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Breed Assignment in Indigenous Portuguese Cattle Breeds As a Tool for Certification of Meat Products

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Breed Assignment in Indigenous Portuguese Cattle Breeds As a Tool for Certification of Meat Products 7th World Congress on Genetics Applied to Livestock Production, August 19-23, 2002, Montpellier, France BREED ASSIGNMENT IN INDIGENOUS PORTUGUESE CATTLE BREEDS AS A TOOL FOR CERTIFICATION OF MEAT PRODUCTS C. Ginja1, M.C.T. Penedo2, J. Mateus1, T. Rangel-Figueiredo1 and J. Matos3 1 University of Trás-os-Montes e Alto Douro, 5000 Vila Real, Portugal 2 Veterinary Genetics Laboratory, University of California, Davis, California, USA 3 INETI/IBQTA/DB/BQII, Lisboa, Portugal INTRODUCTION Domestic animal breeds represent discrete populations, significantly differentiated at molecular level and distinguishable on the basis of phenotypic characteristics (Blott et al., 1999 ; Barker, 2001). Characterization of domestic animal breeds is an important component of conservation efforts to achieve sustainable development, assure environment protection and food security (FAO, 2000). The emergence of markets for high quality beef produced by Portuguese indigenous cattle raised in extensive production systems has significantly contributed to the conservation of these breeds (Rodrigues et al., 1998). Over the last decade, there has been an increase in the number of certified beef products with Protected Denomination of Origin (PDO) that promotes the breed through brand names. In Portugal, 11 among the 13 indigenous cattle breed have a studbook and are maintained as purebred populations by breed associations or State Agencies. Seven of these breeds produce meat with PDO : Alentejana, Arouquesa, Barrosã, Maronesa, Mertolenga, Mirandesa and Preta. Exotic breeds explored in Portugal are mainly Holstein-Friesian, Charolais and Limousine which are raised as purebreds but also used in crossbreeding programs with indigenous stock. Validation of certified products through reliable DNA tests, capable of identifying the source of an individual in terms of breed of origin constitutes an important contribution for its successful marketing. Microsatellites are considered the molecular markers of choice for discrimination between populations, individual identification and genetic diversity analysis (Paetkau et al., 1995 ; MacHugh et al., 1998 ; Blott et al., 1999). Paetkau et al. (1995) developed a method appropriate for individual assignment assuming representative allelic frequency estimates, Hardy-Weinberg equilibrium (HWE) and independence among loci. Multilocus genotypes are used to calculate the probability of an individual genotype in a series of possible source populations based on allele frequencies. The individuals are assigned to the population in which its genotype has the highest probability of occurring. The objective of the present study was to investigate the application of microsatellite-based tests as a tool for breed assignment and certification of meat products of indigenous Portuguese cattle breeds. MATERIALS AND METHODS Sampling and DNA extraction. A total of 584 animals were analysed representing nine indigenous breeds and three exotic breeds raised in Portugal (table 1). Utmost care was taken to select unrelated animals in each breed and from an equal number of males and females. whole blood or hair roots were collected and used as source of DNA that was extracted according to standard procedures. Session 22. Exploitation of molecular information in animal breeding Communication N° 22-10 7th World Congress on Genetics Applied to Livestock Production, August 19-23, 2002, Montpellier, France Microsatellite analysis. Twenty nine microsatellites were used in the assignment test : BM203, BM1818, BM1824, BM2113, BM2613, BRRIBO, CSSM36, CYP21, ETH3, ETH10, ETH152, ETH185, ETH225, HEL9, HEL11, HEL13, ILSTS035, ILSTS065, INRA023, MGTG4B, RM006, RM067, SPS113, SPS115, TGLA53, ETH122, ETH126, ETH227 and ETH345. PCR conditions for amplification were done as described by Penedo et al. (1998) with the following modifications : total volume of reaction was 12.5 µl and MgCl2 concentration was 2.5 mM. The annealing temperature for all markers was 58 °C except for ETH185 (60 °C), HEL9 (52°C) and HEL13 (52 °C). PCR protocols are available upon request. Fluorescently labelled PCR products were run on acrylamide gels using ABI 373 DNA Sequencers (Applied Biosystems, CA, USA). ROX-Genescan 350 (Applied Biosystems) was used as the internal lane standard. Fragment sizes of alleles were determined using STRand software (Hughes, 2000). Statistical analysis and breed assignment. The computer program GENEPOP 3.3 (Raymond and Rousset, 2000) was used to obtain estimates of allelic frequencies, average observed (Ho) and expected (He) heterozygosities and to perform tests for HWE. A Bonferroni correction was used in HWE tests to achieve an experimentwise significance level of 5 % per population and reduce the chances of false rejection of the null hypothesis because of multiple tests. The program DISPAN (Ota, 1993) was used to estimate the Nei's coefficient of genetic diversity (GST). The mean number of alleles (MNA) per locus and per population were determined from allelic frequency tables. Discrimination among populations and individual assignment was performed according to Paetkau et al. (1995) using Doh program. Genotype probabilities were calculated using program defaults. RESULTS AND DISCUSSION Genetic diversity analysis. Results for average Ho, He and MNA per population are shown in table 1. HWE was generally observed across all populations. Statistically significant deviations from HWE (P < 0.0017) were noted for three loci in Brava. The situation for Brava may be indicative of inbreeding or substructuring within the breed. Among the 12 breeds, the average expected heterozygosity ranged from 0.63 to 0.74 with a mean of 0.70. The MNA ranged from 5.59 to 7.83 with an overall mean of 7 alleles/locus. These results indicate that the microsatellites used in this study present a high level of polymorphism. GST estimates (not shown) indicated that, on average, 9 % of the genetic variation was due to differences between breeds. Breed assignment. The test of discrimination and individual assignment was done under the assumption of HWE. Correct assignment of individuals to their population of origin ranged from 91 % in Holstein-Friesian to 100 % in Charolais, Limousine and Maronesa (table 2). The average power of the test was about 97 %. Results agree with those reported by Cañon et al. (2001). These authors assayed 16 microsatellite markers in 18 local breeds from Spain, Portugal and France and obtained correct breed assignment for an average of 96 % of the individuals per breed. In the present study, if clusters defined by a neighbour-joining tree based on genetic distances were considered, 97.8 % of the animals were assigned to the correct cluster (data not shown). These results suggest that a DNA-based test is available for certification of beef products derived from indigenous Portuguese cattle breed. Session 22. Exploitation of molecular information in animal breeding Communication N° 22-10 7th World Congress on Genetics Applied to Livestock Production, August 19-23, 2002, Montpellier, France Table 1. Genetic diversity of indigenous and exotic cattle breeds from Portugal based on 29 microsatellite loci. Numbers shown are for sample size (N), average observed (Ho) and expected (He) heterozygosities, and mean number of alleles (MNA). Standard errors are in parentheses Populations N Ho He MNA Indigenous Alentejana 50 0.648 (0.028) 0.671 (0.027) 6.7 (0.4) Arouquesa 50 0.708 (0.023) 0.727 (0.020) 7.2 (0.5) Barrosã 50 0.665 (0.028) 0.691 (0.026) 6.7 (0.3) Brava 40 0.558 (0.031) 0.642 (0.030) 5.8 (0.3) Galega 50 0.701 (0.025) 0.720 (0.020) 7.6 (0.4) Marinhoa 51 0.668 (0.020) 0.692 (0.017) 6.8 (0.4) Maronesa 50 0.730 (0.022) 0.714 (0.018) 6.8 (0.4) Mertolenga 50 0.690 (0.019) 0.743 (0.019) 7.8 (0.4) Mirandesa 50 0.614 (0.022) 0.631 (0.021) 5.6 (0.2) Exotic Charolais 46 0.679 (0.031) 0.698 (0.026) 7.1 (0.5) Holstein-Friesian 45 0.689 (0.023) 0.723 (0.017) 7.8 (0.5) Limousine 52 0.718 (0.017) 0.728 (0.015) 7.6 (0.6) Mean 0.672 (0.048) 0.698 (0.035) 7.0 (0.7) Table 2. Breed assignment based on 29 microsatellite loci Source populations Al Ar Ba Br Ga Ma Me Mi Mr Ch Fr Lm %* Alentejana (Al) 49 0 0 0 0 1 0 0 0 0 0 0 98 Arouquesa (Ar) 0 48 1 0 1 0 0 0 0 0 0 0 96 Barrosã (Ba) 0 0 49 0 0 0 1 0 0 0 0 0 98 Brava (Br) 0 0 1 38 0 0 1 0 0 0 0 0 95 Galega (Ga) 0 1 0 0 47 0 1 0 1 0 0 0 94 Maronesa (Ma) 0 0 0 0 0 50 0 0 0 0 0 0 100 Mertolenga (Me) 0 1 1 1 0 1 46 0 0 0 0 0 92 Mirandesa (Mi) 0 0 0 0 0 0 0 49 1 0 0 0 98 Marinhoa (Mr) 0 1 0 0 0 0 0 0 50 0 0 0 98 Charolais (Ch) 0 0 0 0 0 0 0 0 0 46 0 0 100 Holstein-Friesian (Fr) 0 0 0 1 0 0 0 0 0 1 41 2 91.1 Limousine (Lm) 0 0 0 0 0 0 0 0 0 0 0 52 100 Mean 96.7 * Percentage of individuals correctly assigned to source population Session 22. Exploitation of molecular information in animal breeding Communication N° 22-10 7th World Congress on Genetics Applied to Livestock Production, August 19-23, 2002, Montpellier, France CONCLUSION This study provides additional evidence that microsatellites are useful markers for genetic diversity analysis and breed assignment of closely related populations. It also contributes to the molecular characterization of Portuguese indigenous breeds. Breed assignment based on multilocus genotype probabilities was successful and indicated that this panel of markers could be implemented as a test for validation of certified meat products.
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