A complete mitochondrial genome sequence from a mesolithic wild aurochs (Bos primigenius)

Abstract

Background: The derivation of domestic cattle from the extinct wild aurochs (Bos primigenius) has been well-documented by archaeological and genetic studies. Genetic studies point towards the Neolithic Near East as the centre of origin for Bos taurus, with some lines of evidence suggesting possible, albeit rare, genetic contributions from locally domesticated wild aurochsen across Eurasia. Inferences from these investigations have been based largely on the analysis of partial mitochondrial DNA sequences generated from modern animals, with limited sequence data from ancient aurochsen samples. Recent developments in DNA sequencing technologies, however, are affording new opportunities for the examination of genetic material retrieved from extinct species, providing new insight into their evolutionary history. Here we present DNA sequence analysis of the first complete mitochondrial genome (16,338 base pairs) from an archaeologically-verified and exceptionally-well preserved aurochs bone sample.

Methodology: DNA extracts were generated from an aurochs humerus bone sample recovered from a cave site located in Derbyshire, England and radiocarbon-dated to 6,738+/-68 calibrated years before present. These extracts were prepared for both Sanger and next generation DNA sequencing technologies (Illumina Genome Analyzer). In total, 289.9 megabases (22.48%) of the post-filtered DNA sequences generated using the Illumina Genome Analyzer from this sample mapped with confidence to the bovine genome. A consensus B. primigenius mitochondrial genome sequence was constructed and was analysed alongside all available complete bovine mitochondrial genome sequences.

Conclusions: For all nucleotide positions where both Sanger and Illumina Genome Analyzer sequencing methods gave high-confidence calls, no discrepancies were observed. Sequence analysis reveals evidence of heteroplasmy in this sample and places this mitochondrial genome sequence securely within a previously identified aurochsen haplogroup (haplogroup P), thus providing novel insights into pre-domestic patterns of variation. The high proportion of authentic, endogenous aurochs DNA preserved in this sample bodes well for future efforts to determine the complete genome sequence of a wild ancestor of domestic cattle.

Figure 1. The identity and distribution of DNA nucleotide mismatches in individual Illumina GA reads compared to the CPC98 consensus mtDNA genome.

(A) The number and proportion of each nucleotide called in the Illumina GA reads (vertical column) compared to the consensus mtDNA sequence (horizontal column) is presented. (B) Mean percentage of discordant nucleotides for each position across all individual Illumina GA sequence reads.

Figure 2. Location of substitutions between the B. taurus reference and the B. primigenius (CPC98) mtDNA genome sequences and evidence of mtDNA heteroplasmy at nucleotide position 16,121 in the CPC98 aurochs sample.

(A) Location of substitutions between the B. taurus reference and the B. primigenius (CPC98) mtDNA genome sequences. (B) Heteroplasmy detected from analysis of individual Illumina GA reads spanning nucleotide position 16,121. (C) Heteroplasmy at nucleotide position 16,121 detected from analysis of Sanger chromatograms. Nucleotide positions according to the bovine mtDNA reference sequence (GenBank accession no. V00654).

Figure 3. Rooted Neighbor-Joining (N–J) phylogenetic tree detailing the relationships among all available complete bovine haplogroup I, P, Q, R and T mtDNA genome sequences and five yak (B. grunniens) mtDNA genome sequences.

Evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Only coding region sequences of the mtDNA genome were used for tree construction (mtDNA nucleotide position 364–15,791). Bootstrap values (1000 replicates) are shown next to the branches. The number of mtDNA sequences within each of the haplogroups is indicated. The haplogroup to which the CPC98 mtDNA genome sequence belongs is highlighted. Five complete mtDNA genome sequences from yak (B. grunniens) were used as outgroups.

Figure 4. Location of substitutions distinguishing the complete CPC98 consensus mtDNA genome sequence and the other complete haplogroup P sequence (GenBank accession no. DQ124389).

Nucleotide positions according to the bovine mtDNA reference sequence (GenBank accession no. V00654).