Productive Performance of the Dairy Cattle Girolando Breed Mediated by the Fat-Related Genes DGAT1 and LEP and Their Polymorphisms ⇑ S.R
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Research in Veterinary Science xxx (2011) xxx–xxx Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc Productive performance of the dairy cattle Girolando breed mediated by the fat-related genes DGAT1 and LEP and their polymorphisms ⇑ S.R. Cardoso, L.B. Queiroz, V. Alonso Goulart, G.B. Mourão, E. Benedetti, L.R. Goulart Laboratório de Nanobiotecnologia, Universidade Federal de Uberlândia, Av. Amazonas, s/n, Bloco 2E, Uberlândia-MG, Brazil article info abstract Article history: Candidate genes have been associated with milk production in bovines, such as the diacylglycerol Received 17 October 2010 O-acyltransferase 1 (DGAT1) and leptin (LEP); however, they have not been simultaneously investi- Accepted 12 February 2011 gated nor have been evaluated in the Brazilian Girolando breed (Gir  Holstein, backcrossed to Available online xxxx Holstein). Our aim was to determine the influence of fat-related genes, DGAT1 and LEP, and their polymorphisms on performance traits of milk production in the Girolando breed. Results indicated Keywords: that the K allele of the DGAT1 gene showed a significant association with total and average daily milk Girolando breed production with additive effect. The LEP gene showed that the A allele and its homozygote are highly Bovine prevalent and almost fixed in this population and may have been favorably selected during backcross- DGAT1 LEP ing for the origin of this breed. The important impact of the K allele of the DGAT1 gene on milk Milk production production corroborates the initiative of performing marker-assisted selections with this gene in Polymorphisms breeding programs of the Girolando breed. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Selection of specific genotypes or alleles from candidate genes in bovines that possess economically favorable traits are advanta- Brazilian bovines breeds have shown low indices of productiv- geous (Williams, 2005) and among them, the diacylglycerol ity, which may be related to climate, nutrition or genetic inheri- O-acyltransferase 1 (DGAT1) and Leptin (LEP) genes have been con- tance. The increasing productive performance of bovines is a sidered of high interest for dairy cattle (Ripoli et al., 2006, 2011). common goal for many cattle breeding programs worldwide. In or- The DGAT1 gene expresses the protein Acyl-CoA Diacylglycerol der to promote a better adaptation of high yield bovine breeds to Acyltransferase, a microsomal enzyme integrating the membrane, adverse environments, Brazilian breeding programs have gener- which acts in the synthesis of triglycerides in animals (Kuhn ated new breeds from zebu and taurine crosses, such as, for exam- et al., 2004; Tantia et al., 2006). As stated by Smith et al. (2000), ple, the Brazilian breeds Girolando (Gir  Holstein), Brangus the absence of the gene DGAT1 in some females, affects the synthe- (Nelore  Angus) and Simbrasil (Guzera  Simental). These breeds sis of Triacylglycerides, contributing to the reduction and/or have shown better adaptation to the hot and humid climate, boost- extinction of milk production in the animal. Furbass et al. (2006) ing dairy cattle productivity (Bicalho et al., 2006). have shown that the gene DGAT1, located in the terminal portion We have decided to investigate the variations of specific gene of the centromere referring to chromosome 14 in bovines, is prob- polymorphisms in the Girolando breed due to some extrinsic and ably a locus with a quantitative profile (QTL). Grisart et al. (2001); intrinsic factors linked to the environment and to the genetic back- Winter et al. (2002) and Roos et al. (2007) demonstrated that the ground, respectively. Javanmard et al. (2005) have explained that oscillation in the QTL profile is often caused by a substitution of both factors play a decisive role in the metabolism of the Girolando non-conservative dinucleotides ApA ? GpC in the exon 8 of the breed in terms of milk synthesis. Amongst these factors, it is worth gene DGAT1, changing from Lysine to Alanine in position 232 highlighting the feeding (Restle et al., 2005), type of management (mutation K232A) in the encoded protein. The increase in fat in (Cerdótes et al., 2004; Khatib et al., 2006; Oliveira and Nogueira, the milk in different breeds of bovine is intimately associated with 2006; López et al., 2007) and genotype (Smith et al., 2000; Lysine (K) in position 232 of the protein encoded by DGAT1 in Komisarek et al., 2004; Grisart et al., 2004; Khatib et al., 2006). bovines (Kaupe et al., 2004), while an Alanine (A) in this position is associated with a low fat content in milk (Winter et al., 2002; Kuhn et al., 2004). ⇑ Corresponding author. Address: Laboratório de Nanobiotecnologia, Instituto de The LEP gene expresses a 16-kDa protein synthesized by the Genética e Bioquímica, Universidade Federal de Uberlândia, Campus Umuarama, Av. Amazonas, s/n, Bloco 2E, Uberlândia-MG, Brazil. Tel./fax: +55 34 32182203. adipose tissue. Its function has been linked to energy homeostasis E-mail address: [email protected] (L.R. Goulart). (Bartha et al., 2005; Chilliard et al., 2005), fertility (Liefers et al., 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.02.006 Please cite this article in press as: Cardoso, S.R., et al. Productive performance of the dairy cattle Girolando breed mediated by the fat-related genes DGAT1 and LEP and their polymorphisms. Res. Vet. Sci. (2011), doi:10.1016/j.rvsc.2011.02.006 2 S.R. Cardoso et al. / Research in Veterinary Science xxx (2011) xxx–xxx 2002, 2005) and immunological properties of the herd (Fruhbreck mix contained 1 lL of genomic DNA, 0.5 U of Taq DNA polymerase et al., 1998; Lord et al., 1998). In bovines, the LEP gene is found on (Platinum, Invitrogen), 10 pmol of each primer, 200 lM of dNTPs, chromosome 4, which consists of 3 exons and 2 introns, although 1, 5 mM of MgCl2, 1X reaction buffer and water. The PCR reaction only 2 exons express the protein (Stone et al., 1996). Lindersson included an initial denaturation of 94 °C for 4 min, followed by et al. (1998) discovered a QTL for the production of milk near the 10 cycles of 60 s at 94 °C, 60 s at 66 °C(1°C.cycleÀ1) and 1 min at LEP gene, at a distance of 82.8 centimorgans (cM). Various poly- 72 °C, and a further 25 cycles 1 min at 94 °C, 2 min at 56 °C and morphisms in the LEP gene, which influence milk production, 1 min at 72 °C, with a final extension of 10 min at 72 °C. reproduction and physiology of nutrients’ ingestion such as three For the LEP genotyping, a PCR product of 400 bp, located in the SNPs in exon 2 (Arg/Cy) and exon 3 (Ala/Val, Glu/Arg), other poly- intron 2 between exons 2 and 3, was used for PCR-RFLP and PCR- morphisms located in the promoter region and nine SNPs in the in- SSCP analyses. The sequences of primers were: sense 50-TGGAGTGGC tron 2, were also found in the bovine genome (Javanmard et al., TTGTTATTTTCTTCT-30 and antisense 50-GTCCCCGCTTCTGGCTACC 2010). Veerkamp et al. (2000), Alfonso (2005) and Williams TAACT-30 near the location of the polymorphism (Liefers et al., (2005) indicated the LEP as a candidate gene for marker-assisted 2002). A 20 lL reaction mix contained 1 lL of genomic DNA, selection, due to it is important physiological role, and its putative 0.5 U of Taq DNA polymerase (Platinum, Invitrogen), 10 pmol of association with the productive performance of dairy cattle, which each primer, 200 lM of dNTPs, 1, 5 mM of MgCl2, 1X reaction buf- was demonstrated elsewhere (Akers, 2006; Liefers et al., 2005). fer and water. The PCR reaction included an initial denaturation This investigation aimed determining the influence of polymor- step of 94 °C for 2 min, followed by 35 cycles of 94 °C, 55 °C and phisms of the two metabolic fat-related genes DGAT1 and LEP in 72 °C (1 min each) and finished with a final extension of 15 min the milk production and its components in the Girolando breed. at 75 °C. Two microlitres of each PCR product was added to 18 lL of Low Ionic Strength – LIS solution (10% sacarose, 0.01% Bromophenol 2. Materials and methods Blue and 0.01% Xylene cyanol) homogenized and heated at 95 °C for 12 min and immediately transferred to an ice bath and loaded 2.1. Sample collection onto a 12% polyacrylamide gel (49:1; acrylamide:bis-acrylamide). Electrophoresis was carried out at room temperature, 15 mA Blood samples were collected from 349 females Girolando (150 W, close to 1000 V) in 1X TBE buffer during 16 h. The PAGE breed (3/8 Gir + 5/8 Holstein). All the animals under analysis were was silver stained and dried, according to the Popescu (1993) milked twice a day. Due to differential frequencies of lactation, the adapted for a porous transparent cellophane paper fixation, per- lactation order greater than seven (8–11) were considered as sev- formed at room temperature for 12 h stretched on the surface of enth order, as registered by the Brazilian Association of Girolando a glass plate. Breeders (ABCG), the official dairy control program monitored by the Dairy Control Service of the Association (SCL). Feeding regimes were grouped into 1 = pasture with a regular mineralized salt sup- 2.3. Statistical analysis plement; 2 = pasture with greater mineralized supplementation and/or confined animals. For the total production, the linear effect The allelic and genotypic frequencies were calculated according of the lactation length (in days) as co-variable was also considered. to Weir (1996). Hardy–Weinberg equilibrium (HWE) for each gene.