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Journal of Heredity Advance Access published online on October 1, 2008
Journal of Heredity, doi:10.1093/jhered/esn080
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© The American Genetic Association. 2008. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

Y Chromosome Haplotype Analysis in Portuguese Cattle Breeds Using SNPs and STRs

Catarina Ginja, Luís Telo da Gama, and Maria Cecilia T. Penedo

From the Instituto Superior de Agronomia, Tapada da Ajuda, Lisboa, Portugal (Ginja); the Departamento de Genética e Melhoramento Animal, Estação Zootécnica Nacional, Fonte Boa, 2005-048 Vale de Santarém, Portugal (Ginja and Telo da Gama); and the Veterinary Genetics Laboratory, University of California, 980 Old Davis Road, Davis, CA 95616 (Ginja and Penedo)

Address correspondence to C. Ginja at the address above, or e-mail: cjginja{at}ucdavis.edu.

DNA samples from 307 males of 13 Portuguese native cattle breeds, 57 males of the 3 major exotic breeds in Portugal (Charolais, Friesian, and Limousin), and 5 Brahman (Bos indicus) were tested for 5 single nucleotide polymorphisms, 1 "indel," and 7 microsatellites specific to the Y chromosome. The 13 Y-haplotypes defined included 3 previously described patrilines (Y1, Y2, and Y3) and 10 new haplotypes within Bos taurus. Native cattle contained most of the diversity with 7 haplotypes (H2Y1, H3Y1, H5Y1, H7Y2, H8Y2, H10Y2, and H12Y2) found only in these breeds. H6Y2 and H11Y2 occurred in high frequency across breeds including the exotics. Introgression of Friesian cattle into Ramo Grande was inferred through their sharing of haplotype H4Y1. Among the native breeds, Mertolenga had the highest haplotype diversity (0.68 ± 0.07), Brava de Lide was the least differentiated. The analyses of molecular variance showed significant (P < 0.0001) differences between breeds with more than 64% of the total genetic variation found among breeds within groups and 33–35% within breeds. The detection of INRA189-104 allele in 8 native breeds suggested influence of African cattle in breeds of the Iberian Peninsula. The presence in Portuguese breeds of Y1 patrilines, also found in aurochs, could represent more ancient local haplotypes.

Key Words: cattle • haplotypes • Y chromosome

Y chromosome–specific markers have been widely used in studies of human origins because the frequency and geographical distribution of Y-haplotypes provide powerful information about migration, sex-specific gene flow, and population relationships (Jorde et al. 2000; Su et al. 2000; Underhill et al. 2000; Hammer et al. 2001; Kayser et al. 2001; Jobling and Tyler-Smith 2003). Relatively low levels of genetic diversity in the Y chromosome have been found in several mammalian species including cattle (Hellborg and Ellegren 2004; Lindgren et al. 2004; Bannasch et al. 2005; Meadows et al. 2006; Li et al. 2007). Y chromosome diversity is lower than for autosomes (The International SNP Map Working Group 2001; Jobling and Tyler-Smith 2003; Hellborg and Ellegren 2004). In the case of domestic animals, the effective Y chromosome contribution tends to be reduced because of common use in breeding schemes of a few selected males that produce a large number of offspring (Hellborg and Ellegren 2004). For example, a demographic analysis of the native Portuguese cattle breed Alentejana indicates that, from an original number of 671 founder sires, only 24 Y chromosomes are currently represented with an effective number of 2.73 males (Carolino and Gama 2008). Despite limitations, studies of male lineages contribute to a better understanding of the origin and relationships among domestic breeds (Edwards et al. 2000; Lindgren et al. 2004; Anderung et al. 2005; Gotherstrom et al. 2005; Li et al. 2007).

Even though information on cattle Y chromosome sequence and polymorphism is limited, microsatellites (STRs) mapped to the nonrecombining region (Bishop et al. 1994; Vaiman et al. 1994; Kappes et al. 1997; Liu et al. 2003) have been used to study domestication and differentiation among bovid species. Some of these markers are useful to detect introgression and to distinguish between Bos taurus and Bos indicus patrilines (Edwards et al. 2000; Giovambattista et al. 2000; Hanotte et al. 2000; Li et al. 2007). Gotherstrom et al. (2005) used Y chromosome single nucleotide polymorphisms (SNPs) with ancient DNA specimens to investigate cattle evolution by comparing this information with that obtained from mtDNA data. This study showed that Y-haplotypes of North European cattle were similar to those found in aurochs and suggested possible hybridization between domestic cattle and male aurochs.

The Iberian Peninsula has numerous breeds of native domestic cattle of which 13 are found in Portugal and are at some level of risk of extinction (FAO 2004). Studies of genetic diversity in Portuguese native cattle based on autosomal STRs (Mateus et al. 2004) showed that these breeds were more diverse (average expected heterozygosity {cong} 0.7) than other European cattle raised under more intensive breeding schemes (Cañon et al. 2001; Ginja 2002; Cymbron et al. 2005). It has been postulated that genetic diversity might have accumulated in the Iberian Peninsula (Hewitt 2001) as a result of human and livestock migrations from Central Europe and Northern Africa (Beja-Pereira et al. 2003, 2006). Characterization and conservation of domestic animal genetic resources is a priority, and efforts are being made to take into account information from nuclear, mitochondrial, and Y chromosome markers to define conservation priorities (European Cattle Genetic Diversity Consortium 2006). The analysis of Y chromosome haplotypes can thus provide additional information for inferring the origins and genetic relationships of Iberian cattle. To our knowledge, extended Y chromosome haplotypes that combine information from STRs and SNPs have not been used to study genetic relationships among cattle breeds. The aim of this work was to investigate haplotype diversity in Portuguese native cattle breeds using a combination of SNPs and STRs specific to the nonrecombinant region of the Y chromosome. We also analyzed breed relationships taking into account geographical distribution and phenotypic characterization.


   Materials and Methods
Top
 Materials and Methods
Results
 Discussion
 Future Perspectives
 Supplementary Material
 Funding
 References
 
Sampling and DNA Extraction
Blood samples were collected from 307 males of 13 native Portuguese cattle breeds: Alentejana (31), Arouquesa (31), Barrosã (33), Brava de Lide (26), Cachena (25), Garvonesa (6), Marinhoa (17), Maronesa (23), Mertolenga (17), Minhota (28), Mirandesa (23), Preta (29), and Ramo Grande (18) and 57 males of the 3 major exotic breeds raised in Portugal: Charolais (13), Friesian (27), and Limousin (17). Semen samples from 5 Brahman (B. indicus) bulls were obtained from a commercial source (Bovine Elite Llc., College Station, TX). To minimize the degree of relationship among individuals, pedigree records were used for selection of nonrelated animals back to the second generation whenever possible. DNA was extracted using the Gentra kit (PUREGENE, Gentra Systems, Inc.) according to the manufacturer's recommendations.

Sequencing and SNP Detection through Pyrosequencing
The Multalign interface (http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html) was used to align Y chromosome sequences for B. taurus, B. indicus, Bos gaurus, Bison bison, and Bubalus bubalis deposited in the National Center for Biotechnology Information database and which contained SNPs (Gotherstrom et al. 2005). The alignments were done for DDX3Y gene (intron 1, previously DBY1, GenBank accession numbers AY928811 [GenBank] , AY928812 [GenBank] , AY928814 [GenBank] , AY928815 [GenBank] , and AY928816 [GenBank] and intron 7, previously DBY7, GenBank accession numbers AY928817 [GenBank] , AY928818 [GenBank] , AY928819 [GenBank] , AY928820 [GenBank] , AY928821 [GenBank] , and AY928822 [GenBank] ), UTY gene (intron 19, GenBank accession numbers AY936539 [GenBank] , AY936540 [GenBank] , AY936541 [GenBank] , AY936542 [GenBank] , and AY936543 [GenBank] ), and ZFY gene (intron 9 in human, previously ZFY4, GenBank accession numbers AY928823 [GenBank] , AY928824 [GenBank] , AY928825 [GenBank] , AY928826 [GenBank] , AY928827 [GenBank] , and AY928828 [GenBank] and intron 10 in human, previously ZFY5, GenBank accession numbers AF241271 [GenBank] , AF465181 [GenBank] , AF032366 [GenBank] , and AY079138 [GenBank] ). Polymerase chain reaction (PCR) primers were designed with Primer3 software (http://fokker.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Y chromosome regions, primer sequences, fragment sizes, and annealing temperatures are shown in Table 1. To confirm specificity of amplicons, 100 ng of genomic DNA from 1 Mirandesa (Northern brown breed group), 1 Mertolenga (Southern red breed group), and 2 Brahmans were amplified in 25 µl reactions containing 1x PCR buffer II (ABgene, Rochester, NY), 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphates (dNTPs), 10 {rho}mol of each primer, and 1 U Taq DNA polymerase (ABgene). DNA and the primers were incubated at 95 °C for 5 min after which the temperature was lowered and held at 80 °C for addition of the remaining reagents. The cycling program continued with 30 cycles at 95 °C for 30 s, 45 s at specific annealing temperatures (Ta) (see Table 1), 1 min at 72 °C, and finished with a final extension at 72 °C for 10 min. In this and all other assays, a negative (no template) and a female DNA were used to control possible PCR contamination and nonspecific amplification. PCR products were cloned with the Topo TA cloning Kit for sequencing (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For each animal and amplified region, 12 colonies were selected for amplification with the universal M13 primers included in the kit. The 50 µl PCRs contained 1x PCR buffer II (ABgene), 2.0 mM MgCl2, 200 µM dNTPs, 2.5 {rho}mol of each primer, and 2.5 U Taq DNA polymerase (ABgene). PCR amplifications were done with 2 cycles of 2 min at 95 °C, 45 s at 55 °C, and 30 s at 72 °C; 30 cycles of 45 s at 95 °C, 45 s at 50 °C, and 45 s at 72 °C; and a final extension at 72 °C for 10 min. For each individual, a minimum of 4 clones per region were sequenced with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and separated by capillary electrophoresis on an ABI 3730 instrument (Applied Biosystems). Sequences were analyzed using the Seqman II software v6.1 (DNASTAR Inc., Madison, WI). Their identities were confirmed by Blast comparison to sequences in GenBank.


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  		Table 1. Locus names, target regions, fragment sizes, annealing temperatures (Ta), and primer sequences for cattle Y chromosome

 
Pyrosequencing assays for SNP detection were designed for the PSQ 96MA 2.1 instrument (Biotage AB, Uppsala, Sweden) with Biotage's Assay Design Software, v. 1.0.6 according to the manufacturer's recommendations. Sequencing primers and the target SNPs are shown in Table 2. PCR fragments were amplified with the primers listed in Table 1 except that one of the primers (indicated in Table 2) was tailed (5'-AGCGCATAACAATTTCACACAGG-3'), and a third biotinylated primer corresponding to the tail was included in each reaction. PCRs were carried out in a total volume of 50 µl containing 200 ng of template, 1x PCR buffer II (ABgene), 2 mM of MgSO4, 200 µM dNTPs, 0.7 M of Betaine (Sigma, St. Louis, MO), 10 {rho}mol of the biotinylated primer, 2 {rho}mol of the tailed primer, 13 {rho}mol of the nontailed primer, and 2 U Taq DNA polymerase (ABgene). DNA and the primers were incubated at 95 °C for 5 min and the temperature lowered and held at 80 °C for addition of the remaining reagents. Amplification continued with 40 cycles at 94 °C for 30 s, specific Ta (see Table 1) for 45 s, and 72 °C for 45 s with a final extension step at 72 °C for 10 min. The PCR products were immobilized on streptavidin-coated Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. The assays were done using the reagents kit from Biotage, and pyrograms were analyzed with the SNP software. Only high-quality sequence peaks (>130 intensity signal) were considered for SNP genotyping.


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  		Table 2. Pyrosequencing primers, SNPs detected, and positions in reference sequences for the cattle Y chromosome

 
Microsatellite Genotyping
Thirteen STR loci (BM861, DDX3Y1STR, INRA124, INRA126, INRA189, UMN0103, UMN0307, UMN0504, UMN0920, UMN2001, UMN2303, UMN2404, and UMN3008) located on the nonrecombining region of the Y chromosome were selected from the literature. Primer sequences, repeat structure, Ta, and references for these markers are shown in Supplementary Table S1. The forward primers were synthesized with tails that matched 1 of 3 different, fluorescence-labeled M13 primers (Fam-TTTCCCAGTCACGACGTTG, Ned-TAAAACGACGGCCAGTGC, and Vic-GCGGATAACAATTTCACACAGG). The QIAGEN multiplex PCR kit (QIAGEN Inc., Valencia, CA) was used to set up 12.5 µl PCRs that contained 60 ng of genomic DNA, 6.25 µl of the QIAGEN PCR master mix, and 3.5 µl of the primer mix (0.63 {rho}mol of the tailed forward primer, 6.26 {rho}mol of the reverse primer, and 1–2 {rho}mol of the M13-labeled primer). The PCR program included an activation step at 95 °C for 15 min; 35 cycles at 94 °C for 30 s, 55/58 °C for 90 s, and 72 °C for 60 s followed by a final extension at 72 °C for 30 min. A negative and a female DNA controls were included in all STR assays. PCR fragments were separated by capillary electrophoresis on ABI 3730 instruments (Applied Biosystems) according to the manufacturer's recommendations. Allele sizes were determined with STRAND software (Hughes 2000) and whenever possible adjusted to match published data (Edwards et al. 2000; Gotherstrom et al. 2005).

Statistical Analysis
SNP and STR alleles were combined into haplotypes. ARLEQUIN v2.0 (Schneider et al. 2000) was used to determine haplotype frequency and diversity (H) for each population, number of shared haplotypes between breeds, pairwise FST values with 10,000 permutations, and significance at the 5% level. The same program was used for analyses of molecular variance (AMOVAs) to estimate the partitioning of the total genetic variation by grouping the native breeds by geographic region as Northern (Arouquesa, Barrosã, Cachena, Marinhoa, Maronesa, Minhota, Mirandesa), Southern (Alentejana, Brava de Lide, Garvonesa, Mertolenga, Preta), and Island (Ramo Grande) and by phenotypic characteristics as Red Convex (Alentejana, Garvonesa, Mertolenga, Minhota), Brown Concave (Arouquesa, Barrosã, Cachena, Marinhoa, Maronesa, Mirandesa), and Black Orthoid (Brava de Lide, Preta). The exotic and Brahman individuals were excluded from AMOVA because we were more interested in analyzing the population structure of the native breeds. This allowed for a hierarchical analysis considering 3 components of the genetic variation as due to differences 1) among haplotypes within populations, 2) among haplotypes in different populations within groups, and 3) among groups (Excoffier et al. 1992). Significance levels for the estimated fixation indices were obtained with 10 000 permutations.

Phylogenetic relationships among haplotypes were investigated using the median-joining (MJ) network program (Bandelt et al. 1995) implemented in NETWORK v4.2.0.1 (Fluxus Technology Ltd, Suffolk, England, 2004–2006). The output from the reduced MJ run was used for the MJ analysis with parameters r = 2 (reduction threshold) and {varepsilon} = 0 (Bandelt et al. 1999). Haplotype components were weighted (w) as dinucleotide repeats w = 2, imperfect repeats w = 5, SNP transitions w = 10, "indel" w = 20, and SNP transversion w = 30 so that the component with the lowest expected mutation rate was assigned the highest weight (Bandelt et al. 2000).


   Results
Top
 Materials and Methods
 Results
Discussion
 Future Perspectives
 Supplementary Material
 Funding
 References
 
The sequences obtained for the 5 intronic Y chromosome regions (GenBank accession numbers EU547258 [GenBank] –EU547277 [GenBank] ) confirmed the SNPs described by Gotherstrom et al. (2005), but we note that the motif for the ZFY_10indel is GT and not the reported AG. Sequence alignments and SNP positions for each gene region are shown in Supplementary Figure S1. Of the 13 STRs screened, INRA126 and UMN2001 were excluded because they amplified alleles with the same size range in females and thus lacked Y specificity. UMN0920, UMN2303, UMN2404, and UMN3008 were also discarded because they showed ladder-like banding pattern that made allele sizing impracticable. This amplification profile is most probably related to the characteristics of the Y chromosome sequence such as highly repetitive palindromic structure, where multiple copies of each gene/sequence might exist along the chromosome (Skaletsky et al. 2003). Among the 7 STRs that were combined with the 6 SNP loci for haplotyping, UMN0504 (monomorphic in our samples) and BM861 amplified female DNA but with different size ranges that did not interfere with the identification of Y-specific alleles. For UMN0103, only 1 fragment was present in all B. taurus samples, whereas 2 fragments (116 and 122 bp) were amplified in the 5 B. indicus. A total of 13 haplotypes were identified from the analysis of 369 individuals (Table 3). To facilitate comparison with published data (Gotherstrom et al. 2005), the nomenclature of Y1, Y2, and Y3 was included in our haplotype notation. The addition of STRs INRA189, UMN0103, and UMN0307 in the analyses allowed detection of additional variability and resulted in the split of Y1 into 4 and of Y2 into 8 distinct haplotypes.


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  		Table 3. Y chromosome haplotypes defined by 13 loci and their overall frequency across breeds

 
Genetic Diversity
Distributions of haplotype frequencies and diversities are shown in Table 4, respectively. Overall, H6Y2 and H11Y2 were the most common and were shared among several breeds. H6Y2 was found in 154 individuals; it was fixed in Garvonesa, Marinhoa, Minhota, and Mirandesa and present with some frequency in the other breeds except Alentejana, Friesian, Maronesa, and Ramo Grande. H11Y2 was found in 103 animals; it was not only fixed in Alentejana and Maronesa but also detected in Arouquesa, Barrosã, Brava de Lide, Cachena, Mertolenga, and Preta. H4Y1 was not only fixed in Friesian but also present in 13 Ramo Grande individuals. H9Y3 was found only in the B. indicus animals. The haplotype diversity within breeds was extremely low and among the breeds where variation was present, Mertolenga was the most diverse (0.68 ± 0.07) and had 4 haplotypes, followed by Barrosã and Ramo Grande (0.61 ± 0.05 each) with 3 and 2 haplotypes, respectively. Brava de Lide had the highest number of haplotypes (5) overall but 3 of these were represented only once, as reflected by the lower haplotype diversity of 0.41 ± 0.12. Among the B. taurus breeds, exclusive haplotypes were found in Arouquesa (H7Y2) and Preta (H8Y2) with frequencies of 0.19 and 0.76, respectively.


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  		Table 4. Frequency and diversity (H ± standard deviation) of Y chromosome haplotypes per breed

 
Differentiation and AMOVA
Pairwise FST values are shown in Table 5 and ranged from nearly zero to 1. The B. taurus breeds were significantly (P < 0.05) differentiated from the B. indicus. For Barrosã, Friesian, Mertolenga, Preta, and Ramo Grande, all pairwise FST values were statistically significant (P < 0.05). Friesian was the most, and Brava de Lide the least, differentiated from all other breeds, with average FST values (±standard deviation) of 0.869 ± 0.191 and 0.384 ± 0.321, respectively. Among the Northern native breeds, Maronesa was the most differentiated with an average FST value of 0.747 ± 0.338 and Arouquesa showed the lowest average FST value (0.407 ± 0.310). Among the Southern native breeds, Alentejana was the most differentiated (average FST of 0.762 ± 0.333) and Garvonesa the least (average FST of 0.446 ± 0.450). Among the native cattle, Ramo Grande was significantly differentiated from all other breeds with an average FST value of 0.630 ± 0.232 (Table 5). The average FST value over all B. taurus breeds was 0.563 ± 0.156, indicating that a considerable amount of the variation is explained by breed differences.


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  		Table 5. Pairwise FST values (below diagonal, values in bold font are statistically significant at the 5% level) and average pairwise FST per breed (FST ± standard deviation)

 
The AMOVA analysis (Table 6) showed significant (P < 0.0001) differences between breeds with more than 64% of the total genetic variation found among breeds within groups and 33–35% within breeds. Geographic distribution of breeds had no significant effect and accounted for only 2% of the total variability. Breed grouping based on traditional phenotypic types had no significant effect, with a negative component of variance.


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  		Table 6. AMOVA for cattle Y chromosome haplotypes

 
Phylogenetic Analysis
The MJ network for Y chromosome haplotypes shows a conservative representation of relationships among haplotypes (Figure 1). Bos indicus and B. taurus haplotypes were clearly separated by their well-defined differences as well as by postulated differences implied by 2 median vectors needed to connect the 2 clusters. The inclusion of Brahman individuals helped confirm previous results for Y chromosome data. Haplotypes H2Y1, H3Y1, H4Y1, and H5Y1 formed one cluster separated by 1 median vector from the cluster of H6Y2, H7Y2, H8Y2, H10Y2, H11Y2, H12Y2, and H13Y2. The Y1 cluster contained southern native breeds (Brava de Lide, Mertolenga, and Preta), Ramo Grande, and the exotic Friesian. H4Y1 was shared only by Ramo Grande and Friesian. Five of the Y2 haplotypes were connected to the 2 most central and frequent H6Y2 and H11Y2 and both Northern and Southern native breeds clustered within the Y2 group. The French breeds Limousin and Charolais shared H6Y2 with several native breeds. H1Y2 found in 3 Brava and 1 Charolais was placed in an intermediate position in the network between B. taurus and B. indicus.


Figure 1
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  		Figure 1. MJ network constructed using Y chromosome haplotype data from 369 animals representing 17 breeds of cattle. Circle sizes are proportional to the haplotype frequency. The black dots represent theoretical median vectors introduced by the network software. Breed groups are represented according to the following: Northern native (hatched), Southern native (white), exotics (light gray), Azores Archipelago (dark gray), and Bos indicus (black).

 

   Discussion
Top
 Materials and Methods
 Results
 Discussion
Future Perspectives
 Supplementary Material
 Funding
 References
 
The combination of 5 SNPs, 1 indel, and 7 STRs identified 13 Y chromosome haplotypes, one of which (H9Y3) was restricted to B. indicus. Several B. taurus breeds showed 2 haplotypes (H6Y2 and H11Y2) in high frequency and thus had a common genetic signature. Because homoplasy is not expected to be common in the nonrecombining region of the Y chromosome (Underhill et al. 2000), this signature likely represents shared ancestry. A similar pattern of frequency distribution in which a few haplotypes are represented in higher frequency across breeds has been described in African cattle based on 5 Y-STRs (Li et al. 2007) and in worldwide sheep breeds based on combined SNP-STR Y-haplotypes (Meadows et al. 2006). Compared with humans in which Y-haplotype diversity levels are >0.89 (Kayser et al. 2001), the low level detected in our breeds (average H of 0.2) is most probably related to the reduced effective male population size characteristic of domestic animal species. In horses, the small number of males used for breeding has been associated with the limited number of patrilines detected based on Y chromosome polymorphisms (Lindgren et al. 2004). This is also the case for our cattle breeds with more intensive breeding programs, such as Charolais, Friesian, and Limousin, and the native Alentejana where artificial insemination is a regular practice. However, in the case of other native breeds such as Cachena, Garvonesa, Marinhoa, Maronesa, Minhota, and Mirandesa, the lack of Y chromosome diversity might also be a consequence of genetic bottlenecks.

In contrast to the low within-breed variation, Y-haplotype diversity was much higher among breeds within groups (64–78% and an overall FST value of 0.580 ± 0.156) than has been reported for autosomal STRs (average FST = 0.09 ± 0.034) (Mateus et al. 2004). Geographical clustering of Y chromosome variability has been described for several species that have male-mediated dispersal and in which differences among population groups are further augmented by genetic drift (Kayser et al. 2001; Jobling and Tyler-Smith 2003; Bannasch et al. 2005; Meadows et al. 2006). The variance explained by geographical groups of native breeds (2%) was not significant and much lower than that found in dogs (11.5%; Bannasch et al. 2005), sheep (12.9%; Meadows et al. 2006), and humans (16.8%; Kayser et al. 2001). Most of these studies considered dispersal across wider regions such as continents, whereas in our case, breeds are dispersed throughout a much smaller territory. Overall, Southern cattle breeds presented higher diversity as the number of haplotypes detected was twice that observed for the Northern group. Although traditional breed groups could be identified through clustering using genetic distances for autosomal STRs (Mateus et al. 2004), this was not the case for Y-haplotypes in which no variation could be attributed to such population grouping. This is consistent with the results obtained for North Ethiopian cattle with similar molecular markers and where traditional breed classification could not explain the substructure of Y chromosome variation (Li et al. 2007).

Among the 3 polymorphic STRs, the amount of variation found for INRA189 was unexpected when 3 alleles (86, 102, and 106) were found in Portuguese cattle that had not been described previously in other B. taurus breeds (Edwards et al. 2000; Li et al. 2007). The network results suggested that H1Y2 with the unusual INRA189-86 allele could represent a more ancestral haplotype of the B. taurus haplogroups. H1Y2 appears to be more related to H6Y2 with at least 2 intermediate theoretical haplotypes separating them. In addition, INRA189-104 allele, which was found in the "West African" taurine N'Dama breed (Edwards et al. 2000), was present in 8 native breeds and in 4 haplotypes of the Y2 group. This allele could be the result of an independent mutation event, and thus, H11Y2 could have derived from the common H6Y2 by gain of a 2-bp repeat and then have given origin to H10Y2 and H12Y2 through additional mutation events. The MJ network suggests that H7Y2, which also has the "104" allele, is derived from H6Y2, but it might be related to the H11Y2 group and have diverged by one mutation event occurring in UMN0103. Alternatively, the haplotypes bearing this allele could be considered further evidence of African cattle influence in breeds of the Iberian Peninsula, as has been suggested by the analysis of nuclear and mtDNA (Cymbron et al. 1999, 2005). The presence of this allele in haplotypes of Southern and Northern breeds would also be consistent with a more complex pattern of introduction of African cattle that could even predate the traditional route associated with the Moorish invasions and occupation (Beja-Pereira et al. 2006; Davis 2008). This is supported by the detection of the 123-bp West African allele at the autosomal STR locus BM2113 in the Southern Portuguese Alentejana and Mertolenga breeds and in the Northern Barrosã breed (Ginja 2002). A more extensive survey of cattle Y-haplotypes with the markers used here and including North African breeds is needed to clarify the origin and patterns of male-mediated dispersion of cattle Y chromosome across the Iberian Peninsula.

The Y-haplotypes that we identified incorporate the Y1 and Y2 patrilines described by Gotherstrom et al. (2005), which are predominant in Northern and Southern European breeds, respectively. Although Y2 haplogroups were the most common among the native breeds, Y1 haplogroups were detected in 3 breeds from the Southern region, that is, Brava de Lide (2 animals), Mertolenga (7 animals), and Preta (1 animal) and in the Ramo Grande (18 animals). Recent introgression of Northern European cattle could explain the presence of Y1 haplotypes in Portuguese breeds, but the most common exotic beef breeds used for crossbreeding, Limousin and Charolais, did not show these haplotypes. The other common exotic breed is Friesian, which appears to be fixed for H4Y1. Among the native breeds, this haplotype was found only in Ramo Grande in high frequency (13 out of 18 animals). Considering that Friesian is the major exotic breed present in the Azores Archipelago as well as the history of the native breed, which has suffered significant reduction in population size, we interpret this finding as evidence of male-mediated introgression of Friesian into the Ramo Grande.

The presence of 3 distinct Y1 patrilines (H2Y1, H3Y1, and H5Y1) in continental native cattle could represent more ancient haplotypes that might have been derived from aurochs. The analysis of ancient bovid remains showed that the Y1 haplotype was present among aurochs from Germany, Italy, and Sweden and in early domesticates (Gotherstrom et al. 2005). Analyses of mtDNA have also suggested hybridization between wild and domestic cattle (Anderung et al. 2005). However, the process of cattle domestication and differentiation is complex, and this hypothesis needs to be confirmed by the analysis of ancient specimens from the Iberian Peninsula and a more thorough survey of worldwide breeds.


   Future Perspectives
Top
 Materials and Methods
 Results
 Discussion
 Future Perspectives
Supplementary Material
 Funding
 References
 
The establishment of a reference database and a common nomenclature for Y chromosome haplotypes in cattle, similar to that available for humans (Jobling and Tyler-Smith 2003), is important in order to standardize results and implement future comparison studies. Complementing our Y chromosome study with information from ancient cattle remains of the Iberian Peninsula, with worldwide breed surveys, and with mtDNA data would enhance our understanding of evolution and origins of domestic cattle. The Y-haplotypes described here can be used to investigate the demographic expansion of Iberian breeds which were spread throughout different countries during colonial periods and which are considered the ancestors of the Creole cattle of the Western Hemisphere.


   Supplementary Material
Top
 Materials and Methods
 Results
 Discussion
 Future Perspectives
 Supplementary Material
Funding
 References
 
Supplementary Figure S1 and Table S1 can be found at http://www.jhered.oxfordjournals.org/.


   Funding
Top
 Materials and Methods
 Results
 Discussion
 Future Perspectives
 Supplementary Material
 Funding
References
 
Fundação para a Ciência e a Tecnologia (Ref. SFRH/BD/13502/2003) to C.G.; Veterinary Genetics Laboratory, University of California, Davis.


   Acknowledgments
 
We gratefully acknowledge the collaboration and assistance of the Portuguese breed associations for the sampling of animals. We thank Elisabete Pires for helpful comments on the manuscript. The sample collection and establishment of the DNA bank were supported by Direcção Geral de Veterinária and Sociedade Portuguesa de Recursos Genéticos Animais.


   Footnotes
 
Corresponding Editor: Jill Pecon-Slattery

Received March 25, 2008
Revised August 28, 2008
Accepted September 3, 2008


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