Bioarchaeology aids the cultural understanding of six characters in search of their agency (Tarquinia, ninth-seventh century BC, central Italy).

Etruria contained one of the great early urban civilisations in the Italian peninsula during the first millennium BC, much studied from a cultural, humanities-based, perspective, but relatively little with scientific data, and rarely in combination. We have addressed the unusual location of twenty inhumations found in the sacred heart of the Etruscan city of Tarquinia, focusing on six of these as illustrative, contrasting with the typical contemporary cremations found in cemeteries on the edge of the city. The cultural evidence suggests that the six skeletons were also distinctive in their ritualization and memorialisation. Focusing on the six, as a representative sample, the scientific evidence of osteoarchaeology, isotopic compositions, and ancient DNA has established that these appear to show mobility, diversity and violence through an integrated bioarchaeological approach. The combination of multiple lines of evidence makes major strides towards a deeper understanding of the role of these extraordinary individuals in the life of the early city of Etruria.

Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel.

Sequencing ancient DNA to high coverage is often limited by sample quality and cost. Imputing missing genotypes can potentially increase information content and quality of ancient data, but requires different computational approaches than modern DNA imputation. Ancient imputation beyond humans has not been investigated. In this study we report results of a systematic evaluation of imputation of three whole genome ancient Sus scrofa samples from the Early and Late Neolithic (∼7,100-4,500 BP), to test the utility of imputation. We show how issues like genetic architecture and, reference panel divergence, composition and size affect imputation accuracy. We evaluate a variety of imputation methods, including Beagle5, GLIMPSE, and Impute5 with varying filters, pipelines, and variant calling methods. We achieved genotype concordance in most cases reaching above 90%; with the highest being 98% with ∼2,000,000 variants recovered using GLIMPSE. Despite this high concordance the sources of diversity present in the genotypes called in the original high coverage genomes were not equally imputed leading to biases in downstream analyses; a trend toward genotypes most common in the reference panel is observed. This demonstrates that the current reference panel does not possess the full diversity needed for accurate imputation of ancient Sus, due to missing variations from Near Eastern and Mesolithic wild boar. Imputation of ancient Sus scrofa holds potential but should be approached with caution due to these biases, and suggests that there is no universal approach for imputation of non-human ancient species.

The phylogenetic position of the 'giant deer' Megaloceros giganteus.

The giant deer, or 'Irish elk', has featured extensively in debates on adaptation, sexual selection, and extinction. Its huge antlers--the largest of any deer species, living or extinct--formed a focus of much past work. Yet the phylogenetic position of the giant deer has remained an enigma. On the basis of its flattened antlers, the species was previously regarded as closely related to the living fallow deer. Recent morphological studies, however, have challenged that view and placed the giant deer closer to the living red deer or wapiti. Here we present a new phylogenetic analysis encompassing morphological and DNA sequence evidence, and find that both sets of data independently support a sister-group relationship of giant and fallow deer. Our results include the successful extraction and sequencing of DNA from this extinct species, and highlight the value of a joint molecular and morphological approach.

Variation in chicken populations may affect the enzymatic activity of lysozyme.

The chicken lysozyme gene encodes a hydrolase that has a key role in defence, especially in ovo. This gene was resequenced in global chicken populations [red, grey, Ceylon and green jungle fowl (JF)] and related bird species. Networks, summary statistics and tests of neutrality indicate that although there is extensive variation at the gene, little is present at coding sites, with the exception of one non-synonymous site. This segregating site and a further fixed non-synonymous change between red JF and domestic chicken populations are spatially close to the catalytic sites of the enzyme and so might affect its activity.

High concordance of bovine single nucleotide polymorphism genotypes generated using two independent genotyping strategies.

Single nucleotide polymorphisms (SNPs) represent the most common form of DNA sequence variation in mammalian livestock genomes. While the past decade has witnessed major advances in SNP genotyping technologies, genotyping errors caused, in part, by the biochemistry underlying the genotyping platform used, can occur. These errors can distort project results and conclusions and can result in incorrect decisions in animal management and breeding programs; hence, SNP genotype calls must be accurate and reliable. In this study, 263 Bos spp. samples were genotyped commercially for a total of 16 SNPs. Of the total possible 4,208 SNP genotypes, 4,179 SNP genotypes were generated, yielding a genotype call rate of 99.31% (standard deviation ± 0.93%). Between 110 and 263 samples were subsequently re-genotyped by us for all 16 markers using a custom-designed SNP genotyping platform, and of the possible 3,819 genotypes a total of 3,768 genotypes were generated (98.70% genotype call rate, SD ± 1.89%). A total of 3,744 duplicate genotypes were generated for both genotyping platforms, and comparison of the genotype calls for both methods revealed 3,741 concordant SNP genotype call rates (99.92% SNP genotype concordance rate). These data indicate that both genotyping methods used can provide livestock geneticists with reliable, reproducible SNP genotypic data for in-depth statistical analysis.

Maternal and paternal genealogy of Eurasian taurine cattle (Bos taurus).

Maternally inherited mitochondrial DNA (mtDNA) has been used extensively to determine origin and diversity of taurine cattle (Bos taurus) but global surveys of paternally inherited Y-chromosome diversity are lacking. Here, we provide mtDNA information on previously uncharacterised Eurasian breeds and present the most comprehensive Y-chromosomal microsatellite data on domestic cattle to date. The mitochondrial haplogroup T3 was the most frequent, whereas T4 was detected only in the Yakutian cattle from Siberia. The mtDNA data indicates that the Ukrainian and Central Asian regions are zones where hybrids between taurine and zebu (B. indicus) cattle have existed. This zebu influence appears to have subsequently spread into southern and southeastern European breeds. The most common Y-chromosomal microsatellite haplotype, termed here as H11, showed an elevated frequency in the Eurasian sample set compared with that detected in Near Eastern and Anatolian breeds. The taurine Y-chromosomal microsatellite haplotypes were found to be structured in a network according to the Y-haplogroups Y1 and Y2. These data do not support the recent hypothesis on the origin of Y1 from the local European hybridization of cattle with male aurochsen. Compared with mtDNA, the intensive culling of breeding males and male-mediated crossbreeding of locally raised native breeds has accelerated loss of Y-chromosomal variation in domestic cattle, and affected the contribution of genetic drift to diversity. In conclusion, to maintain diversity, breeds showing rare Y-haplotypes should be prioritised in the conservation of cattle genetic resources.

Cytochrome b sequences of ancient cattle and wild ox support phylogenetic complexity in the ancient and modern bovine populations.

Mitochondrial DNA has been the traditional marker for the study of animal domestication, as its high mutation rate allows for the accumulation of molecular diversity within the time frame of domestic history. Additionally, it is exclusively maternally inherited and haplotypes become part of the domestic gene pool via actual capture of a female animal rather than by interbreeding with wild populations. Initial studies of British aurochs identified a haplogroup, designated P, which was found to be highly divergent from all known domestic haplotypes over the most variable portion of the D-loop. Additional analysis of a large and geographically representative sample of aurochs from northern and central Europe found an additional, separate aurochs haplotype, E. Until recently, the European aurochs appeared to have no matrilinear descendants among the publicly available modern cattle control regions sequenced; if aurochs mtDNA was incorporated into the domestic population, aurochs either formed a very small proportion of modern diversity or had been subsequently lost. However, a haplogroup P sequence has recently been found in a modern sample, along with a new divergent haplogroup called Q. Here we confirm the outlying status of the novel Q and E haplogroups and the modern P haplogroup sequence as a descendent of European aurochs, by retrieval and analysis of cytochrome b sequence data from twenty ancient wild and domesticated cattle archaeological samples.

Variance components for bovine tuberculosis infection and multi-breed genome-wide association analysis using imputed whole genome sequence data.

Bovine tuberculosis (bTB) is an infectious disease of cattle generally caused by Mycobacterium bovis, a bacterium that can elicit disease humans. Since the 1950s, the objective of the national bTB eradication program in Republic of Ireland was the biological extinction of bTB; that purpose has yet to be achieved. Objectives of the present study were to develop the statistical methodology and variance components to undertake routine genetic evaluations for resistance to bTB; also of interest was the detection of regions of the bovine genome putatively associated with bTB infection in dairy and beef breeds. The novelty of the present study, in terms of research on bTB infection, was the use of beef breeds in the genome-wide association and the utilization of imputed whole genome sequence data. Phenotypic bTB data on 781,270 animals together with imputed whole genome sequence data on 7,346 of these animals' sires were available. Linear mixed models were used to quantify variance components for bTB and EBVs were validated. Within-breed and multi-breed genome-wide associations were undertaken using a single-SNP regression approach. The estimated genetic standard deviation (0.09), heritability (0.12), and repeatability (0.30) substantiate that genetic selection help to eradicate bTB. The multi-breed genome-wide association analysis identified 38 SNPs and 64 QTL regions associated with bTB infection; two QTL regions (both on BTA23) identified in the multi-breed analysis overlapped with the within-breed analyses of Charolais, Limousin, and Holstein-Friesian. Results from the association analysis, coupled with previous studies, suggest bTB is controlled by an infinitely large number of loci, each having a small effect. The methodology and results from the present study will be used to develop national genetic evaluations for bTB in the Republic of Ireland. In addition, results can also be used to help uncover the biological architecture underlying resistance to bTB infection in cattle.

Mitochondrial DNA variation and evolution of Japanese black cattle (Bos taurus).

This article describes complete mitochondrial DNA displacement loop sequences from 32 Japanese Black cattle and the analysis of these data in conjunction with previously published sequences from African, European, and Indian subjects. The origins of North East Asian domesticated cattle are unclear. The earliest domestic cattle in the region were Bos taurus and may have been domesticated from local wild cattle (aurochsen; B. primigenius), or perhaps had an origin in migrants from the early domestic center of the Near East. In phylogenetic analyses, taurine sequences form a dense tree with a center consisting of intermingled European and Japanese sequences with one group of Japanese and another of all African sequences, each forming distinct clusters at extremes of the phylogeny. This topology and calibrated levels of sequence divergence suggest that the clusters may represent three different strains of ancestral aurochs, adopted at geographically and temporally separate stages of the domestication process. Unlike Africa, half of Japanese cattle sequences are topologically intermingled with the European variants. This suggests an interchange of variants that may be ancient, perhaps a legacy of the first introduction of domesticates to East Asia.

Microsatellite DNA variation and the evolution, domestication and phylogeography of taurine and zebu cattle (Bos taurus and Bos indicus).

Genetic variation at 20 microsatellite loci was surveyed to determine the evolutionary relationships and molecular biogeography of 20 different cattle populations from Africa, Europe and Asia. Phylogenetic reconstruction and multivariate analysis highlighted a marked distinction between humpless (taurine) and humped (zebu) cattle, providing strong support for a separate origin for domesticated zebu cattle. A molecular clock calculation using bison (Bison sp.) as an outgroup gave an estimated divergence time between the two subspecies of 610,000-850,000 years. Substantial differences in the distribution of alleles at 10 of these loci were observed between zebu and taurine cattle. These markers subsequently proved very useful for investigations of gene flow and admixture in African populations. When these data were considered in conjunction with previous mitochondrial and Y chromosomal studies, a distinctive male-mediated pattern of zebu genetic introgression was revealed. The introgression of zebu-specific alleles in African cattle afforded a high resolution perspective on the hybrid nature of African cattle populations and also suggested that certain West African populations of valuable disease-tolerant taurine cattle are under threat of genetic absorption by migrating zebu herds.

Paging through history: parchment as a reservoir of ancient DNA for next generation sequencing.

Parchment represents an invaluable cultural reservoir. Retrieving an additional layer of information from these abundant, dated livestock-skins via the use of ancient DNA (aDNA) sequencing has been mooted by a number of researchers. However, prior PCR-based work has indicated that this may be challenged by cross-individual and cross-species contamination, perhaps from the bulk parchment preparation process. Here we apply next generation sequencing to two parchments of seventeenth and eighteenth century northern English provenance. Following alignment to the published sheep, goat, cow and human genomes, it is clear that the only genome displaying substantial unique homology is sheep and this species identification is confirmed by collagen peptide mass spectrometry. Only 4% of sequence reads align preferentially to a different species indicating low contamination across species. Moreover, mitochondrial DNA sequences suggest an upper bound of contamination at 5%. Over 45% of reads aligned to the sheep genome, and even this limited sequencing exercise yield 9 and 7% of each sampled sheep genome post filtering, allowing the mapping of genetic affinity to modern British sheep breeds. We conclude that parchment represents an excellent substrate for genomic analyses of historical livestock.

Ancient Ethiopian genome reveals extensive Eurasian admixture throughout the African continent.

Characterizing genetic diversity in Africa is a crucial step for most analyses reconstructing the evolutionary history of anatomically modern humans. However, historic migrations from Eurasia into Africa have affected many contemporary populations, confounding inferences. Here, we present a 12.5× coverage ancient genome of an Ethiopian male ("Mota") who lived approximately 4500 years ago. We use this genome to demonstrate that the Eurasian backflow into Africa came from a population closely related to Early Neolithic farmers, who had colonized Europe 4000 years earlier. The extent of this backflow was much greater than previously reported, reaching all the way to Central, West, and Southern Africa, affecting even populations such as Yoruba and Mbuti, previously thought to be relatively unadmixed, who harbor 6 to 7% Eurasian ancestry.

Mitochondrial sequence variation suggests an African influence in Portuguese cattle.

A total of 49 samples from indigenous Portuguese cattle breeds were analysed for sequence variation in the hypervariable region of the mitochondrial DNA D-loop. Sequence comparison and phylogenetic analyses revealed that haplotypes fell into two distinct groups. These corresponded with two separate haplotype clusters into which, respectively, all African, or alternatively all sequences of European origin, have previously been shown to fall. Here, the majority of sequences of African type were encountered in three southern, as compared to three northern breeds. This pattern of African influence may reflect an intercontinental admixture in the initial origins of Iberian breeds, or it is perhaps an introgression dating from the long and influential Moorish occupation of the south of the Iberian peninsula.

Microsatellite DNA variation within and among European cattle breeds.

Microsatellite markers offer great potential for genetic comparisons within and between populations. We report the analysis of 12 microsatellite loci in six breeds of European cattle. This yielded a wide spectrum of variability with observed heterozygosities ranging from 0.00 to 0.91. Deviations from Hardy-Weinberg equilibrium were noted for some locus-population combinations, particularly at a microsatellite located within the prolactin gene. Also, significant linkage disequilibrium was detected between two microsatellite loci located within the bovine major histocompatibility complex, and this association was maintained across breeds, providing evidence for marker stability during short-term evolution. The mode of mutation was investigated by comparing the observed data with that expected under the infinite alleles model of neutral mutation, and six of the microsatellite loci were found to deviate significantly, suggesting that a stepwise mutation model may be more appropriate. One indication of marker utility is that, when genetic distance estimates were computed, the resultant dendrogram showed concordance with known breed histories.

Ancient DNA suggests a recent expansion of European cattle from a diverse wild progenitor species.

A total of 11 Bos primigenius and Bos taurus bones from archaeological sites between 500 and 12000 years old were examined for the presence of DNA. It was possible to amplify and sequence mitochondrial control region DNA extracted from seven of the 11 samples, including two Pleistocene B. primigenius samples. We compared the results with published data by constructing phylogenetic networks. The two B. primigenius samples clustered with the extant B. taurus samples in the networks. The similarity between B. primigenius and modern taurine cattle confirms that these should be considered members of a single species. The sequences obtained from the B. taurus specimens were either identical to the reference sequence for modern European cattle or closely related to it. They included two sequences not previously documented. The network analysis of the ancient data highlights the intermediary nature of the B. primigenius sequences between modern European and African B. taurus and the proximity of the ancient DNA B. taurus sequences to modern European B. taurus. Further analysis of the extant data in the light of the ancient DNA results suggests that a degree of Pleistocene diversity survives in the extant European Bos population that is mainly derived from a more recent population expansion.

Diagnosis of susceptibility to malignant hyperthermia with flanking DNA markers.

OBJECTIVE: To define the region on human chromosome 19 carrying the gene for malignant hyperthermia susceptibility and to evaluate the use of flanking DNA markers in diagnosing susceptibility. DESIGN: Prospective molecular genetic linkage studies in a large malignant hyperthermia pedigree. SETTING: Irish malignant hyperthermia testing centre. SUBJECTS: A large Irish malignant hyperthermia pedigree. MAIN OUTCOME MEASURES: Routine diagnosis of susceptibility to malignant hyperthermia with in vitro contracture test on muscle biopsy specimens and genetic linkage between susceptibility and polymorphic DNA markers in a malignant hyperthermia family. RESULTS: Genetic typing of polymorphic DNA markers in a large Irish malignant hyperthermia pedigree generated a lod score of greater than 3 for the marker D19S9 and showed that the gene for susceptibility is flanked by the markers D19S9 and D19S16. These tightly linked flanking markers allowed non-invasive presymptomatic diagnosis of susceptibility in five untested subjects in the large pedigree with an accuracy of greater than 99.7%. CONCLUSIONS: DNA markers flanking the gene for susceptibility to malignant hyperthermia can be used with high accuracy to diagnose susceptibility in subjects in large known malignant hyperthermia pedigrees and may replace the previous in vitro contracture test for diagnosing this inherited disorder in large families with malignant hyperthermia.

Genome-wide association study for calving traits in Holstein-Friesian dairy cattle.

Dystocia and perinatal mortality are quantitative traits that significantly impact animal productivity and welfare. Their economic importance is reflected by their inclusion in the national breeding goals of many cattle populations. The genetic architecture that influences these traits, however, has still yet to be thoroughly defined. Regions of the bovine genome associated with calving difficulty (direct and maternal) and perinatal mortality were detected in this study using a Bayesian approach with 43 204 single nucleotide polymorphisms (SNPs) on up to 1970 Holstein-Friesian bulls. Several SNPs on chromosomes 5, 6, 11, 12, 17,18 and 28 were detected to be strongly associated with these calving performance traits. Novel genomic regions with previously reported associations with growth, stature, birth weight and bone morphology were identified in the present study as being associated with the three calving performance traits. Morphological abnormalities are a known contributor to perinatal mortality and the most significantly associated SNP for perinatal mortality in the present study was located in a region in linkage disequilibrium with the gene SLC26A7. This gene, SLC26A7, has similarities and colocalises with SLC4A2, which has previously been associated with osteoporosis and mortality in cattle populations. The HHIP gene that is known to be associated with stature in humans was strongly associated with direct calving difficulty in the present study; large calves are known to, on average, have a greater likelihood of dystocia. A stemloop microRNA, bta-mir-1256, on chromosome 12, involved in post-transcriptional regulation of gene expression was associated with maternal calving difficulty. Previously reported quantitative trait loci associated with calving performance traits in other populations were again identified in this study; with one genomic region on chromosome 18 supporting very strong evidence of an underlying causative mutation and accounting for 2.1% of the genetic variation in direct calving difficulty. Overlapping genomic regions associated with one or more of the calving traits were also detected substantiating the known genetic covariances existing between these traits. Moreover, some genomic regions were only associated with one of the calving traits implying the selective genomic breeding programs exploiting these regions could help resolve genetic antagonisms.

Bayesian inference of population expansions in domestic bovines.

The past population dynamics of four domestic and one wild species of bovine were estimated using Bayesian skyline plots, a coalescent Markov chain Monte Carlo method that does not require an assumed parametric model of demographic history. Four domestic species share a recent rapid population expansion not visible in the wild African buffalo (Syncerus caffer). The estimated timings of the expansions are consistent with the archaeological records of domestication.

Differentiation of European cattle by AFLP fingerprinting.

The Neolithic introduction of domestic cattle into Europe was followed by differential adaptation, selection, migration and genetic isolation, leading ultimately to the emergence of specialized breeds. We have studied the differentiation of European cattle by amplified fragment length polymorphism (AFLP) fingerprinting. Combining AFLP data sets from two laboratories yielded 81 biallelic polymorphic markers scored in 19-22 individual animals from 51 breeds. Model-based clustering differentiated Podolian cattle as well as French and Alpine breeds from other European cattle. AFLP genetic distances correlated well with microsatellite-based genetic distances calculated for the same breeds. However, the AFLP data emphasized the divergence of taurine and indicine cattle relative to the variation among European breeds and indicated an Eastern influence on Italian and Hungarian Podolian breeds. This probably reflects import from the East after the original introduction of domestic cattle into Europe. Our data suggest that Italian cattle breeds are relatively diverse at the DNA sequence level.