Journal of Heredity Advance Access published online on November 5, 2007
Journal of Heredity, doi:10.1093/jhered/esm096
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Genetic Diversity and Relationships of Endangered Spanish Cattle Breeds
From the Laboratorio de Genética Bioquímica, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain (Martín-Burriel, Rodellar, Sanz, Cons, Osta, and Zaragoza); the Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands (Lenstra); Instituto Técnico y de Gestión Ganadera Navarra, S.A., Carretera del Sadar, s/n. Ed. El Sarrio 2a, 31006 Pamplona, Spain (Reta); CENSYRA de Torrelavega. Consejería de Ganadería, Agricultura y Pesca. Gobierno de Cantabria, Sierrapando s/n 39300 Torrelavega, Spain (Argüello); and the Centro de Investigación y Tecnología Agroalimentaria. Gobierno de Aragón, Avenida Montañana 930, 50059 Zaragoza, Spain (Sanz)
Address correspondence to I. Martín-Burriel at the address above, or e-mail: minma{at}unizar.es.
Information on the genetic structure and variability of autochthonous livestock breeds is essential for effective conservation programs. Here we present a molecular characterization on the basis of 30 microsatellite markers of 5 Spanish endangered cattle breeds Betizu (BET), Mallorquina (MAL), Menorquina, Monchina (MON), and Serrana de Teruel (ST) and of 2 fighting bull populations, Casta Navarra (CN) and Casta Vistahermosa. The feral and critically endangered BET is divided into 2 subpopulations, one of which has exceptionally low diversity values. A low number of alleles was also observed in the island population MAL. Although the small population size and genetic drift have caused a considerable divergence between the breeds, phylogenetic analysis is in accordance with historical and geographical data. The 2 northern Spanish feral breeds BET and MON cluster together. The local fighting breed CN is relatively close to the more inbred Casta Vistahermosa, which is the progenitor of most other fighting bulls in Spain. Comparison with nonendangered breeds suggests admixture of Alpine and/or Pyrenean mountain cattle in the ST, which may contribute to the high level of linkage disequilibrium in this population.
During the last century, many indigenous domestic breeds became extinct by replacement or crossbreeding with exotic breeds, substitution of draft animals by technology, or unfavorable marketing (Köhler-Rollefson 2000). About one-third of the world's recognized 5000 livestock and poultry breeds are endangered (FAO/UNEP 1995) but represent in both developed and developing countries a unique resource to meet present and future breeding objectives. Molecular characterization of animal genetic resources may contribute to a rational approach to conservation (Hanotte and Jianlin 2005; European Cattle Genetic Diversity Consortium 2006) by giving a high priority to breeds that are taxonomically most distinct (Barker 1999).
Molecular markers like microsatellites are now commonly used for the estimation of genetic diversity, calculation of genetic distances and detection of admixture, genetic bottlenecks, and inbreeding (Sunnucks 2000). Several reports have described the genetic relationships between Spanish cattle breeds on the basis of microsatellites (Martin-Burriel et al. 1999; Cañon et al. 2001; Beja-Pereira et al. 2003; Rendo et al. 2004). Here we analyze 2 Balearic island breeds (Mallorquina [MAL] and Menorquina [MEN]), one rustic breed (Serrana de Teruel [ST]), 2 feral or semiferal populations (Betizu [BET] and Monchina [MON]), and 2 fighting bull populations (Casta Navarra [CN] and Casta Vistahermosa [TL]). All these populations are endangered except Casta Vistahermosa fighting bull (TL). BET lives in small nuclei in Basque Country, Navarra, and in the neighboring French Atlantic Pyrenees and is considered as a lowly developed and distant relative of the Pyrenean Turdetano breed group. MON is supposed to be influenced by Red Turdetano and Blond-Brown Cantabrico cattle. CN represents a distinct subpopulation of fighting bull. It has been isolated from the other subpopulations for hundreds of years, whereas TL is the progenitor caste of most other Spanish fighting bulls. Due to similar wild behavior and morphological characteristics, MON and BET are often thought to be related to CN cattle. The aim of this work is to characterize the endangered populations in terms of genetic variability and to analyze their genetic relationships with other breeds (Martín-Burriel et al. 1999; Schmid et al. 1999).
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Materials and Methods |
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A total of 291 animals from the MAL (28), MEN (50), ST (44), CN (50), TL (44), MON (59), and BET (60) breeds were sampled. However, only one farmer breeds MEN cattle, whereas BET animals come from 3 farms. The unusual high number of loci in disequilibrium (see Results) led to a subdivision of BET in 2 subpopulations, BETA (n = 37) and BETB (n = 23), according to the farm of origin. TL was sampled from 10 different locations.
DNA was extracted from blood using standard protocols and the GFX Genomic Blood DNA purification kit (Amersham Biosciences, now GE Healthcare, Chalfont St. Giles, UK). Thirty microsatellite loci recommended by the Food and Agriculture Organization (http://www.projects.roslin.ac.uk/cdiv/) for genetic diversity studies were analyzed: INRA063*, INRA005*, ETH225*, ILSTS005*, HEL5*, HEL1*, INRA35*, ETH152*, INRA023*, ETH10*, HEL9*, CSSM66, INRA032*, ETH3*, BM2113*, BM1824*, HEL13*, INRA037*, BM1818, ILSTS006, MM12E6, CSRM60, ETH185, HAUT24, HAUT27, TGLA227*, TGLA126*, TGLA122*, TGLA53*, and SPS115 (asterisks indicate the 21 microsatellites that were also used for comparison with surrounding breeds). Microsatellite allele sizes were visualized by using 
[32P]-dCTPs in the polymerase chain reaction and 6% denaturing polyacrylamide gel electrophoresis or by using the ABI PRISM 3130 Genetic Analyser (Applied Biosystems, Foster City, CA). The internal size standard GeneScan-500LYS (Applied Biosystems, Warrington, United Kingdom) was used for sizing alleles. Three control samples were shared with participants of the EU RESGEN CT 98-118 cattle genetic diversity project in order to ensure compatibility of allele sizes with other research groups.
Genotypes for the 21 common microsatellites of Asturiana de los Valles (AV), Asturiana de las Montañas, Morenas del Noroeste (MNO), Rubia Gallega (RG), Pirenaica (PIR), and Swiss Brown (SWB) have been published previously (Martín-Burriel et al. 1999; Schmid et al. 1999).
Allele frequencies for each locus were calculated by direct count using GENEPOP (Raymond and Rousset 1995). This program was also used to test possible deviations from the Hardy–Weinberg proportion using a Markov chain method to estimate the P value. Genotypic linkage disequilibrium (LD) for all 2-locus pairs in all populations was calculated using GENEPOP. The probability (P) of rejecting the null hypothesis of random association between loci was calculated using a Markov chain method. LD between 2 loci was considered significant for P values less than 0.05.
Mean number of alleles per locus, unbiased and direct count estimated heterozygosities, and the allelic richness or corrected mean number of alleles per population based on minimum sample size (10) were calculated using FSTAT (Goudet 2002). Inbreeding coefficients (F) were calculated for each population on the basis of expected versus observed heterozygosity.
Weir and Cockerham (1984) estimators of FIT, FST, and FIS were calculated for each locus and overall using FSTAT (Goudet 2002). The DISPAN program (Ota 1993) was used to estimate DA (Nei et al. 1983) genetic distances on the basis of 21 microsatellites and to construct the neighbor-joining (NJ) trees.
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Results and Discussion |
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Fixation of single alleles was observed for markers SPS115 and HEL13 in MAL and INRA63, ILSTS005, INRA32, and CSSM66 in the subpopulation BETA, which also displayed the lower mean expected unbiased heterozygosity value (0.41). Conversely, CN and MON showed the highest diversity measures (see Table 1).
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All populations showed statistically significant deviations from Hardy–Weinberg equilibrium (HWE) at one or more loci (Table 1). Twenty-one loci were in disequilibrium in the BET population, 20 of which because of a deficit of heterozygosity. This departure from HWE could be a consequence of the Wahlund effect as in populations that contain subpopulations; there are fewer homozygotes than in the average for the set of subdivided populations. Then, there may seem to be more homozygotes in BET than expected from HWE principle as a result of pooling of the separate subpopulations. In fact, when the 2 BET subpopulations were considered independently, only one and 5 loci were found in disequilibrium in BETA and BETB, respectively. The subdivision found in the BET cattle confirms the observation of Rendo et al. (2004), who found a large FIS value attributed to geographic isolation among BET herds. The same subpopulations corresponding to the farms of origin were identified by model-based clustering (Pritchard et al. 2000; European Cattle Genetic Diversity Consortium 2006) and by Amplified Fragment Length Polimorphism profiling (Negrini et al. 2007). The other feral breed, MON, has the highest mean number of alleles and expected heterozygosity. Different ecotypes based on coat color have been described for this population, which may explain the relatively high number of HWE deviations (16%).
Diversity measures for CN, a fighting bull subpopulation, are higher than those previously observed for other fighting bull breeds (Kidd et al. 1980; Martin-Burriel et al. 1999). The fighting bull is partitioned into several lines, traditionally bred on farms that impose reproductive isolation. This breeding strategy has led to divergence among farms and even to morphological differences. However, CN breeders are reputed to allow a higher gene flow between farms. Furthermore, by their isolation from the remaining castes for centuries and possibly also by admixture of other breeds, CN has characteristics not found in other fighting bulls, like small size, mainly red coat, lyre-shaped horns, and a quick and vivacious disposition. The high inbreeding coefficient of TL (Table 1) is probably explained by genetic isolation of separate breeding farms and suggests that crossing animals from different farms would counteract possible negative health effects of the inbreeding.
The proportion of marker pairs with significant LD ranged from 0.062 in CN to 0.166 in BETA, with the exception of the pooled BET population (0.80). Most of the breeds analyzed displayed LD proportions similar to those reported for nonsyntenic loci in other cattle populations (Vallejo et al. 2003). However, the percentage of LD was higher in ST and BETA. LD can be generated by drift (Hill and Roberston 1968; Ohta and Kimura 1969) or admixture (Stephens et al. 1994; Farnir et al. 2000). For BETA, the reduced effective size and reproductive isolation are the most likely causes of LD and admixture for ST. However, in spite of its endangerment, ST has high diversity values as well as a low inbreeding coefficient. This breed is reputed to be influenced not only by Negra Avileña from Central Spain (http://www.tiho-hannover.de/einricht/zucht/eaap/) but also by other mountain type breeds of northern Spain. Probably, these admixtures have contributed to the present variability and the observed LD.
Inbreeding coefficients (FIS) were close to zero for all markers except INRA035. With the coancestry coefficients (FST) per locus ranging from 0.045 to 0.202, the overall differences between breeds explained 9.6% of total genetic variability. Mean expected heterozygosities of these cattle populations, as well as the F-statistics differentiation values, are similar to those previously reported for other European or Iberian cattle populations (MacHugh et al. 1997; Moazami-Gourdarzi et al. 1997; Martin-Burriel et al. 1999; Edwards et al. 2000; Kantanen et al. 2000; Cañón et al. 2001; Mateus et al. 2004).
Figure 1 shows a phylogenetic tree constructed on the basis of Nei's DA pairwise distances. A similar tree but with lower bootstrapping values was obtained with DS distances (not shown). Although most bootstrap values are low, the clustering of breeds is in accordance with historical data. For example, the island population MAL is clearly differentiated from the other breeds due to a strong genetic drift but clusters with MEN cattle. Both breeds belong to the Red Convex Iberian cattle group and have been strongly influenced by Brown Ultra-convex African cattle (Puigserver et al. 2000). However, their origin appears to be obscured by the degree of inbreeding (Takezaki and Nei 1996), as noted previously for Jersey island cattle (Medjugorac et al. 1994). The 2 fighting bull populations TL and CN are also clustered, indicative of a common origin. In agreement with the breeding practice, TL has more diverged from the other Spanish breeds than CN.
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ST forms a cluster with SWB and appears also to be relatively close to PIR, confirming direct or indirect gene flow between the mountain cattle breeds and ST. PIR and AV are known to be influenced by SWB (Sanchez Belda 1984; Felius 1992; Sierra Alfranca 1998), which explains their relatively short genetic distance. MNO and RGA are both from North-western Spain and also appear to be relatively close. Finally, the BETA population is diverged by strong genetic drift but is clustered with BETB in the NJ tree. Their closest relative is the other feral breed MON, which has similar behavior and morphology and also shares with BET a common geographical origin in the north of Spain.
Different genetic markers are being used to elucidate the relationships between modern cattle populations. Mitochondrial variation in Spanish cattle shows the influence of African cattle (Miretti et al. 2004; Beja-Pereira et al. 2006). However, as in cattle only a few major mitochondrial DNA lineages exist (Bruford et al. 2003), microsatellites give more insight into the recent demographic history of domestic breeds. Nevertheless, the divergence of Balearic breeds observed in our microsatellite study is in accordance with analysis based on Y chromosome data that have shown that MEN displays a North European haplotype instead of the one found in other Iberian and Mediterranean populations (Götherström et al. 2005).
The present study of Spanish native cattle breeds contributes to a genetic characterization of indigenous populations. Genetic divergence of the Spanish cattle breeds and within-population genetic diversity are the combined result of breed origin, founder effects, population size, genetic drift, and admixture. As shown here, this may lead to inbreeding and low genetic diversity. Exchanging individuals between isolated nuclei and avoiding further inbreeding by pedigree analysis may increase the genetic variability and viability of the autochthonous endangered breeds.
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Funding |
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The European Community (European Project RESGEN CT 98-118); Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria projects PET07-05-C03-03 and RZ-00003-C02-02.
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Acknowledgments |
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The authors thank Gaudenz Dolf (Berne) for making available the genotypes of the Swiss Brown, L. Bericat for her technical assistance, G. Segui and S. Segui for the Menorquina blood samples, and G. Puigserver for the Mallorquina samples. The content of this publication does not represent the views of the European Commission or its services.
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Corresponding Editor: James Womack
Received November 6, 2006
Accepted August 6, 2007
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