of the boiled mixture was measured at both 500 nm (A₅₀₀) and 650 nm (A₆₅₀) and the SpTM value was taken as the A₅₀₀reading. The ratio of the values for A₆₅₀ : A₅₀₀ was taken to represent the ratio of eumelanin : total melanin (SpEM : SpTM) in the plasma sample (Ozeki et al., 1996b).

For all plasma samples, duplicate analyses were made.

Functional domain prediction was effected using an online server (http://www.bioinfotllo.org/domac.html). Protein motif prediction was conducted using the PROSITE motif search tool (http://www.cbs.dtu.dk/services/NetNGlyc/). The likelihood of the non-synonymous coding single nucleotide polymorphisms causing a putative functional impact on the protein was predicted using the Protein Variation Effect Analyzer (PROVEAN) software volume 1.1 (http://provean.jcvi.org/seq_submit.php).

Genotypic and allelic frequencies were calculated by direct count. Differences in the distribution of genotypes and alleles between the three groups of sheep and deviation from the Hardy–Weinberg equilibrium were assessed using the χ² test.

Population genetic diversity parameters, including heterozygosity (He), homozygosity (Ho), effective allele numbers (Ne) and polymorphism content (PIC), were calculated as described by Nei and Li (1979).

The association between genotype and melanin-related traits was analyzed using the general linear model (GLM) procedure (SAS Institute Inc., Cary, NC, USA) according to the following relationship:

where Y_ij represents the observation of the melanin-related traits, μ is the least squares mean, P_i is the effect of its population (i being either the NPBB, NPN or RM sheep), G_j is the effect of the particular genotype and e_ij is the random residual.

Values for all traits have been presented as means ± SE (standard errors) of the means.

Statistical significance was determined using the Duncan's new multiple range test and a P value of 0.05 was taken to denote statistical significance.

The activity of tyrosinase in the plasma of the NPBB sheep was significantly higher (P < 0.01) than in the plasma of the NPN sheep, which had a significantly higher (P < 0.01) activity of plasma tyrosinase than for the RM sheep (Fig. 4a).

Values of SpPM, SpASM and SpTM, as well as the ratio of SpEM : SpTM, were consistently and significantly (P < 0.01) higher for the NPBB than RM sheep. Although the values of SpPM were similar for NPBB and NPN sheep, the concentrations of SpASM, SpTM and the ratio of SpEM : SpTM were significantly lower (P < 0.01) for the NPN than NPBB sheep. Values of SpTM were similar for the NPN and RM sheep but values of SpTM, SpASM and the ratio of SpEM : SpTM were significantly lower (P < 0.01) for the RM than NPN sheep (Fig. 4b).

Table 2Association analysis of the TYRP1 gene with black traits (least squares mean value ±1 SE; SE indicates the standard errors of the means) in the three flocks of sheep and all sheep sampled.

${}^{\mathrm{a},\mathrm{b},\mathrm{c}}$ Values in the same column indicate that either the three populations or all sheep with different superscripts differ (p < 0.05).

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Figure 2The genotype and gene frequencies of the SNPs c.203C > T (a) and c.1202T > C (b) from the TYRP1 gene in Nanping black-boned (NPBB), Nanping normal (NPN) and Romney Marsh (RM) sheep.

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Figure 3Genetic diversity parameters at the SNPs c.203C > T and c.1202T > C from the TYRP1 gene in Nanping black-boned (NPBB), Nanping normal (NPN) and Romney Marsh (RM) sheep. (a) Ho: gene homozygosity; He: gene heterozygosity; Ne: effective allele numbers. (b) PIC: polymorphism information content.

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Associations between genotype and melanin-related traits for the SNPs c.203C > T and c.1202T > C are presented in Table 2. In the case of SNP c.203C > T there were no significant (P > 0.05) associations between genotype and melanin-related traits. When the data for all sheep were pooled, in the case of SNP c.203C > T, sheep with the CC genotype showed significantly higher (P < 0.05) values for tyrosinase activity, SpPM, SPTM and the ratio of SpEm : SpTM than sheep with the CT and TT genotypes for which similar values were measured for all traits. In the case of the SNP c.1202T > C, no significant associations were measured for each of the genotypes and melanin-related traits.

In the case of coat color, polymorphism of the site c.203C > T had a significant effect (P < 0.01) on coat color in the NPN group of sheep. No significant effects of polymorphism on coat color was measured for SNP 1202T > C even though the majority of the NPBB sheep of each of the three genotypes had black or brown coats (Table 3).

Data are available upon reasonable request from the corresponding author.

The authors declare that they have no conflict of interest.