OPTIMIZATION OF THE METHOD OF DNA ISOLATION FROM FOSSILS
Authors: Mokhnachova N. B. , Starodub L. F. , Dobryanska M. L. 
Published: November 2020

The history of the origin and domestication of farm animals has always interested mankind. However, these issues are covered in the literature in great detail only from the time when herds of domestic animals have already formed. Most often, the genesis of individual species, the original forms that formed the basis of domestication, remain unclear. [2] An example is the history of domestication of the horse, as the horse played a central role among other domestic animals in the development of human society. In the study of mammal fauna of the Pleistocene-Holocene of Europe there is a problem of studying the origin of the domestic horse Eguus cabalus L., ie, the establishment of wild ancestors of domesticated breeds, place, time and process of their domestication. Analysis of literature data on paleontological and archaeological finds in Ukraine showed that most researchers believe that the first domesticated horses began to recognize horses, the remains of which were found during archaeological excavations of the settlement of the third millennium BC. BC in Botai (Northern Kazakhstan), but from which taxon the opinions of scientists differ. Some believe that it could be Tarpan, however, there is an opinion that a large horse could not come from a small tarpan and Przewalski's horse. Therefore, preference was given to the hypothesis of the origin of the domestic horse from the ancient Pleistocene. At present, the problem of the origin of the domestic horse does not go beyond hypotheses and assumptions, and this is primarily due to the slight difference between the bones of the domestic and wild horse. The plasticity of the skeleton of the genus Eguus is very weak and this explains the problems faced by paleontologists in trying to develop the evolutionary history of horses. Thus, to understand the processes of domestication of this animal, in addition to archaeological and paleontological research methods, it is necessary to use tools from other fields of science, such as molecular genetic analysis of DNA samples. One of the variants of test systems for studying genetic polymorphism is the use of ISSR markers, which allow to analyze DNA fragments and make certain phylogenetic connections in the studied groups.

In the laboratory of genetics of the Institute of Breeding and Genetics of Animals named after M.V.Zubets NAAS began research in the field of paleogenetics, namely – the study of the molecular genetic component in the fossils of ancient members of the genus Eguus using ISSR-markers. Inverted repeats are of particular interest because they are unevenly distributed throughout the genome and do not require prior knowledge of the nucleotide sequence of the test DNA. A significant point in the selection of research methods for us was that intermicrosatellite polymorphism is used to study interspecific and intraspecific genetic variability. It is believed that DNA fragments obtained by ISSR analysis can be species- and breed-specific, and this method is widely used by researchers in the study of breed groups.

The purpose of our work is to develop a new method of DNA isolation from fossil remains (bones) of ancient horses and the production of ISSR-PCR with isolated DNA samples in the laboratory of genetics IRGT. M.V.Zubets NAAS according to the available reagents and existing protocols.

The research was carried out on samples of fossil bones of horses of the Pleistocene period (about 10 thousand years BC). One bone was found in the village. Beeches of Zhytomyr region in a career. Excavations were carried out in 1960, the metacarpal bone (os. Tarsicentral). Another bone was found in Novgorod-Siversky, Chernihiv region. in a career. Excavations were conducted by Boriskovsky PI in 1935. A tooth found in the village of Tarpan was used to study a wild tarpan horse (4.5 thousand years BC). Skibnytsia, Trostyanets district, Vinnytsia region. Excavations were conducted in 1959 by VM Danylenko. The paleontological material for the study was provided by the Kyiv National Museum of Natural History of the National Academy of Sciences of Ukraine, Department of Paleontology.

As a result of this work for the first time in the Department of Genetics and Biotechnology IRGT. Research on paleogenetics has been started by M.V.Zubets. We optimized the method of extracting genetic material from fossils and obtained DNA from the bones of a horse of the Pleistocene period (about 10 thousand years BC) and the tooth of a wild horse tarpan (4.5 thousand years BC). Also, the optimal conditions for PCR were selected to work with DNA obtained from fossil remains, to study polymorphism with ISSR markers, and electrophoregrams of amplification products were obtained.