Rabokon A. Genetic polymorphism evaluation in plants using introns of β-tubulin genes at intraspecies and interspecies levels.

Introns are universal and convenient tool for molecular genetic research in a wide range of organisms because of its length polymorphism. A systematic assessment of the application possibility and effectiveness of one of the modern molecular marker systems based on the presence of an intron-specific DNA polymorphism of the tubulin family in plants (tubulin-based polymorphism, TBP) was carried out in the present work. Because it is not yet widely used, this method requires verification of its effectiveness for genotyping plants at different taxonomic levels. The previous data were reviewed and new application examples of the TBP analysis (TBP method – I intron length polymorphism assessment) and its modifications (сТВР method - II intron length polymorphism assessment, and hТВРmethod – I+II introns length polymorphism assessment) in monocotyledonous and dicotyledonous plants assessment at the interspecies and interspeces level were presented. Following plants were tested: finger millet (E. coracana) varieties and the genotypes of Indian goosegrass (E. indica), Crimean populations of aegilops (A. biuncialis), Antarctica island populations of hair grass (D. antarctica), wheat (T. aestivum) and barley cultivars (H. vulgare); varieties and cultivars of camelina (C. sativa), flax species (L. bienne, L. angustifolium, L. usitatissimum, L. humile, L. perenne), various cultivars of flax and Belarusian flax landraces. Range of amplicon size variation (introns of the tubulin genes) was obtained for all investigated plants, UPGMA-denograms were constructed between populations, varieties and genotypes based on the values of the genetic similarities of Ney and Lee; PIC values (Polymorphism Information Content) were calculated. In addition, a bioinformation analysis of the exon-intron structure of β-tubulin genes in wheat, barley and flax was conducted. To obtain a specific TBP profile of all studied samples, a polymerase chain reaction was performed with known primer sequences and the resulting reaction products were separated using a non-denaturing polyacrylamide gel followed by silver nitrate visualization. The high application efficiency of the β-tubulin gene intron length polymorphism assessment was shown for fingerprinting of various plant genotypes, that can be used both for plant molecular genetics and for purposeful application in molecular selection, for example, during creating and testing new varieties. The obtained results allow us to recommend this β-tubulin gene’s intron length evaluation method and its modifications for the molecular genetic differentiation of monocotyledonous and dicotyledonous plant representatives, as well as to offer it for application in molecular genetic research of other plant phylogenetic groups, because this method combines reliability and high output data obtaining speed with data analysis simplicity.