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Growth Hormone Alui Polymorphism Analysis in Eight Portuguese Bovine Breeds

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Growth Hormone Alui Polymorphism Analysis in Eight Portuguese Bovine Breeds GROWTH HORMONE ALUI POLYMORPHISM ANALYSIS IN EIGHT PORTUGUESE BOVINE BREEDS ANçLISIS DEL POLIMORFISMO ALUI DE LA HORMONA DE CRECIMENTO EN OCHO RAZAS BOVINAS PORTUGUESAS Reis, C.1, D. Navas2, M. Pereira3 and A. Cravador1 1Universidade do Algarve. UCTA. Campus de Gambelas. 8000-810 Faro. Portugal. E-mail: [email protected] / [email protected] 2Esta•‹o ZootŽcnica Nacional, Departamento de Bovinicultura. Fonte Boa. 2000-763 Vale de SantarŽm. Portugal. E-mail: [email protected] 3Esta•‹o ZootŽcnica Nacional. Departamento de Ovinicultura. Fonte Boa. 2000-763 Vale de SantarŽm. Portugal. E-mail: [email protected] ADDITIONAL KEYWORDS PALABRAS CLAVE ADICIONALES Somatotropin. Polymorphism. Meat production. Somatotropina. Polimorfismo. Producci—n de car- PCR-RFLP. ne. PCR-RFLP. SUMMARY RESUMEN A total of 195 bulls of eight Portuguese beef Un total de 195 bovinos pertenecientes a cattle breeds (Alentejana, Arouquesa, Barros‹, ocho razas productoras de carne portuguesas Maronesa, Marinhoa, Mertolenga, Mirandesa (Alentejana, Arouquesa, Barros‹, Maronesa, and Preta) were genotyped for the GH AluI Marinhoa, Mertolenga, Mirandesa y Preta) fue- polymorphism by the polymerase chain reaction ron genotipados utilizando PCR-RFLP para el and restriction length polymorphism (PCR- polimorfismo CH AluI. Se determinaron el geno- RFLP). The genotype and gene frequencies for tipo y las frecuencias gŽnicas para cada raza each breed were determined and shown to be mostrando una gran variabilidad entre razas. quite variable among the breeds. The overall Las frecuencias gŽnicas globales para L y V gene frequencies for L and V were 0.759 and fueron 0,759 y 0,241 respectivamente. Se esta- 0.241, respectively. The relation between the bleci— en 168 de los animales analizados la re- bGH-AluI polymorphism and growth performan- laci—n entre el polimorfismo bGH-AluI y los re- ces was ascertained in 168 of the animals analysed. According to our results there is a sultados de crecimiento. De acuerdo con significant association between the genotypes nuestros resultados hay una asociaci—n signifi- LL and LV of the bGH and the average live cativa entre los genotipos LL y LV de bGH y el body weight of the animals of the breeds Alen- peso vivo de los animales en las razas Alente- tejana, Marinhoa and Preta. jana, Marinhoa y Preta. Arch. Zootec. 50: 41-48. 2001. REIS, NAVAS, PEREIRA AND CRAVADOR INTRODUCTION This transversion enables the genoty- ping at this particular locus using the The use of polymorphic specific ge- endonuclease AluI since this enzyme nes as molecular detectable markers is does not recognize its target sequence a promising alternative to the current when a G is present instead of a C. The methods of trait selection, once these AluI (+/-) polymorphism is believed to genes are proven to be associated with be related to plasma levels of GH as traits of interest in animals. suggested by Schlee (1994b). This au- The bovine growth hormone (bGH) thor observed that genotype LL was is a 22 KDa single-chain polypeptide usually associated with higher circula- hormone produced in the anterior pitui- ting concentrations of GH when com- tary gland. The encoding gene is appro- pared to genotype LV. Chrenek (1998) ximately 1800 base pairs (bp) and con- reported an association between bGH- sists of five exons separated by four AluI polymorphism and meat produc- intervening sequences (Woychick et al., tion traits in Slovak Simmental bulls. 1982; Gordon et al., 1983). It is well This hormone was shown to be poly- known that it plays an important role in morphic in many breeds, being the dis- biological processes such as mammary tribution of GH variants (LL, LV, VV) development, lactation, growth and me- and their frequencies different among tabolism regulation (reviewed by Ether- each breed. The study of the effects of ton, 1998), being therefore a promising growth hormone genotypes on growth candidate gene marker for improving traits is of great interest in the breeds milk and meat production in cattle. analysed in this study, as their main Recently several studies have inves- purpose is meat production. tigated associations between genetic The objectives of the present study polymorphisms at the bGH locus with were: (1) to reveal GH-AluI polymor- phism in the eight major indigenous production traits, namely to milk pro- Portuguese cattle breeds and estimate tein percentage (Lagziel et al., 1996 the gene frequencies, (2) to look for an and Vukasinovic et al., 1999, and refe- association between growth performan- rences therein). A TaqI RFLP, using a ces and GH-AluI variants. complementary DNA (cDNA) probe for GH, has been associated with the birth-weight of beef cattle (Rocha, MATERIAL AND METHODS 1991). A polymorphism in the fifth exon, responsible for two alternative ANIMALS forms of the hormone, was reported by A total of 195 bulls of the following Lucy (1991). A substitution of a citosi- indigenous breeds were included in the ne (C) for a guanine (G) at position present report: Alentejana (AL, n=22), 2141 (Zang, 1992); [designation from Arouquesa (AR, n=24), Barros‹ (BA, the sequence in work of Gordon n=23), Marinhoa (MO, n=32), Marone- (1983)] causes an amino acid change sa (MA, n=24), Mertolenga (ME, from leucine (L, codon CTG) to a vali- n=22), Mirandesa (MI, n=21) and Preta ne (V, codon GTG) at the residue 127. (PR, n=27). Animals born between Archivos de zootecnia, vol 50, nœm 189-190, p. 42. GROWTH HORMONE ALUI POLYMORPHISM IN PORTUGUESE BOVINE BREEDS April 1996 and January 1997 came lation kit from Puregene. from various herds in Portugal and we- Twenty-five ml polymerase chain re- re purchased through Associations of actions (PCR) were carried out in a Bio- Breeders. Rearing was made at the fee- metra UNO II 48 thermalcycler, using dlot of the Esta•‹o ZootŽcnica Nacio- PCR beads Ready-To-Go (Amersham nal (SantarŽm, Portugal), being each Pharmacia Biotec) with 50 ng of bovine breed physically isolated from each genomic DNA and 16 pmol of each pri- other. Initial average age (IAW) and in- mer. The primers GH5F (5«- itial weights were, respectively: GCTGCTCCTGAGGGCCCTTC-3«) 237.5 ± 11.3 d and 248.4 ± 8.8 kg and GH5R (5«CATGACCCTCAGG- for AL; 247.3 ± 10.6 d and 221.9 ± 8.2 TACGTCTCCG-3«) flanked a 211 base kg for AR; 217.0 ± 10.2 d and 178.7 ± pair (bp) fragment, consisting of 49 bp 7.9 kg for BA; 173.6 ± 10.2 d and of the fourth intron and 162 bp from the 190.6 ±7.9 kg for MO; 247.3 ± 9.9 d fifth exon according to the published se- and 207.2 ± 7.7 kg for MA; 246.5 ± quence by Gordon et al. (1983). After a 11.8 d and 195.7 ± 9.2 kg for ME; first denaturation step at 95¼ for 5 min, 261.8 ± 11.9 d and 277.2 ± 9.2 kg for the samples were amplified for 30 MI and 294.6 ± 10.3 d and 217.0 ± 8.0 cycles: denaturation 95¼ x 30 s; primer kg for PR. annealing 62¼ x 30 s; primer extension Animals were all fed with the same 72¼ x 30 s; followed of a 5 min final ex- feeding ration (maize silage and con- tension step at 72¼. Amplification pro- centrate). The control of body weights ducts (8.5 ml) were digested at 37¼ for at was made at the arrival of each animal least 14 hours with 5 Units of AluI and subsequently each 21 days. Among [AG|CT] (Gibco BRL, Life Technolo- the eight races, four are currently consi- gies) and separated on a 3,5 p.100 aga- dered to be small breeds (Arouquesa, rose gel containing 0.1 mg/ml EtBr. Barros‹, Maronesa and Mertolenga) re- aching a mature weight of 700 kilos STATISTICAL ANALYSIS and four are considered to be heavy Allele frequencies were calculated breeds (Alentejana, Marinhoa, Miran- by allele counting. desa, Preta) reaching largest mature Of the 195 animals, 27 were exclu- weights of 1000 kg. A first group of the ded of the statistical approach due to animals was slaughtered when reaching missing values or unreliable data. The approximately 50 p.100 of the expected data collected regarding the weights mature weight (P2). A second group was analysed with the SAS procedure was slaughtered at 70 p.100 of the ex- (SAS system for Windows 6.12, 1996 pected mature weight (P3) and finally a SAS Institute INC.) with mixed proce- third group was weighted until the ma- dure according to the following statisti- ture weigh (P4). cal model: _ Y = m + a + b + g (x - x) + GENOTYPING OF BULLS ijk _i j ijk + (x - x)2 + Animal + DNA was extracted from periphe- g ijk ijk eijk ral blood leukocytes using DNA Iso- where: Archivos de zootecnia, vol 50, nœm 189-190, p. 43. REIS, NAVAS, PEREIRA AND CRAVADOR Yijk: phenotypic value of the weight of mals gave a three-band (211, 159 and the animal k of the i breed with a ge- 52 bp) pattern (figure 1). notype j Considering the 195 bulls analysed, m: overall mean the overall genotype frequencies were ai: fixed effect of the breed 0.600 for LL, 0.318 for LV and 0.082 bj: fixed effect of the genotype (j = LL, for VV. Gene frequencies of alleles L LV, VV)_ and V were 0.759 and 0.241, respecti- g (xijk - x_): linear effect of covariate age vely. Significant differences were ob- g (xijk - x)2: quadratic effect of covariate served among breeds (table I). Genoty- age pe VV for example, was absent in the Animalijk: random effect analysed populations of Marinhoa, eijk: random error Mertolenga, Mirandesa and Preta bre- This model was firstly adjusted for the heavy breeds and then for the small breeds considering 3 distinct periods of 1 2 3 4 5 6 growth.
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