THE EFFECTIVENESS OF THE USE OF VARIOUS ISSR-MARKERS IN THE STUDY OF HORSES
Abstract
The article presents the results of molecular genetic studies performed on samples of biological material from ancient horses (DNA was isolated from fossils of Pleistocene horse and tarpan) and three aboriginal breeds of modern horses (Polish horse, Hutsul breed, Arabian breed). The research was conducted in the laboratory of genetics IRGT. M.V.Zubets NAAS. Purpose of research. This work was carried out to compare the effectiveness of each of the ISSR-markers and then select the optimal combination in the study of polymorphism of the genetic structure of horses To study the DNA polymorphism of horses on ISSR markers, we used eight primers that are considered the most informative (AG)8CA, (AG)9C, (GA)6CC, (GA)9C, (AG)8CG, (GAG)6C, (ACC)6G, (CTC)6C.
Research methods. DNA from the blood was isolated using a set of reagents "DNA sorb B". From fossil remains of horses, DNA was isolated by an optimized method using proteinase K and dithiothreitol. The PCR mixture contained: 1 μl of buffer for Tag polymerase, 1 μl of a mixture of triphosphates ("Amplisens", RF), 0.8 μl of the appropriate primer, 0.2 μl of DNA polymerase ("Fermentas", Lithuania), water for PCR 3 μl. Genomic DNA was added in an amount of 4 μl. The total volume of the DNA mixture was 10 μl. Amplification was performed on a programmed four-channel thermocycle "Tertsyk" ("DNA technology", Russia). The amplification program included primary denaturation (95°C, 2 min); 30 cycles of denaturation (95°C, 30 s), hybridization of primers (54–64°C, 30 s) and elongation (72°C, 1 min), finishing elongation (72°C, 5 min).
To more accurately estimate the lengths of the detected amplification fragments, a universal scale was used, where the gradation of DNA fragments by molecular weights was used. Depending on the zone ("heavy", "medium" and "light" fragments), a certain step from 10 to 200 bp was used. As a result, 38 zones with a fixed interval were identified, which allow to accurately determine the molecular weight for amplification products of different lengths and standardize the results of this study.
Obtained results and conclusions. In the study of the obtained spectra of amplification products, we found that the largest number of loci was obtained by using as primers the sequences (GA) 6CC and (GAG) 6C – 9 and 8 loci. At the same time, primers (AG) 8CA – 0.27, (AG) 9C – 0.21 and (ACC) 6G – 0.21 were the most polymorphic in terms of PIC. It should be noted that when using primer (GA) 6CC in the study of genetic polymorphism of horses was obtained a significant range of amplification products – 9 loci, with a polymorphism index of 0.16, which allows it to be used quite effectively for research. Using the sequence (AG) 8CG was characterized by the lowest PIC index and was 0.02. As a result, we found that the most effective for the detection of polymorphism in the PIC index in horses was the use of primers sequences (AG) 8CA, (AG) 9C, and (ACC) 6G. To obtain the largest range of amplification fragments in horses by ISSR-PCR, the most effective was the use of the sequence (GA) 6CC and (GAG) 6C.
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