Page 124 - Zbornik_Konf_2019

https://doi.org/10.15414/2019.9788055220703

4 International Scientific Conference Abstracts Book
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GENOMIC FINGERPRINTING OF LINUM USITATISSIMUM L. CULTIVARS USING
INTRON LENGTH POLYMORPHISM OF γ‐TUBULIN
Yaroslav Pirko, Denys Buy, Anastasiia Rabokon, Anastasiia Postovoitova,
Lubov Kalafat, Yaroslav Blume

Institute of Food Biotechnology and Genomics of the NAS of Ukraine
Kyiv, Ukraine; E-mail.: rabokonnastya@gmail.com

Linum usitatissimum L. is one of the major oilseed and fiber crops in the world, and its
cultivation history spans many thousands of years. Accurate identification of flax genotypes is
very useful at all stages of selection, starting with the selection of parental lines and ending
with the final use of the obtained raw materials. Currently, various molecular markers are
used to identify crop cultivars, including flax. Although some marker systems (such as RAPD,
AFLP, ISSR, and SSR) have already been developed for flax, they are sometimes characterized
by low reproducibility. A new type of molecular-genetic markers, which is becoming
increasingly practical, is the estimation of the intron length polymorphism of genes (Intron
Length Polymorphism, ILP). The universality and simplicity of ILP markers allow DNA
profiling and genotyping of plants. г-tubulin is critically necessary for the microtubule
nucleation, therefore, its amino acid sequence is highly conserved among phylogenetically
diverse organisms. Relies on this, we have developed and proposed a new marker system,
which is based on the intron length polymorphism of г-tubulin genes. The aim of this work
was to verify the possibility of using this marker system for genetic identification of Ukrainian
flax cultivars.
A search for conservative exon γ-tubulin sites on the border with introns (in flax and
other plant species) and design of degenerated primers (F: 5′-
GAYGTBTTYTTTTACCARGCKGA-3; R: 5′-GAGTTGTARGGYTGGACRAC-3) were done. In the
case of flax, the amplification covers the region of the 1 and 2 intron, together with the 2
st
nd
nd
exon. Genomic DNA was extracted from fresh leaf tissue and after EPIC-PCR amplification,
performed with the one pair of degenerated primers, the products were analyzed on non-
denaturing acrylamide gels.
According to the results of the electrophoretic analysis, it was found that bands were
formed in a range of 500–600 and 700–900 bp, wherein 4 bands were detected for each
cultivar in most cases. It is significant that Globus is most different from other cultivars and
characterized by 5 bands about 498 bp, 563 bp, 590 bp, 752 bp, 845 bp. However, this flax
cultivar is poorly differentiated from other samples using TBP and SSR analysis, as we showed
earlier. Miandr and Kameniar cultivars also differ from other samples in the bands set of DNA
profile, as in the case of using TBP and SSR markers.
Thus, this proposed method, based on the intron length polymorphism of γ-tubulin gene,
can be easily and successfully used in molecular genetic studies of flax, because it allows to
identify, differentiate and evaluate genetic polymorphism of cultivars according to obtained
DNA profiles.
Keywords: Linum usitatissimum, molecular-genetic markers, γ-tubulin genes, introns.

Acknowledgments
This work was supported by the National Academy of Sciences of Ukraine, carried out as part of a
grant for young scientists “The implementation of the intron length polymorphism of gamma-tubulin
genes method for the molecular-genetic differentiation of plants” (2019–2020).

|124 4 International Scientific Conference Agrobiodiversity Nutrition, Health and Quality of Human and Bees Life
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September 11–13, 2019