Periodic Reporting for period 1 - EARLYRIDERS (Tracking the temporal and geographic origins of modern domestic horses)
Periodo di rendicontazione: 2018-11-01 al 2020-10-31
The aim of the project was to advance our understanding of the horse domestication process. Horse domestication has been transformative for the history of human civilizations and promoted the first stage of globalization. It was also hypothesized to be involved in the spread of Indo-European languages and culture, as the expansion of these societies could be heavily linked to the emergence of horse riding, among other technological breakthroughs. As such, the results of the project will be influential for understanding this major breakthrough in human history. Moreover, animal domestication, and the genomic changes underlying this process, represent an essential model in evolutionary biology, offering an opportunity to better understand the evolutionary process of selection.
Secondly, the project was aimed at testing ambitious but risky hypotheses with regards to the spread of important human diseases during domestication. More specifically, recent work has revealed that the plague, caused by Yersinia pestis, was already affecting Early Bronze Age populations. As this was also characterized by the intensification of human-horse relationships and massive population movements facilitated by the horse, we aimed at determining, against conventional views, whether the horse could be the source or vector of this disease.
Finally, the project was aimed at developing novel DNA-capture protocols, allowing to enrich the ancient genomic libraries for the DNA horse fragments. Besides important applications for basic research, such methods have also potential applications of importance for the horse industry through the innovative target-enrichment methods for a fast, cost-effective genotyping of horses, even from damaged samples and using minute amounts of tissue.
Secondly, the project was aimed at testing ambitious but risky hypotheses with regards to the spread of important human diseases during domestication. More specifically, recent work has revealed that the plague, caused by Yersinia pestis, was already affecting Early Bronze Age populations. As this was also characterized by the intensification of human-horse relationships and massive population movements facilitated by the horse, we aimed at determining, against conventional views, whether the horse could be the source or vector of this disease.
Finally, the project was aimed at developing novel DNA-capture protocols, allowing to enrich the ancient genomic libraries for the DNA horse fragments. Besides important applications for basic research, such methods have also potential applications of importance for the horse industry through the innovative target-enrichment methods for a fast, cost-effective genotyping of horses, even from damaged samples and using minute amounts of tissue.
Ancient DNA (aDNA) extracts are often characterized by low amount of focal (endogenous) DNA, caused by degradation and contamination by DNA of exogenous origins. We thus performed extensive experiments to adapt and optimize protocols for enriching the DNA libraries for fragments of interest and, therefore, focus sequencing efforts and resources on relevant DNA targets. The method, called hyRAD (hybridization RAD), previously used mostly for museum samples of less than 100 years old, was originally by Tomasz Suchan when he was a PhD student. It allows inexpensive production of molecular probes from fresh DNA, that allow selective enrichment of the aDNA of interest. The main modification introduced consisted of the synthesis of RNA probes, as opposed to the DNA probes produced by the original protocol and was proved to decrease contamination risk, enable producing the probes at larger scales, and increase capture efficacy. The protocol has been tested with samples of varying endogenous DNA content, preservation contexts, and age, in order to establish the method suitability to the broader range of samples, and obtained excellent enrichment rates in samples with low endogenous DNA content.
As the host lab was the first to report that aDNA methylation patterns can detected in aDNA, we have also tested the potential of using methylation sensitive and non-sensitive restriction enzymes for selective targeting unmethylated parts of the genome. The probes obtained with methylation-sensitive enzyme were shown to capture more fragments that mapped uniquely on the genome and showing lower post-mortem damage rates. This result marked an important step forward for the selective characterization of genomic regions with different methylation patterns and reducing the sequencing error rates in aDNA studies.
Another important breakthrough was to characterize how the hyRAD could be applied to more distant species. We prepared molecular probes from horse and donkey (that diverged around 4-4.5 mya), used to capture aDNA libraries of horses, donkeys, and mules (which are the offspring of a horse mare and a donkey jack). We then carefully analyzed if capturing the libraries of one species against the probes produced with another one introduces any sort of allelic bias, such as for example increasing or decreasing the chances of detecting target alleles. Our preliminary analyses show, that the hyRAD does not introduce significant bias for the captures performed on the probes prepared from relatively close relatives, which gauges for the method’s future popularity and utility in deeper phylogenetic scales. Additional analyses are being finalized and the submission of the article is planned for mid-May 2021.
We have also bioinformatically screened previously obtained ancient horse genomes for the presence of pathogens, despite considerable sequencing efforts (over 10 billion read pairs), we could not identify the presence of Yersinia pestis in the data. We thus used target-enrichment procedures to test for the presence of the pathogen in horse samples. Unfortunately, none of the horse tested supported the presence of the pathogen. However, the absence of a proof cannot be confused as the proof of the absence, as the lack of Y. pestis DNA in our data could, also indicate limited detectability in our assays. Thus the negative results obtained could not be used conclusively at that stage.
One of the aims of the project was to identify the center of origin of the domestic horses. We have obtained and sequenced crucial samples, that filled the important spatial (Central-Eastern Europe) and temporal (Eneolithic/Bronze Age) gaps in the host lab dataset of ancient horse genome sequences. The results of the abovementioned analyses are included in an article covering the origin of horse, authored by more than 100 specialists in many fields. The article solves the question of the temporal and geographic origins of horse domestication, and has strong consequences for our understanding of human mobility during the third millennium Before Common Era.
Overall, the work will be published in three scientific articles presenting: 1) the first application presenting the optimizations of the hyRAD protocol for aDNA from osseous remains several thousand years old; 2) second methodological paper assessing the potential biases of the method; 3) identifying the domestication centre of the horse, underlying the spread of the domestic horse in the third millennium BCE. Additionally, the effects of the project will disseminated during at the 9th International Symposium for Biomolecular Archaeology in June 2021, the most important conference in the field. For communicating the results to a general audience, the outreach activities took the form of lectures and lab visits organized, including within Fête de la Science events in 2019 and 2020.
As the host lab was the first to report that aDNA methylation patterns can detected in aDNA, we have also tested the potential of using methylation sensitive and non-sensitive restriction enzymes for selective targeting unmethylated parts of the genome. The probes obtained with methylation-sensitive enzyme were shown to capture more fragments that mapped uniquely on the genome and showing lower post-mortem damage rates. This result marked an important step forward for the selective characterization of genomic regions with different methylation patterns and reducing the sequencing error rates in aDNA studies.
Another important breakthrough was to characterize how the hyRAD could be applied to more distant species. We prepared molecular probes from horse and donkey (that diverged around 4-4.5 mya), used to capture aDNA libraries of horses, donkeys, and mules (which are the offspring of a horse mare and a donkey jack). We then carefully analyzed if capturing the libraries of one species against the probes produced with another one introduces any sort of allelic bias, such as for example increasing or decreasing the chances of detecting target alleles. Our preliminary analyses show, that the hyRAD does not introduce significant bias for the captures performed on the probes prepared from relatively close relatives, which gauges for the method’s future popularity and utility in deeper phylogenetic scales. Additional analyses are being finalized and the submission of the article is planned for mid-May 2021.
We have also bioinformatically screened previously obtained ancient horse genomes for the presence of pathogens, despite considerable sequencing efforts (over 10 billion read pairs), we could not identify the presence of Yersinia pestis in the data. We thus used target-enrichment procedures to test for the presence of the pathogen in horse samples. Unfortunately, none of the horse tested supported the presence of the pathogen. However, the absence of a proof cannot be confused as the proof of the absence, as the lack of Y. pestis DNA in our data could, also indicate limited detectability in our assays. Thus the negative results obtained could not be used conclusively at that stage.
One of the aims of the project was to identify the center of origin of the domestic horses. We have obtained and sequenced crucial samples, that filled the important spatial (Central-Eastern Europe) and temporal (Eneolithic/Bronze Age) gaps in the host lab dataset of ancient horse genome sequences. The results of the abovementioned analyses are included in an article covering the origin of horse, authored by more than 100 specialists in many fields. The article solves the question of the temporal and geographic origins of horse domestication, and has strong consequences for our understanding of human mobility during the third millennium Before Common Era.
Overall, the work will be published in three scientific articles presenting: 1) the first application presenting the optimizations of the hyRAD protocol for aDNA from osseous remains several thousand years old; 2) second methodological paper assessing the potential biases of the method; 3) identifying the domestication centre of the horse, underlying the spread of the domestic horse in the third millennium BCE. Additionally, the effects of the project will disseminated during at the 9th International Symposium for Biomolecular Archaeology in June 2021, the most important conference in the field. For communicating the results to a general audience, the outreach activities took the form of lectures and lab visits organized, including within Fête de la Science events in 2019 and 2020.
The project helped to illuminate the temporal and spatial origins of horse domestication, a process that has been transformative for the history of human civilizations and promoted the first stage of globalization. By helping to understand horse domestication, the project contributed for the development of a robust, necessary, unifying theory about the biological and evolutionary processes underlying animal domestication, but also for understanding human history.
Moreover, the methodological advancements developed in the course of the project will influence ancient DNA research, museomics, and molecular ecology, by providing an inexpensive and fast way of analyzing degraded samples with minute amounts of the DNA of interest.
Moreover, the methodological advancements developed in the course of the project will influence ancient DNA research, museomics, and molecular ecology, by providing an inexpensive and fast way of analyzing degraded samples with minute amounts of the DNA of interest.