THE MODIFIED CTAB METHOD FOR DNA ISOLATION FROM TILIA CORDATA MILL. TO DETECT MULTILOCUS POLYMORPHISM

Abstract

Small-leaved linden ( Tilia cordata Mill.) is the main honey plant in the Republic of Bashkortostan (RB). Linden forests, forming the basis of the entire forest fund of the RB, grow in a wide range of climatic and geographical conditions. This suggests the presence of many linden populations in RB, which, moreover, may differ in nectar quality and productivity. Therefore, studies to identify the genetic polymorphism and population structure of T. cordata in RB using various methods of molecular genetics seem to be relevant. Dried leaves are most suitable for DNA isolation of various plants, but they contain a large amount of PCR inhibitors in linden. Based on this, the purpose was to select and test the optimal method for isolating high-quality total DNA from dry leaves of T. cordata . The quality of the isolated DNA was assessed spectrophotometrically, by agarose gel electrophoresis, and by ISSR-PCR. Of the six DNA extraction methods tested, the most suitable for T. cordata was the method modified by us using CTAB, which differs from the standard approaches by using mercaptoethanol, polyvinylpyrrolidone, and ascorbic acid in the extraction buffer, homogenize the leaves in the cold, and partial sampling of the aqueous phase during treatment with chloroform. Using this method, DNA was isolated from 24 samples of T. cordata from different regions of the RB, and ISSR analysis was performed using eight primers. According to the results of the analysis, two large groups of populations of small-leaved linden were identified, the northern group was associated with the Ufa River basin, and the southern one with the Belaya River basin.