Mitochondrial control region genetic diversity and maternal ancestry of a Brangus-Ibage cattle populations

ANIMAL GENETICS

RESEARCH ARTICLE

Mitochondrial control region genetic diversity and maternal ancestry of a Brangus-Ibage cattle populations

Luiz Ernani Henkes^{I, II}; Wilson Araújo Silva Jr^III; José Carlos Ferrugem Moraes^IV; Tania Azevedo Weimer^{I, V}

^IUniversidade Federal do Rio Grande do Sul, Departamento de Genética, Porto Alegre, RS, Brazil

^IIVincent Centre for Reproductive Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

^IIICentre for Cell-Based Therapy, Ribeirão Preto, SP, Brazil

^IVEmpresa Brasileira de Pesquisa Agropecuária, Bagé, RS, Brazil

^VUniversidade Luterana do Brasil, Hospital Veterinário, Laboratório de Biotecnologia Animal, Canoas, RS, Brazil

^{Correspondence}

ABSTRACT

The genetic diversity of 277 nucleotides in the mitochondrial DNA control region (nt 15,964 to 16,240 in reference sequence) was analyzed in crossbreed beef cattle (Brangus-Ibage, 5/8 Bos primigenius taurus x 3/8 Bos primigenius indicus) as well as in some Nellore samples (B. p. indicus). Fifty-seven mutations were found in Brangus-Ibage comprising 18 haplotypes (haplotype diversity, h = 0.851 ± 0.041 and nucleotide diversity, ntd = 0.009 ± 0.006) and 66 in Nellore (h = 1.00 ± 0.27, ntd = 0.014 ± 0.012). These data indicated sequence identities of 99.6 and 92.1% between the B. p. taurus' reference sequence and Brangus-Ibage and Nellore, respectively. The comparison of our data with sequence data for 612 individuals recovered from GenBank showed a total of 205 haplotypes defined by 99 polymorphic sites. Most of the variability (53%) was due to differentiation within breeds. The phylogenetic tree constructed using the neighbor-joining method showed clearly the well-known dichotomy between B. p. taurus and B. p. indicus. The Brangus-Ibage clustered with B. p. taurus lineages; however, the displacement of Nellore from B. p. indicus branch probably indicates a substantial B. p. taurus maternal ancestry in some Nellore samples (obtained from GenBank) and reflects the primarily male-driven introduction of this breed in Brazil.

Key words: bovine mtDNA, maternal lineage, sequence analysis, beef cattle, genetic diversity.

Introduction

Mitochondria are maternally inherited organelles of eukaryotic cells, which play an important role in the cell energy provision. Vertebrate mtDNA includes, in addition to coding regions, a non-coding segment: the displacement loop (D-loop), which is the major control region for mtDNA expression (Taanman, 1999). Therefore, sequence differences in mtDNA D-loop may alter the transcription and/or replication rates (Schutz et al., 1994). However, despite its functional importance, this region has a rate of nucleotide substitution five to ten times higher than that of nuclear DNA (Brown et al., 1979).

The rapid rate of sequence divergence of mtDNA makes it suitable for the analysis of short-term evolutionary phenomena, while the maternal mode of inheritance allows the evolutionary relationships between lineages to be defined in terms of their phylogenetic divergence without the ambiguities caused by recombination. Therefore, mtDNA polymorphisms have been widely used to investigate the structure of populations, interspecies variability, the evolutionary relationships between populations or species and for the identification of maternal lineages (Bradley and Cunningham, 1999; Cymbron et al., 1999; Magee et al., 2002; Troy et al., 2001). Additionally, there has been an increasing interest in its potential use in the development of new biotechnologies (Smith et al., 2000).

In livestock species, mtDNA variability has been studied in connection with maternally inherited physiological parameters and it has been suggested that bovine mtDNA may affect milk production as well as some other productive traits (Schutz et al., 1992; Schutz et al., 1994; Mannen et al., 2003; Henkes et al., 2004).

In this study, we provide mtDNA D-loop sequencing data for a Brangus-Ibage cattle population (5/8 Bos primigenius taurus x 3/8 Bos primigenius indicus) as well as for some Nellore samples (B. p. indicus). We also analyzed these data pooled with the majority of cattle D-loop sequences available in GenBank in an attempt to advance our understanding of the origins of the maternal lineages that contributed to the formation of this Brangus-Ibage population.

Material and Methods

Brangus-Ibage is a composite beef cattle breed resulting from the crossing of Aberdeen Angus cows (ABG) and Nellore bulls (NEL). In Brazil, the early work crossing Nellore and Aberdeen Angus cattle was done by the Brazilian Agricultural Research Corporation (EMBRAPA - CPPSUL) and started about 1945 (Oliveira et al., 1998).

Samples of whole blood were collected from 49 Brangus-Ibage cows from the Brazilian Agricultural Research Corporation and DNA was extracted by the method of Plante et al. (1992). To validate the supposed B. p. taurus origin of the Brangus-Ibage mtDNA, three DNA samples of Nellore (B. p. indicus) animals were also investigated. These samples were kindly provided by Dr. F.V. Meirelles (for details of these samples see Meirelles et al., 1999).

A 277 nt fragment of the control region (positions 15964-16240) was analyzed from an amplified product obtained using primers designed from the reference sequence of B. p. taurus mtDNA (Anderson et al., 1982) as follows:

MTR 11, 5' CCT ACG CAA GGG GTA ATG TA 3' (positions 15,949-15,968) and

MTR 12, 5' CCT GAA GAA AGA ACC AGA TG 3' (positions 16,265-16,285).

PCR amplification was carried out by two reactions: the first used 100 ng of DNA in 25 mL reaction volume, with 1.25 mM of each deoxynucleotide, 1.25 mM of each primer and 1 unit of Taq DNA Polymerase (Pharmacia Biotech) for 30 cycles. Each cycle consisted of denaturation at 95 °C for 1 min, annealing at 45 °C for 50 s and extension at 72 °C for 1 min. These double-stranded amplification products were then purified with the Wizard PCR Preps DNA Purification System (Promega Corporation). Aliquots of these products were later used for asymmetric amplification (Ward et al., 1991) using a 1:10 ratio of the same primers. The single-strand amplification products were then purified using a SEPHADEX column and phenol/chloroform (1:1) extraction. The purified fragments were sequenced using a T7SequencingTM Kit (Pharmacia Biotech) according to the manufacturer's instructions. Reaction products were separated by electrophoresis on 6% polyacrylamide gels containing 8 M urea. Gels were fixed in 5% acetic acid and 15% methanol for 10-15 min, dried and exposed to a Kodak XAR film for 24-48 h. Sequences were analyzed in at least two independent gels, aligned and compared using the BIOEDIT computer package (Hall, 1999). Insertions/deletions were introduced in order to minimize substitutions. The variant sequences observed in this study were submitted to GenBank under accession numbers AF308591 and AF309100-AF3091113 (Brangus-Ibage) and AF309097-AF309099 (Nellore).

Intra-populational variation was estimated by computing haplotype and nucleotide diversities (Nei and Tajima, 1981; Nei, 1989). The last analysis was corrected for among-site heterogeneity (Tamura and Nei, 1993).

The present sequence data were compared to those of all other breeds for which, at that time, there were at least three available sequences in GenBank (n = 612, GenBank accession numbers AB003793-AB003801, AB044587-AB 044592, AB065119-AB065131, AB079300-AB079365, AB085922, AB085923, AF016060-AF016071, AF01607 9-AF016097, AF022916, AF022918-AF022924, AF034439, AF034441, AF034442, AF034444, AF034445, AF083354, AF209124, AF209126, AF308591, AF309095, AF309096, AF336383-AF336744, AF516713, AF516714, AF531383, AF531412, AF531413, AJ295936, AY119666, AY235731-AY378140, L27720, L27721, L27732, L27733, U51806-U51842 and U92230-U92244). This comparison was restricted to 210 nucleotides to accommodate the shorter published sequences. Data for all individuals were available only between nucleotides 16,031 and 16,240. The total analysis was comprised of 664 individuals (49 Brangus-Ibage, 3 Nellore and 612 from GenBank) from 54 different cattle breeds.

Population genetic structure indexes were estimated by analyses of molecular variance (AMOVA, Excoffier et al., 1992) using the substitution model of Tamura and Nei (1993). The significance of these analyses was tested using a non-parametric permutation procedure (Excoffier et al., 1992). All of these analyses were performed using the Arlequin software (Schneider et al., 2000).

The phylogenetic relationships between populations were determined using the neighbor-joining (NJ) algorithm (Saitou and Nei, 1987). The robustness of the tree was evaluated by resampling the data by the bootstrap test (Felsenstein, 1985) with 2,000 replicates (Hedges, 1992). These analyses were conducted using MEGA version 2.1 (Kumar et al., 2001). The relatedness of the Brangus-Ibage mtDNA haplotypes was assessed through reduced median networks (Bandelt et al., 1995) constructed with Network software (www.fluxus-engineering.com).

Results

Sequence analysis of mtDNA was based on a 277 bp fragment of the D-loop region (nt 15,964 to 16,240 in reference sequence). Sixteen polymorphic sites were identified in Brangus-Ibage mtDNA sequences (Table 1). A total of 18 mitochondrial lineages were verified; the most frequent occurred in 17 individuals and was equal to the B. p. taurus' reference sequence (Anderson et al., 1982). Most haplotypes were scored only once or twice (Table 2), resulting in a high total haplotype diversity, h = 0.851 ± 0.041. The nucleotide diversity was estimated at 0.009 ± 0.006. Most of the variable sites were transitions (transition/transversion rate of 15) and deletion/insertion was identified in one position only (16,191). The mean number of pairwise differences among sequences was 1.91 ± 1.11. Nellore samples showed 3 different mitochondrial lineages defined by 25 polymorphic sites (Table 1), when compared with B. p. taurus' sequence (Anderson et al., 1982). One deletion was identified at position 16,143 and an A insertion was observed at one site (16,200). The Nellore haplotype diversity was estimated at 1.00 ± 0.27 and the nucleotide diversity was 0.014 ± 0.012. The mean number of pairwise differences among sequences was 4.00 ± 2.72.

In the whole sample, 57 mutations were found in Brangus-Ibage and 66 in Nellore. These data showed average sequence identities of 99.6% between the B. p. taurus' reference sequence (Anderson et al., 1982) and Brangus-Ibage and of 92.1% between B. p. taurus' sequence (Anderson et al., 1982) and Nellore.

Pooling our mitochondrial sequences with those obtained from GenBank defined 99 polymorphic sites (Table 3) and a total of 205 haplotypes. Eighty-seven of the substitutions were transitions, 11 were transversions (transitions/transversions rate = 7.9) and insertions/deletions were identified at 9 positions. More than two nucleotides were detected only at positions 16,057, 16118, 16156 and 16230 (Table 3). The average pairwise sequence divergence within breeds varied from 0.67 ± 0.67 (Charolais and Friesian) to 4.00 ± 2.73 (Tharparkar, B. p. indicus). Two mutations are being described here for the first time, at position 16177 (T ® C) in Brangus-Ibage and at site 16221 (G ® A) in Nellore.

Hierarchical analysis of haplotype diversity (Table 4) indicated that 53% of the variability was due to differentiation within the breed. The validity of this partition tested by the permutation test was highly significant (p < 0.001).

The phylogenetic tree constructed using the neighbor-joining method (Figure 1) shows clearly the two main branches separating B. p. taurus and B. p. indicus. Brangus-Ibage is clustered with B. p. taurus lineages. The reduced mean network (Figure 2) shows that all haplotypes in Brangus-Ibage root back to the phylogeny through the primary B. p. taurus haplotypes (Troy et al., 2001). The Nellore haplotypes from B. p. indicus origin are clearly dispersed from the main node.

Discussion

As expected Brangus-Ibage sequences were similar to those of B. p. taurus animals (99.6% of sequence identity) and distinct from those of Nellore. These data confirm no female contribution of B. p. indicus to the composition of the maternal lineages of Brangus-Ibage. However, we have detected some samples displaying mutations at positions 16050 and 16113 which, together with the substitution at position 16255, would be an indication of the presence of haplogroup T1, characteristic of African B. p. taurus (Troy et al., 2001). It is well-known that most European modern cattle breeds were introduced to South America at the beginning of the last century and most imported animals were males, which were mated to local cows (cows previously introduced in South America). The Brazilian Aberdeen Angus did not escape this rule (http://www.angus.org.br). Some cows of our herd might be descendants from cattle introduced in America by the first Portuguese and Spanish settlers. Since there is evidence of African B. p. taurus influence in Portuguese cattle (Cymbron et al., 1999) and since we found three haplotypes that could be of African origin (Iba-3, Iba-4 and Iba-5), we extended our analysis of these three haplotypes in order to investigate the position 16255. Only one of these haplotypes (Iba-3) presented the mutation T ® C at this position, characteristic of African B. p. taurus. The presence of an African B. p. taurus haplotype that had survived might suggest some adaptive value in this specific environment. Interestingly, Brangus-Ibage is situated at an intermediate position between the European and African grouping on the phylogenetic tree (Figure 1).

The degree of mtDNA genetic diversity of this sample is equal to that of Aberdeen Angus sequences and is between the range of variation observed for the other cattle samples whose nucleotide diversity ranges from 0.003 (Charolais and Friesian) to 0.019 (B. indicus Sahiwal and Tharparkar).

The three Nellore sequences we reported are unique and did not match any sequence of this breed so far described in GenBank. Our three sequences clustered with B. p. indicus branch on the phylogenetic tree. However, polled with the GenBank published Nellore sequences, this breed clustered in an intermediate position between the major B. p. indicus and B. p. taurus branches, corroborating earlier findings that Brazilian Nellore has a substantial B. p. taurus maternal ancestry (Meirelles et al., 1999).

The strong transitional bias verified here is a characteristic of mtDNA evolution and has been observed not only in cattle but also in other mammalian species (Loftus et al., 1994; Simonsen et al., 1998; Wood et al., 1996).

Three new mutations were observed in Brangus-Ibage and two in Nellore. Among them, the C deletion at position 16,191 and the transition T ® C at 16,177 are in Box F of the conserved sequence box, a region of remarkable sequence identity between vertebrates (Steinborn et al., 1998). According to these authors, mutations in this region might be associated with functional constraint. However, we verified that maternal lineages presenting the 16,191 deletion showed significantly higher calf birth weight than animals without this mutation (Henkes et al., 2004). Furthermore, considering the high variation in Nellore mtDNA, it might be interesting to compare the performance of Brangus originated from an opposite crossing (ABG bulls and Nellore cows), carrying B. p. indicus mitochondria with the performance of the Brangus-Ibage investigated here (originating from the crossing between Nellore bulls and ABG cows) in different environments in order to uncover any specific mitochondrial influence.

Loci with large mutation rates such as D-loop mtDNA frequently exhibit higher population gene diversities than loci with low mutation rates (Chakraborty and Jim, 1992). In the present analysis, 47% of the diversity is due to differentiation among breeds, but this value drops to 14% if only B. p. taurus samples are compared. In this last case the variability due to differentiation within population is as high as those verified in other species (Bortolini et al., 1998; Simonsen et al., 1998). Therefore, this very high level of differentiation among breeds verified herein results from the highly divergent B. p. indicus sequences.

Acknowledgements

Thanks are due to the Fundação Hemocentro de Ribeirão Preto, SP (FHCRP) in the person of Dr. Marco Antônio Zago for the facilities provided. We are also grateful to Dr. João F. Oliveira for help in the collection of the Brangus-Ibage samples and Dr. Flavio V. Meirelles for providing Nellore samples. This study was supported by Financiadora de Estudos e Projetos (FINEP/ PRONEX), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Pró-Reitoria de Pesquisa e Pós-Graduação da Universidade Federal do Rio Grande do Sul (PROPESQ/UFRGS), FHCRP, and EMBRAPA/CPPSUL.

Received: November 11, 2003; Accepted: September 4, 2004.

Associate Editor: Sérgio Furtado dos Reis

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Publication Dates

• Publication in this collection
  08 Sept 2005
• Date of issue
  Mar 2005

History

• Received
  11 Nov 2003
• Accepted
  04 Sept 2004