J. Anim Sci. 2008. 86:1291-1295. doi:10.2527/jas.2007-0498
© 2008 American Society of Animal Science
Technical note: A novel method for routine genotyping of horse coat color gene polymorphisms1
L. J. Royo*,
I. Fernández*,
P. J. Azor
,
I. Álvarez*,
L. Pérez-Pardal* and
F. Goyache*,2
* Servicio Regional de Investigación y Desarrollo Agroalimentario del Principado de Asturias-Somió, C/Camino de los Claveles 604, E-33203 Gijón (Asturias), Spain; and
Departamento de Genética, Universidad de Córdoba, Campus de Rabanales, E-14071, Córdoba, Spain
 |
Abstract
|
|---|
The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.
Key Words: horse • real-time polymerase chain reaction • coat color • melanocortin-1 receptor • Agouti-signaling peptide • membrane-associated transporter protein
|
INTRODUCTION
|
|---|
Coat color in mammals depends on the relative amount of eumelanin (black-brown) and phaeomelanin (yellow-red), which are controlled, in turn, by the Extension (E) and Agouti (A) loci (Searle, 1968
), encoded, respectively, by the melanocortin-1 receptor (MC1R) and the Agouti-signaling peptide (ASIP; Bultman et al., 1992). In horses, various mutations responsible for the major coat phenotypes have been identified: in the MC1R gene, Marklund et al. (1996) identified a chestnut (e) allele, and further, Wagner and Reissmann (2000) identified a second chestnut allele (ea). In the ASIP gene, Rieder et al. (2001) reported an 11-bp deletion that, in homozygosis, is completely associated with recessive black coat color. Mariat et al. (2003) identified the causal mutation of the dilution in the membrane-associated transporter protein (MATP) gene.
The interest in coat color inheritance in horses has a long history. Its inheritance can be explained from a qualitative perspective (Bowling, 2000; Toth et al., 2006). Besides major phenotypes (bay, chestnut, and black), many coat colors in horses, such as buckskin, perlino, palomino, or cremello, are dilutions of the basic colors. The design of an easy and quick method for the diagnosis of mutations responsible for major coat color in horses is of interest to breeders to improve color classification and to enable genetic counseling for more efficient mating strategies for color production.
The aim of this note is to report a reliable, fast, and cost-effective method for routine genotyping of mutations responsible for most coat color variation in horses using real-time PCR. This methodology has been shown to be useful to fulfill the need of genotyping hundreds or thousands of individuals in standard-equipped molecular genetic laboratories (Van Poucke et al., 2005).
|
MATERIALS AND METHODS
|
|---|
All procedures involving animals were approved by the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria.
DNA Isolation
Genomic DNA used in this research was isolated from blood or hair roots using standard procedures (Sambrook et al., 1989).
PCR Primer and Probe Design
Primers and dual-labeled allele-specific oligonucleo-tide probes were designed with Beacon Designer software version 2.0 (Bio-Rad, Barcelona, Spain) and synthesized by Sigma-Genosys (Haverhill, UK) and Euro-gentec (Liege, Belgium). Three primer pairs were designed to amplify fragments of the MC1R, ASIP, and MATP genes containing the polymorphisms described by Marklund et al. (1996), Wagner and Reissmann (2000), Rieder et al. (2001), and Mariat et al. (2003). A dual-labeled allele-specific oligonucleotide probe was designed for each of the 7 allelic variants (3 for MC1R, 2 for ASIP, and 2 for MATP, respectively), in which the SNP is localized approximately in the middle of the sequence. Two PCR protocols were designed. In PCR protocol 1, the 3 allelic variants of the MC1R locus were determined simultaneously with 3 allele-specific probes. Two reactions were performed to identify the allelic variants of the MATP and ASIP loci using PCR protocol 2. In this protocol, MATP and ASIP variants were determined with 4 allele-specific probes, each containing a different fluorophore. A combination of the fluorophores FAM, HEX, Texas Red, and Cy5 was chosen, as described by Ugozzoli et al. (2002). The fluoro-phores FAM and HEX were quenched with BHQ1, and Texas Red and Cy5 were quenched with BHQ2. Ampli-con sizes, primer and probe sequences, and DNA melting temperature are listed in Table 1.
View this table:
[in this window]
[in a new window]
|
Table 1. Oligo and probe melting temperature (Tm) and sequence, dual labeling (only for probes), as well as amplicon sizes (only for each primer pair) for diagnostics of the MC1R, ASIP, and MATP alleles included in the protocol
|
|
The PCR assays were performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using optically clear PCR plates and sealing films (Bio-Rad Laboratories), in a total volume of 15 µL containing iQ Supermix (50 mM KCl, 20 mM Tris-HCl, pH 8.4, 0.8 mM deoxynucleoside triphos-phate, 0.375 U of iTaq DNA polymerase, 3 mM MgCl2, and stabilizers; Bio-Rad Laboratories), 160 nM of MC1R primers and 400 nM of each MATP and ASIP primers and probes, and ~150 ng of DNA. The real-time PCR program for both reactions consisted of an iTaq DNA polymerase activation and DNA denaturation step (3 min at 95°C), followed by 40 amplification cycles, that in PCR 1 were: denaturation for 20 s at 95°C and annealing-elongation for 40 s at 70°C; and in PCR 2 were: denaturation for 20 s at 95°C and annealing-elongation for 40 s at 60°C. The fluorescent signals were detected in real-time during the annealing-elongation step.
The data were analyzed using the iCycler iQ Real-Time PCR Detection System Software version 3.1 (Bio-Rad Laboratories). For every probe, relative fluores-cence unit values were measured every cycle and after background normalization plotted against the cycle number. Based on these real-time amplification plots, Ct-values were calculated in the PCR Quantification tab of the Data Analysis module by user-defined assignation of the baseline cycles and the threshold position. The data obtained, Ct-values for detection and N/A for nondetection, were copied to a Microsoft Excel spreadsheet to semiautomatically determine the genotype using an Excel macro developed by the authors that can be downloaded free of charge from http://www.igijon.com/personales/fgoyache/software_i.htm (HorseColor; both last accessed on February 26, 2008).
Each assay included 6 samples of known genotypes representing all combinations of the polymorphisms tested on each locus as positive controls (EE-Aa-CcrCcr; Ee-AA-CCcr; Eea-aa-CC; ee-Aa-CcrCcr; eaea-aa-CCcr; and eea-AA-CC) and one no-template control. Positive controls were sequenced using the dye-terminator V3.1 protocol (Applied Biosystems, Foster City, CA) in an ABI 310 sequencer (Applied Biosystems), to confirm the presence of the polymorphisms described. Genotypes of unknown samples were considered reliable only if the results for all reference samples were correct.
Validation of the Method
The performance of the assay was demonstrated via a cross-validation study: i) the wild type-E, chestnut-e (C901T) and chestnut-ea (C901T and G903A) MC1R alleles were determined by restriction enzyme digestion with TaqI to resolve E and e (Marklund et al., 1996) and allele-specific PCR for E and ea based on the sequence reported by Wagner and Reissmann, 2000; ii) the wild type-A and black-a (ADEx2) ASIP alleles were determined by size difference of the PCR product, including the 11-bp deletion in ASIP exon 2, as described by Rieder et al. (2001); and iii) wild type-C and cream-Ccr (G457A) MATP alleles were determined by restriction enzyme digestion using MseI.
|
RESULTS AND DISCUSSION
|
|---|
The method has been demonstrated on a sample of 156 Spanish Purebred (Andalusian) horses, with coat colors recorded by the authors (Table 2). The obtained amplification curves are shown in Figure 1. The correlation between the genotypes of 30 random nongray samples generated with the dual-fluorescent multiprobe assay and the reference method was 100%.
View this table:
[in this window]
[in a new window]
|
Table 2. Genotypes of 156 Spanish Purebred horses analyzed for the 3 genes that affect coat color variation, described according to their phenotype
|
|
View larger version (84K):
[in this window]
[in a new window]
|
Figure 1. Amplification plots (from panel A to panel G) obtained with the dual-fluorescence multiprobe assay for horse coat color genotyping. For all amplification plots, DNA purified from hair roots was used as the template. Amplification plots are shown for a homozygous positive (+/+), a heterozygote (+/–), a homozygous negative (–/–), and a no-template control with: (A) E-probe (FAM-labeled) in PCR 1; (B) e-probe (HEX-labeled) in PCR 1; (C) ea-probe (Texas Red-labeled) in PCR 1; (D) C-probe (FAM-labeled) in the first reaction of PCR 2; (E) Ccr-probe (HEX-labeled) in the first reaction of PCR 2; (F) A-probe (Texas Red-labeled) in the second reaction of PCR 2; (G) a-probe (Cy5-labeled) in the second reaction of PCR 2; and (H) an amplification plot for 30 test samples and 6 control samples for the Ccr-probe. RFU = relative fluorescence units.
|
|
In the Spanish Purebred horses, the recorded coat colors could be explained by different combinations of the genotyped allelic variants of the MC1R, ASIP, and MATP genes, except for the gray color. Note that graying has dominant inheritance, and its casual mutation remains unknown (Pielberg et al., 2005).
Although sequencing is still the gold standard for detecting polymorphisms, it is a time-consuming and expensive technique, not suitable for the routine typing of a large number of samples. The assay described here is an alternative method for routine typing of a defined number of polymorphisms, although it is absolutely necessary to include positive controls as well as 1 no-template control in every assay. Furthermore, the assay is robust enough to be performed with DNA purified from hair roots as a template for PCR. The allelic variants are detected in real time, and no post-PCR manipulations are required, therefore reducing time, costs, and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype.
In conclusion, we have developed and validated a dual-fluorescence multiprobe assay for robust, reliable, and reproducible genotyping of horse coat color genes in a fast, simple, and cost-effective way.
|
Footnotes
|
|---|
1 This research was partially funded by grants from the Instituto Nacional de Invesatigación y Technología Agraria y Alimentaria No. RZ03-011 and RZ2004-00023. We thank Javier Alba (Bio-Rad Laboratories, Hercules, CA) for his excellent technical help and comments, as well as Asociación Nacional de Criadores de Caballos de Pura Raza Española (http://www.ancce.com), Asociación de Criadores de Ponis de Raza Asturcón (http://www.asturcones.com/), and Asociación García-Dory for their kind collaboration. 
2 Corresponding author: fgoyache{at}serida.org
Received for publication August 6, 2007.
Accepted for publication February 15, 2008.
|
LITERATURE CITED
|
|---|
Bowling, A. T. 2000. Genetics of colour variation. Pages 53–68 in The Genetics of the Horse. A. T. Bowling and A. Ruvinsky, ed. CABI Publ., Wallingford, Oxon, UK.
Bultman, S. J., E. J. Michaud, and R. P. Woychik. 1992. Molecular characterization of the mouse agouti locus. Cell 71:1195–1204.[CrossRef][Medline]
Mariat, D., S. Taourit, and G. Guérin. 2003. A mutation in the MATP gene causes the cream coat colour in the horse. Genet. Sel. Evol. 35:119–133.[CrossRef][Medline]
Marklund, L., M. Johansson Moller, K. Sandberg, and L. Anderson. 1996. A missense mutation in the gene for melanocyte-stimulating hormone receptor (MC1R) is associated with the chestnut coat color in horses. Mamm. Genome 7:895–899.[CrossRef][Medline]
Pielberg, G., S. Mikko, K. Sandberg, and L. Andersson. 2005. Comparative linkage mapping of the Grey coat colour gene in horses. Anim. Genet. 36:390–395.[CrossRef][Medline]
Rieder, S., S. Taourit, D. Mariat, B. Langlois, and G. Guérin. 2001. Mutations in the agouti (ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus). Mamm. Genome 12:450–455.[CrossRef][Medline]
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY.
Searle, A. G. 1968. Comparative genetics of coat colour in mammals. Logos Press, London, UK.
Toth, Z., M. Kaps, J. Sölkner, I. Bodo, and I. Curik. 2006. Quantitative genetic aspects of coat color in horses. J. Anim. Sci. 84:2623–2628.[Abstract/Free Full Text]
Ugozzoli, L. A., D. Chinn, and K. Hamby. 2002. Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations. Anal. Biochem. 307:47–53.[CrossRef][Medline]
Van Poucke, M., J. Vandesompele, M. Mattheeuws, A. Van Zeveren, and L. J. Peelman. 2005. A dual fluorescent multiprobe assay for prion protein genotyping in sheep. BMC Infect. Dis. 5:13.[CrossRef][Medline]
Wagner, H.-J., and M. Reissmann. 2000. New polymorphism detected in the horse MC1R gene. Anim. Genet. 31:289–290.[CrossRef][Medline]
Top
Abstract
INTRODUCTION
