Comparative study of different methods of DNA extraction from cattle bones specimens maintained in a craniological collection

The development of molecular-genetic methods allows elucidating the origin and demographic history of breeds of farm animals. Samples of bones and teeth maintained in craniological collections can serve as a source of DNA for such studies. The work with historical samples is complicated by the presence of a very low quantity of DNA, the high degree of its degradation and by the contamination of samples by PCR inhibitors. The aim of this work was the comparison of the efficiency of various methods of DNA extraction from historical cattle skulls, suitable for molecular genetic studies. The material was teeth extracted from historical skulls of cattle of the Yaroslavl and Kholmogor breeds stored in the craniological collection of the Liskun Museum of Livestock (Timiryazev Russian State Agrarian University-Moscow Agrarian Academy). At the first stage, we compared various DNA isolation methods implemented in the form of commercial kits, i.e. Prep Filer™ BTA Forensic DNA Extraction Kit («Thermo Fisher Scientific Inc.», USA), COrDIS Extract decalcine («GORDIZ» LLC, Russia), M-sorb-bone («Syntol» LLC, Russia), QIAamp DNA Investigator Kit («Qiagen», USA), with the modification of the amount of bone material and conditions of lylis. Based on preliminary research results, we selected for more detailed studies two kits, the QIAamp DNA Investigator Kit («Qiagen», USA) which implements the technology of column with silica gel membrane, and Prep Filer™ BTA Forensic DNA Extraction Kit («Thermo Fisher Scientific Inc.», USA) which is based on using magnetic particles. The quantitative and qualitative characteristics of the obtained DNA were evaluated by measuring the concentration of double-stranded DNA using a Qubit™ fluorimeter («Invitrogen, Life Technologies», USA) and determining the ratio of the absorption at 260 nm and 280 nm (OD260/280) on a NanoDrop 8000 instrument («Thermo Fisher Scientific, Inc.», USA). The suitability of the obtained DNA extracts for molecular genetic studies was assessed based on the multiplex analysis of 11 microsatellite loci (TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA122, INRA23, TGLA126, BM1818, ETH225, BM1824) as well as genome-wide genotyping on high-density DNA chips containing 777 thousand SNPs (Bovine HD BeadChip, «Illumina, Inc.», USA). Concentrations of double-stranded DNA (dsDNA) obtained using QIAamp DNA Investigator Kit and Prep Filer™ BTA Forensic DNA Extraction Kit ranged from 0.146 ng/µl to 2.060 ng/µl and from 0.110 ng/µl to 13,600 ng/µl, respectively, and averaged 0.83±0.23 ng/µl and 2.75±1.33 ng/µl. The correlation coefficient ( r ) between the concentrations of dsDNA in isolations DNA obtained by two different methods was 0.84. Analysis of microsatellites showed that each of the samples has its own unique genotype which differs from other historical and modern samples of individuals. Efficiency of SNP genotyping (Call Rate) of the historical samples was 0.533-0.878 and 0.958-0.977 for DNA preparations produced using QIAamp DNA Investigator Kit и Prep Filer™ BTA Forensic DNA Extraction Kit, respectively. The results of microsatellite analysis and SNP genotyping, on the one hand, indicate the suitability of the obtained DNA for polymorphism research, on the other hand, confirm the compliance of the laboratory in which this analysis was performed with the authenticity criteria for working with ancient DNA. Conducting large-scale studies of historical samples using different types of DNA markers will clarify the origin and demographic history of domestic cattle breeds and develop effective programs for their conservation.