Short communication: An association analysis between one missense polymorphism at the SREBF1 gene and milk yield and composition traits in goats

S HORT C OMMUNICATION : An association analysis between one missense polymorphism at the SREBF1 gene and milk yield and composition traits in goats A. Manunza 1 , A. Zidi 1 , V. M. Ferna ´ndez-Cabana ´s 2 , J. Jordana 3 , J. Carrizosa 4 , E. Belaifa 1 , B. Urrutia 4 , O. Polvillo 2 , P. Gonza ´lez-Redondo 2 , M. Amills 1,3,6 , and J. M. Serradilla 5,6 1 Departament de Gene `tica Animal, Centre de Recerca en Agrigeno `mica (CRAG), Universitat Auto `noma de Barcelona, 08193 Bellaterra, Spain ; 2 Departamento de Ciencias Agroforestales, Escuela Te ´cnica Superior de Ingenierı´a Agrono ´mica, Universidad de Sevilla, Carretera Utrera, km. 1, 41013 Sevilla, Spain ; 3 Departament de Cie `ncia Animal i dels Aliments, Universitat Auto `noma de Barcelona, 08193 Bellaterra, Spain ; 4 Instituto Murciano de Investigacio ´n y Desarrollo Agrario y Alimentario (IMIDA). Estacio ´n Sericı´cola, 30150-La Alberca, Murcia, Spain; and 5 Departamento de Produccio ´n Animal, Campus de Rabanales, Universidad de Co ´rdoba, 14071 Co ´rdoba, Spain . Received 7 October 2011, accepted 7 February 2012. Manunza, A., Zidi, A., Ferna ´ndez-Cabana ´s, V. M., Jordana, J., Carrizosa, J., Belaifa, E., Urrutia, B., Polvillo, O., Gonza ´lez-Redondo, P., Amills, M. and Serradilla, J. M. 2012. S HORT C OMMUNICATION : An association analysis between one missense polymorphism at the SREBF1 gene and milk yield and composition traits in goats. Can. J. Anim. Sci. 92: 167173. Sterol regulatory element binding transcription factor 1 (SREBF1) regulates the expression of genes involved in the biosynthesis of fatty acids and cholesterol. Herewith, we have sequenced the near-complete coding region and part of the 3?UTR of the goat SREBF1 gene. In doing so, we have detected a missense c.353CT polymorphism causing a proline to leucine substitution at position 118 (P118L). An association analysis with milk composition traits recorded in Murciano- Granadina goats only revealed a statistical tendency linking SREBF1 genotype and milk omega-3 fatty acid content. The lack of significant associations suggests that the P118L substitution does not involve a functional change. Key words: Goat SREBF1 gene, milk fat content and composition Manunza, A., Zidi, A., Ferna ´ndez-Cabana ´s, V. M., Jordana, J., Carrizosa, J., Belaifa, E., Urrutia, B., Polvillo, O., Gonza ´lez-Redondo, P., Amills, M. et Serradilla, J. M. 2012. C OMMUNICATION B RE `VE : Analyse d’association entre un polymorphisme non synonyme dans le ge` ne SREBF1 et la production et la composition laitie` res chez les che` vres. Can. J. Anim. Sci. 92: 167173. Le facteur de transcription de ´nomme ´Sterol regulatory element binding transcription factor 1 (SREBF1) re ´gule l’expression des ge `nes implique ´s dans la biosynthe `se des acides gras et du choleste ´rol. Dans cette e ´tude, nous avons se ´quence ´la quasi-totalite ´de la re ´gion codante et une partie du la re ´gion 3?UTR du ge `ne SREBF1 de la che `vre. Ce travail, nous a permis d’identifier un polymorphisme non-synonyme c.353CT causant la substitution d’une Proline en Leucine a ` la position 118. L’e ´tude d’association avec la composition du lait enregistre ´e en che `vres Murciano-Granadina, a re ´ve ´le ´ seulement une tendance statistique reliant SREBF1 ge ´notype et l’acide gras ome ´ga-3 du lait. L’absence d’associations significatives sugge `re que la substitution P118L n’implique pas un changement fonctionnel. Mots cle ´s: Ge `ne SREBF1de la che `vre, composition de la matie `re grasse du lait Sterol regulatory element binding transcription factor 1 (SREBF1) plays a key role in energy homeostasis by regulating the expression of lipogenic genes (Brown and Goldstein 1997). At a cellular level, inactive SREBF1 remains anchored to either the nuclear envelope or the endoplasmic reticulum, while the N- and C- terminal ends localize to the cytoplasm (Pa ´rraga et al. 1998). Low levels of cholesterol activate SREBF1 through the proteolytic cleavage of the N-terminal segment by site-1 and site-2 proteases (Shimano 2001). Subsequently, SREBF1 is translocated to the nucleus where it forms homodimers and upregulates the expression of as many as 30 genes involved in cholesterol and FA biosynthesis (Pa ´rraga et al. 1998; Shimano 2001). In lactating cows, measurement of SREBF1 mRNA levels has shown that they increase after parturition, a finding consistent with the key role of this molecule in the coordination of the mammary gland lipogenic pathways (Harvatine and Bauman 2006; Rudolph et al. 2007; Viturro et al. 2009). The main goal of the current work was to characterize the genetic variability of the caprine SREBF1 coding region and find out if it is associated with phenotypic variation of milk fat content and composition traits in dairy goats. Total RNA was extracted from liver samples obtained from three Murciano-Granadina and two Malaguen ˜a A. Manunza and A. Zidi contributed equally to this work. 6 Corresponding authors: Marcel Amills (e-mail: Marcel. [email protected]t) and Juan Manuel Serradilla (e-mail: [email protected]). Can. J. Anim. Sci. (2012) 92: 167173 doi:10.4141/CJAS2011-111 167 Can. J. Anim. Sci. Downloaded from pubs.aic.ca by 87.222.191.14 on 10/11/15 For personal use only. goats following protocols reported by Zidi et al. (2008), and cDNA was synthesized with the ThermoScript RTPCR kit (Invitrogen S.A., Barcelona, Spain). The amplification of the coding region and part of the 3?UTR of the goat SREBF1 gene was performed with six primer pairs, following the protocols detailed at Supplementary Table 1. After enzymatic purification (ExoSAP-IT, Amersham Biosciences), PCR products were sequenced by using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequencing reaction products were purified with the Montage SEQ 96 cleanup kit (Millipore Corporation, Billerica, MA) and electrophoresed in an ABI PRISM 3730 DNA Analyser (Applied Biosystems, Foster City, CA). Molecular analyses yielded the near-complete sequence of the coding region and part of the 3?UTR of the goat SREBF1 gene, consisting of 3561 bp and encoding a 1139 amino acid protein (GenBank accession number: HM443643, Supplementary Fig. 1). Partial sequences had previously been submitted to GenBank, but assembled together they just covered half of the coding region of this gene (GenBank accession numbers: DQ487874, DQ455606, DQ480338 and DQ483057). Blast analysis of the goat SREBF1 cDNA sequence showed that it was very similar to its bovine (96% identity), porcine (88%) and human (86%) orthologs. Functional domains were inferred according to the UniProtKB entry for human SREBF1 (http://www. uniprot.org/uniprot/P36956). The caprine SREBF1 consisted of an N-terminal transcription factor portion, including a basic region/helixloophelix/leucine zipper (bHLHZ) DNA-binding motif, a hydrophobic region with two membrane spanning regions, and a C-terminal regulatory segment. The helix-loop-helix (HLH) domain of SREBF1 displayed a 60100 amino acid residue motif, encoding two amphipathic ahelices separated by a variable loop that is essential for forming homodimers (Pa ´rraga et al. 1998). This motif is flanked at its N- terminal and C-terminal ends by a highly conserved basic region that binds to DNA, and a leucine zipper motif, respectively (Supplementary Fig. 1). Finally, it is worth highlighting the existence of an acidic transactivation domain, which it is fundamental to activate transcription, adjacent to a proline-rich region. Alignment of goat SREBF1 cDNA sequences allowed us to detect a c.353CT missense polymorphism that involved a proline to leucine replacement at position 118 (Supplementary Fig. 2). A primer-extension based protocol was implemented to genotype it. Genomic DNA isolation and amplification reactions (PCR composition and thermal cycling) were performed as reported in Zidi et al. (2010), with the only exception of the annealing temperature (T ann 648C). Primer Express software (Applied Biosystems, Foster city, CA) was employed to design primers to amplify the polymorphic region as well as to make the extension reaction (Supplementary Table 1). The primer-extension reaction was carried out by following manufacturer instructions and using the following thermal cycling profile: 25 cycles of 10 s at 968C, 5 s at 508C and 30 s at 608C. Extension products were run in an ABI PRISM 3730 capillary electrophoresis device (Applied Biosystems, Foster City, CA) and analyzed with the ABI PRISM GeneMapper software v.4.0 (Applied Biosystems, Foster city, CA). In silico prediction of amino acid substitutions was carried out with the Polyphen software (Ramensky et al. 2002). This tool allows one to infer the functional consequences of an amino acid substitution by building a multiple alignment of the query protein sequence and related sequences and calculating a profile-matrix. Elements of this matrix (PSIC scores) are logarithmic ratios of the likelihood of given amino acid occurring at a particular position to the likelihood of this amino acid occurring at any position. Subsequently, PolyPhen calculates the absolute value of the difference between PSIC scores of both alleles in the variable site. If this difference is high (i.e., above 1.5), it means that the analyzed substitution is rarely or never observed in the protein family. According to this criterion, substitutions are classified as benign (do not involve a functional change) or possibly/probably damaging (they may involve a functional change). Genotypic frequencies of c.353CT polymorphism were calculated in a sample of 427 Murciano-Granadina goats. Frequencies were 0.39, 0.46 and 0.15, for CC, CT and TT genotypes, respectively (C and T allele frequencies: 0.62 and 0.38, respectively). Hardy-Weinberg equilibrium was assessed with an online Microsoft Excel tool available at http://www.tufts.edu/mcourt01/ Documents (Court Lab-HW calculator file). This approach allowed us to demonstrate that genotype frequencies matched the Hardy Weinberg equilibrium expectation (x 2 0.155, Pvalue: 0.693). The effects of this mutation were predicted in silico with the Polyphen software (Ramensky et al. 2002), obtaining a PSIC score difference of 2.55. This result suggested that this polymorphism might be likely damaging. With the aim of gaining additional insights into the biological effects of this substitution, we decided to investigate if the goat SREBF1 genotype is associated with milk yield and quality traits. In order to achieve this goal, we used as animal material two subsamples obtained from the 427 Murciano-Granadina population mentioned above (Badaoui et al. 2007; Zidi et al. 2010). Subsample 1 consisted of 116 goats distributed in three herds. These goats had records for dairy traits i.e., milk yield, milk protein, fat, lactose and dry extract contents and logarithm of the total somatic cell count (logSCC). The management of this population and phenotype recording are fully described in Badaoui et al. (2007). Subsample 2 was composed of 174 goats belonging to a single herd. For each goat, whole milk in the first of the two daily milkings was collected beginning 3 mo after parturition (average 97.7 d) and with a periodicity of 168 CANADIAN JOURNAL OF ANIMAL SCIENCE Can. J. Anim. Sci. Downloaded from pubs.aic.ca by 87.222.191.14 on 10/11/15 For personal use only. every 2 mo (65.5 d average sampling interval). Protein, fat, lactose, dry extract contents and somatic cells were measured with a CombiFoss 600 FC instrument, as reported by Zidi et al. (2010). A small sample of milk from each goat was freeze-dried and stored at 208C until it was used for the gas chromatography analyses of FA. Samples were weighted before performing all milk FA composition analyses. Separation and quantification of the FA methyl esters were carried out using a gas chromatograph Agilent 6890N Network GS System (Agilent, Santa Clara, CA), as reported by Zidi et al. (2010). As a whole, the number of goats with registers for dairy traits (milk yield, milk protein, fat, lactose and dry extract contents and logSCC) was 290 (116 goats from subsample 1 and 174 from subsample 2), while for milk FA composition, we had data from only 174 goats (subsample 2). A mixed model for repeated measurements (Littell et al. 1998) implemented with the SAS software (SAS 9.2 Institute, Inc., Cary, NC) was used to detect associations between SREBF1 genotypes and traits under study. This model included SREBF1 genotype, ordinal number of lactation, number of offspring born, month of lactation, season of sampling and herd as fixed effects; the random animal effect and the residual error term. Milk FA composition phenotypes were obtained in goats belonging to a single herd, so this fixed effect was not considered in the statistical model. In contrast, season of sampling was included as a fixed effect because there is a seasonal effect on lipid composition of plant pastures (Mel’uchova ´et al. 2008). Besides, logSCC was used as a covariate in the statistical model. In this sense Jensen (2002) suggested that udder health might affect milk FA composition. As shown in Tables 1 and 2, the association analysis did not yield significant results. We just found a statistical tendency for omega-3 FA content. This latter result is consistent with the key role of SREBF1 in the synthesis of unsaturated FA. In this way, increased expression of SREBF1 in the mammary gland of rats has been shown to induce an augmentation in the expression of stearoyl-CoA desaturases (Rodrı´guez- Cruz et al. 2006). Interestingly, when comparing previous studies aimed to associate SREBF1 genotype with fat content and composition (Hoashi et al. 2007; Ohsaki et al. 2009; Barton ˇet al. 2010; Matsuhashi et al. 2011), contrary results were noticed. For instance, Hoashi et al. (2007) identified an association between a 84 bp-indel at intron 5 of the SREBF1 gene and muscle monounsaturated FA content in Japanese Black cattle. Similarly, Barton ˇet al. (2010) found an association between this indel and muscle C14:1 cis-9 FA content in Fleckvieh cattle. In contrast, Ohsaki et al. (2009) analyzed the segregation of the 84 bp-indel in two groups of Japanese Black cattle and, overall, they did not find any significant association with an array of seven carcass and fourteen FA composition traits (although significant associations for myristic, palmitic and elongation index were observed in one group of cows). Similarly, Matsuhashi et al. (2011) performed an association study between the aforementioned 84-bp indel and FA composition of the longissimus thoracis muscle and carcass and meat quality traits in 480 commercial Japanese Black cattle and did not find any significant relationship. Our study is closely aligned with the work carried out by Ohsaki et al. (2009) and Matsuhashi et al. (2011) Table 1. Association between c.353CTSREBF1 genotype and dairy traits in Murciano-Granadina goats Data available CC CT TT Total Number of goats 114 135 41 290 Number of records 361 437 142 940 Traits Least square means9SE Pvalue Milk yield (kg d 1 ) 1.7090.11 1.7290.11 1.8090.13 0.58 Protein (%) 3.6190.05 3.5990.05 3.6590.06 0.51 Fat (%) 5.7490.18 5.6790.18 5.6790.20 0.78 Lactose (%) 4.9290.04 4.9290.04 4.9790.04 0.36 LogSCC 2.7190.07 2.7490.07 2.7390.08 0.75 Table 2. Association between c.353CTSREBF1 genotype and milk fatty acid composition traits in Murciano-Granadina goats Data available CC CT TT Total Number of goats 71 80 23 174 Number of records 194 224 65 483 Trait z Least square means9SE Pvalue SFA 76.1590.44 76.3490.42 76.5690.56 0.79 MUFA 19.8690.40 19.6890.38 19.7990.50 0.82 PUFA 4.1590.07 4.4190.06 3.9790.10 0.26 Ratio PUFA/MUFA 0.2090.008 0.2190.007 0.1990.01 0.40 Total CLA 0.7290.02 0.7390.02 0.6890.04 0.51 cis-9, trans-11 CLA 0.3990.03 0.4090.03 0.3690.04 0.61 C18:1 n9c 13.8490.25 14.0390.23 14.090.38 0.79 Omega 3 FA 0.3190.007 0.3190.007 0.2990.01 0.07 Omega 6 FA 3.0390.05 3.090.05 2.9090.08 0.42 z Fatty acid content was expressed as the percentage of total methyl esters. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; CLA, conjugated linoleic acid. MANUNZA ET AL. *GOAT SREBF1 AND MILK QUALITY 169 Can. J. Anim. Sci. Downloaded from pubs.aic.ca by 87.222.191.14 on 10/11/15 For personal use only. in the sense that we did not find a significant association between goat SREBF1 genotype and milk yield and quality traits. Notably, the results of the association analyses outlined in Table 1 and Table 2 challenge the in silico prediction obtained with PolyPhen that indicated that the P118L might have functional consequences. Recently, Rincon et al. (2011) identified a non-synonymous SNP at exon 14 of the bovine SREBF1 gene, which, according to in silico analyses with SIFT (Ng and Henikoff 2003) and PolyPhen, involved a functional change. Interestingly, this SNP displayed significant associations with milk fat and polyunsaturated FA content in cows suffering milk fat depression, but not in their healthy counterparts. As a whole, these results illustrate the complexity of assessing the functional effects of SNP on production traits through the performance of association studies, since results can be strongly affected by a plethora of biological (linkage phase between markers and causal mutations, genotype by environment interactions, dominant and epistatic effects, etc.) and experimental (sample size, number of records, phenotype recording etc.) factors. This circumstance stresses the need of validating associations between genetic markers and phenotypes in multiple populations and breeding environments in order to reach meaningful conclusions. This research was funded by projects AGL2007-66161-C02-01 and AGL2007-66161-C02-02. A. Zidi received fellowships of the Spanish Agency of International Cooperation and Development and of the Centre for Research in Agrigenomics (CRAG). Arianna Manunza received a postdoctoral fellowship from the Centre for Research in Agrigenomics (CRAG). Badaoui, B., Serradilla, J. M., Toma` s, A., Urrutia, B., Ares J. L., Carrizosa, J., Sa` nchez, A., Jordana, J. and Amills, M. 2007. Goat acetyl-coenzyme A carboxylase a: molecular characterization, polymorphism, and association with milk traits. J. Dairy Sci. 90: 10391043. Barton ˇ, L., Kott, T., Bures ˇ, D., .R ˇeha´ k, D., Zahra´ dkova´ , R. and Kottova´ , B. 2010. The polymorphisms of stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes and their association with the fatty acid profile of muscle and subcutaneous fat in Fleckvieh bulls. Meat Sci. 85:1520. Brown, M. S. and Goldstein, J. L. 1997. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: 331340. Harvatine, K. J. and Bauman, D. E. 2006. SREBP1 and thyroid hormone responsive spot 14 (S14) are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA. J. Nutr. 136: 24682474. Hoashi, S., Ashida, N., Ohsaki, H., Utsugi, T., Sasazaki, S., Taniguchi, M., Oyama, K., Mukai, F. and Mannen, H. 2007. Genotype of bovine sterol regulatory element binding protein- 1 (SREBP-1) is associated with fatty acid composition in Japanese Black cattle. Mamm. Genome 18: 880886. Jensen, R. G. 2002. The composition of bovine milk lipids: January 1995 to December 2000. J. Dairy Sci. 85: 295350. Littell, R. C., Henry, P. R. and Ammerman, C. B. 1998. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. 76: 12161231. Matsuhashi, T., Maruyama, S., Uemoto, Y., Kobayashi, N., Mannen, H., Abe, T., Sakaguchi, S. and Kobayashi, E. 2011. Effects of bovine fatty acid synthase, stearoyl-coenzyme A desaturase, sterol regulatory element-binding protein 1, and growth hormone gene polymorphisms on fatty acid composition and carcass traits in Japanese Black cattle. J. Anim. Sci. 89:1222. Mel’uchova´ , B., Blas ˇko, J., Kubinec, R., Go´ rova´ , R., Dubravska´ , J., Margetı´n, M. and Soja´ k, L. 2008. Seasonal variations in fatty acid composition of pasture forage plants and CLA content in ewe milk fat. Small Rum. Res. 78:5665. Ng, P. C. and Henikoff, S. 2003. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 31: 38123814. Ohsaki, H., Tanaka, A., Hoashi, S., Sasazaki, S., Oyama, K., Taniguchi, M., Mukai, F. and Mannen, H. 2009. Effect of SCD and SREBP genotypes on fatty acid composition in adipose tissue of Japanese Black cattle herds. Anim. Sci. J. 80: 225232. Pa´ rraga, A., Bellsolell, L., Ferre´ -D’Amare´ , A. R. and Burley S. K. 1998. Co-crystal structure of sterol regulatory element binding protein 1a at 2.3-A ˚resolution. Structure 6: 661672. Ramensky, V., Bork, P. and Sunyaev, S. 2002. Human nonsynonymous SNPs: server and survey. Nucleic Acids Res. 30: 38943900. Rincon, G., Islas-Trejo, A., Castillo, A. R., Bauman, D. E., German, B. J. and Medrano, J. F. 2012. Polymorphisms in genes in the SREBP1 signalling pathway and SCD are associated with milk fatty acid composition in Holstein cattle. J. Dairy Res. (in press). Rodrı´guez-Cruz, M., Tovar, A. R., Palacios-Gonza´ lez, B., Del Prado, M. and Torres, N. 2006. Synthesis of long-chain polyunsaturated fatty acids in lactating mammary gland: role of D5 and D6 desaturases, SREBP-1, PPARa, and PGC-1. J. Lipid Res. 47: 553560. Rudolph, M. C., McManaman, J. L., Phang, T., Russell, T., Kominsky, D. J., Serkova, N. J., Stein, T., Anderson, S. M. and Neville, M. C. 2007. Metabolic regulation in the lactating mammary gland: a lipid synthesizing machine. Physiol. Genom. 28: 323336. Shimano, H. 2001. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog. Lipid Res. 40: 439452. Viturro, E., Koenning, M., Kroemer, A., Schlamberger, G., Wiedemann, S., Kaske, M. and Meyer, H. H. D. 2009. Cholesterol synthesis in the lactating cow: Induced expression of candidate genes. J. Steroid Biochem. Mol. Biol. 115: 6267. Zidi, A., Sa` nchez, A., Obexer-Ruff, G. and Amills, M. 2008. Sequence analysis of goat major histocompatibility complex class I genes. J. Dairy Sci. 91: 814817. Zidi, A., Serradilla, J. M., Jordana, J., Carrizosa, J., Urrutia, B., Polvillo, O., Gonza´ lez-Redondo, P., Gallardo, D., Amills, M. and Ferna´ ndez-Cabana´ s, V. M. 2010. Polymorphism of the caprine malic enzyme 1 (ME1) gene: lack of significant association with milk production and composition traits in Murciano-Granadina goats. Animal 4: 19531957. 170 CANADIAN JOURNAL OF ANIMAL SCIENCE Can. J. Anim. Sci. Downloaded from pubs.aic.ca by 87.222.191.14 on 10/11/15 For personal use only. Supplementary Table 1. Sequences of primers employed to amplify the goat SREBF1 gene cDNA and to genotype the c.353CT polymorphism. Primer sequence z T ann y (8C) Init-Fw, 5?-ATGGACGAGCCACCCTTCA-3?64 Init-Rw, 5?-GCTGGCTCCTCCTTGATCC-3? F1- Fw, 5?-TGCTTCAGCTCATCAACAACCAAG-3?64 F1-Rw, 5?-GTTGATGGGCAGCTTGTCAGTGT-3? F2-Fw, 5?-CTGCAGCCCCACTTCATCAAGGC-3?64 F2-Rw, 5?-GACAGAGGAAGACGAGCGCGCA-3? F3-Fw, 5?-CCCTCCCAGAGCAGCCCCTTGT-3?61 F3-Rw, 5?-CTGGGCTGGGCCACGCAATT-3? F4-Fw, 5?-TTGCCATGCAGTGGCTCTGC-3?64 F4-Rw, 5?-AAGGAGCAGGTCACACAGGAGCA-3? Fin-Fw, 5?-GCCTCTCTGACAGCTGTGGT-3?66 Fin-Rw, 5?-CAGGGGCCAGCACTATCCT-3? F5, 5?-ACCCACCCCCCTGAAGATGTA-3?64 R2, 5?-AATGGGGGCAGAGCTGAACT-3? S2, 5?-TTGATCCCAGGCCCC-3? z Primers Init, F1, F2, F3, F4 and Fin were used to amplify and sequence the goat SREBF1 coding region. F5 and R2 primers were employed to amplify the region containing the c.353CT polymorphism, whilst S2 was used to carry out the extension reaction. y Annealing temperature. The amplification conditions for all fragments were as follows: an initial denaturation step at 948C for 5 min, followed by 35 cycles of denaturation at 948C for 1 min, annealing temperature for 1 min and extension at 728C for 2 min, plus a final extension step at 728C for 10 min. Amplification reactions were performed by using a reaction mixture containing 1.5 mM MgCl 2 , 100 mM of each dNTP, 0.5 mM of each primer, 2 mL of reverse transcription reaction, and 1.25 U of Taq DNA polymerase (Ecogen S.R.L., Barcelona, Spain) in a final volume of 25 mL. MANUNZA ET AL. *GOAT SREBF1 AND MILK QUALITY 171 Can. J. Anim. Sci. Downloaded from pubs.aic.ca by 87.222.191.14 on 10/11/15 For personal use only. Supplementary Fig. 1. Functional domains of the goat SREBF1 transcription factor according to the UniProtKB database (entry P36956) for human SREBF1: Acidic domain (underlined), putative transmembrane domains (double underlined), basic DNA- binding domain (black box), helix-loop-helix (HLH) motif (grey box), leucine zipper region (*) and protease cleavage sites (boxed arrows). 172 CANADIAN JOURNAL OF ANIMAL SCIENCE Can. J. Anim. Sci. Downloaded from pubs.aic.ca by 87.222.191.14 on 10/11/15 For personal use only. Supplementary Fig. 2. Electropherograms showing CC (lane A), CT (lane B) and TT (lane C) SREBF1 genotypes detected by primer-extension analysis. MANUNZA ET AL. *GOAT SREBF1 AND MILK QUALITY 173 Can. J. Anim. Sci. Downloaded from pubs.aic.ca by 87.222.191.14 on 10/11/15 For personal use only.