Performance and automation of ancient DNA capture with RNA hyRAD probes

DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analys- ing ancient DNA (aDNA) extracts, which are often dominated by exogenous environ- mental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic re- striction of fresh DNA extracts to produce RNA probes that cover only a fraction ofthe genome and can serve as baits for capturing homologous fragments from aDNA li- braries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5-fold on-target enrichment for aDNA extracts showing ex- tremely low endogenous content (20%¿30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensi- tive to the original endogenous DNA content. Procedures based on two instead of a single round of capture increased on-target coverage up to 3.6-fold. Additionally, we used methylation-sensitive restriction enzymes to produce probes targeting hypo- methylated regions, which improved data quality by reducing post-mortem DNA dam- age and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture process- ing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples.This project received funding from: the University Paul Sabatier IDEX Chaire d’Excellence (OURASI); the CNRS Programme de Recherche Conjoint (PRC); the CNRS International Research Project (IRP AMADEUS); the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 797449; the Russian Foundation for Basic Research, project No. 19-59-15001 “Horses and their importance in the life of the ancient population of Altai and adjacent territories: interdisciplinary research and reconstruction”; and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681605)

The origins and spread of domestic horses from the Western Eurasian steppes

This is the final version. Available on open access from Nature Research via the DOI in this recordData availability: All collapsed and paired-end sequence data for samples sequenced in this study are available in compressed fastq format through the European Nucleotide Archive under accession number PRJEB44430, together with rescaled and trimmed bam sequence alignments against both the nuclear and mitochondrial horse reference genomes. Previously published ancient data used in this study are available under accession numbers PRJEB7537, PRJEB10098, PRJEB10854, PRJEB22390 and PRJEB31613, and detailed in Supplementary Table 1. The genomes of ten modern horses, publicly available, were also accessed as indicated in their corresponding original publications57,61,85-87.NOTE: see the published version available via the DOI in this record for the full list of authorsDomestication of horses fundamentally transformed long-range mobility and warfare. However, modern domesticated breeds do not descend from the earliest domestic horse lineage associated with archaeological evidence of bridling, milking and corralling at Botai, Central Asia around 3500 BC. Other longstanding candidate regions for horse domestication, such as Iberia and Anatolia, have also recently been challenged. Thus, the genetic, geographic and temporal origins of modern domestic horses have remained unknown. Here we pinpoint the Western Eurasian steppes, especially the lower Volga-Don region, as the homeland of modern domestic horses. Furthermore, we map the population changes accompanying domestication from 273 ancient horse genomes. This reveals that modern domestic horses ultimately replaced almost all other local populations as they expanded rapidly across Eurasia from about 2000 BC, synchronously with equestrian material culture, including Sintashta spoke-wheeled chariots. We find that equestrianism involved strong selection for critical locomotor and behavioural adaptations at the GSDMC and ZFPM1 genes. Our results reject the commonly held association between horseback riding and the massive expansion of Yamnaya steppe pastoralists into Europe around 3000 BC driving the spread of Indo-European languages. This contrasts with the scenario in Asia where Indo-Iranian languages, chariots and horses spread together, following the early second millennium BC Sintashta culture

Traces of Late Bronze and Early Iron Age Mongolian Horse Mitochondrial Lineages in Modern Populations

The Mongolian horse is one of the most ancient and relatively unmanaged horse breeds. The population history of the Mongolian horse remains poorly understood due to a lack of information on ancient and modern DNA. Here, we report nearly complete mitochondrial genome data obtained from five ancient Mongolian horse samples of the Khereksur and Deer Stone culture (late 2nd to 1st third of the 1st millennium BC) and one ancient horse specimen from the Xiongnu culture (1st century BC to 1st century AD) using target enrichment and high-throughput sequencing methods. Phylogenetic analysis involving ancient, historical, and modern mitogenomes of horses from Mongolia and other regions showed the presence of three mitochondrial haplogroups in the ancient Mongolian horse populations studied here and similar haplotype composition of ancient and modern horse populations of Mongolia. Our results revealed genetic continuity between the Mongolian horse populations of the Khereksur and Deer Stone culture and those of the Xiongnu culture owing to the presence of related mitotypes. Besides, we report close phylogenetic relationships between haplotypes of the Khereksur and Deer Stone horses and the horses of indigenous breeds of the Middle East (Caspian and Iranian), China (Naqu, Yunnan, and Jinjiang), and Italy (Giara) as well as genetic similarity between the Xiongnu Mongolian horses and those of the most ancient breeds of the Middle East (Arabian) and Central Asia (Akhal-Teke). Despite all the migrations of the Mongolian peoples over the past 3000 years, mitochondrial haplogroup composition of Mongolian horse populations remains almost unchanged

High genetic diversity of ancient horses from the Ukok Plateau.

A growing number of researchers studying horse domestication come to a conclusion that this process happened in multiple locations and involved multiple wild maternal lines. The most promising approach to address this problem involves mitochondrial haplotype comparison of wild and domestic horses from various locations coupled with studies of possible migration routes of the ancient shepherds. Here, we sequenced complete mitochondrial genomes of six horses from burials of the Ukok plateau (Russia, Altai Mountains) dated from 2.7 to 1.4 thousand years before present and a single late Pleistocene wild horse from the neighboring region (Denisova cave). Sequencing data indicates that the wild horse belongs to an extinct pre-domestication lineage. Integration of the domestic horse data with known Eurasian haplotypes of a similar age revealed two distinct groups: the first one widely distributed in Europe and presumably imported to Altai, and the second one specific for Altai Mountains and surrounding area

The origins and spread of domestic horses from the Western Eurasian steppes.

Domestication of horses fundamentally transformed long-range mobility and warfare1. However, modern domesticated breeds do not descend from the earliest domestic horse lineage associated with archaeological evidence of bridling, milking and corralling2-4 at Botai, Central Asia around 3500 BC3. Other longstanding candidate regions for horse domestication, such as Iberia5 and Anatolia6, have also recently been challenged. Thus, the genetic, geographic and temporal origins of modern domestic horses have remained unknown. Here we pinpoint the Western Eurasian steppes, especially the lower Volga-Don region, as the homeland of modern domestic horses. Furthermore, we map the population changes accompanying domestication from 273 ancient horse genomes. This reveals that modern domestic horses ultimately replaced almost all other local populations as they expanded rapidly across Eurasia from about 2000 BC, synchronously with equestrian material culture, including Sintashta spoke-wheeled chariots. We find that equestrianism involved strong selection for critical locomotor and behavioural adaptations at the GSDMC and ZFPM1 genes. Our results reject the commonly held association7 between horseback riding and the massive expansion of Yamnaya steppe pastoralists into Europe around 3000 BC8,9 driving the spread of Indo-European languages10. This contrasts with the scenario in Asia where Indo-Iranian languages, chariots and horses spread together, following the early second millennium BC Sintashta culture11,12