Ist and IIIrd intron length polymorphism of actin genes as a tool for flax DNA-profiling
Aim. The purpose of the work was to evaluate the possibility of using the polymorphism of the Ist and IIIrd introns of actin genes for DNA plant genotyping using flax varieties as model. Methods. 16 varieties of Ukrainian flax were analyzed. PCR was conducted using self-developed species-specific primers for the Ist and IIIrd introns of flax actin genes. DNA fragments were separated by electrophoresis in a 6% polyacrylamide gel and visualized by silver stains. Results. As a result of the evaluation of the Ist and IIIrd intron length polymorphism of actin genes, the species-specific DNA profiles of 16 flax varieties containing the target amplicons were obtained. The 7 allele phenotypes (PIC = 0.62) were detected for the Ist introns of the actin genes, and 3 allelic phenotypes (PIC = 0.32) for the IIIrd intron of actin genes. The highest level of polymorphism in the flax varieties was detected by evaluating the Ist intron length polymorphism of actin genes. Conclusions. Evaluation of the polymorphism of the Ist and IIIrd introns of actin genes allows genotyping and obtaining DNA profiles of flax varieties, which demonstrates the feasibility of further using both approaches for molecular genetic analysis of plants.
Keywords: gene introns, length polymorphism, actin genes, flax (Linum usitatissimum L.).
Wang X., Zhao X., Zhu J., Wu W. Genome-wide investigation of intron length polymorphisms and their potential as molecular markers in rice (Oryza sativa L.). DNA Res. 2005. Vol. 12. Р. 417–427. doi: 10.1093/dnares/dsi019.
Braglia L., Manca A., Mastromauro F., Breviario D. cTBP: A successful intron length polymorphism (ILP)-based genotyping method targeted to well defined experimental needs. Diversity. 2010. Vol. 2. P. 572–585. doi: https://doi.org/10.3390/d2040572.
Huang L., Cao H., Yang L., Yu Y., Wang Y. Large-scale development of PIP and SSR markers and their complementary applied in Nicotiana. Russ. J. Genet. 2013. Vol. 49. P. 827–838.
Xia X., Luan L.L., Qin G., Yu L.F., Wang Z.W., Dong W.C., Song Y., Qiao Y., Zhang X.S., Sang Y.L., Yang L. Genome-wide analysis of SSR and ILP markers in trees: diversity profiling, alternate distribution, and applications in duplication. Sci. Rept. 2017. Vol. 7 (1). P. 1–10. doi: 10.1038/s41598-017-17203-6.
Rabokon A.N., Pirko Ya.V., Demkovych A.Ye., Blume Ya.B. Comparative analysis of the efficiency of intron-length poly-morphism of в-tubulin genes and microsatellite loci for flax varieties genotyping. Cytol. Genet. 2018. Vol. 52 (1). P. 3–15.
Postovoitova A.S., Yotka O.Yu., Pirko Ya.V., Blume Ya.B. Molecular genetic evaluation of Ukrainian flax cultivars homoge-neity based on intron length polymorphism of actin genes and microsatellite loci. Cytol. Genet. 2018. Vol. 52 (6). P. 448–460.
Bardini M., Lee D., Donini P., Mariani A., Giani S., Toschi M., Lowe C., Breviario D. Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species. Genome. 2004. Vol. 47 (2). Р. 281–291. doi: 10.1139/g03-132.
Breviario D., Baird W.V., Sangoi S., Hilu K. High polymorphism and resolution in targeted fingerprinting with combined в-tubulin introns. Mol. Breed. 2007. Vol. 20. P. 249–259.
Rabokon A.N., Pirko Ya.V., Demkovych A.Ye., Blume Ya.B. Studing of в-tubulin gene intron length polymorphizm of Triti-cum aestivum L. and Hordeum vulgare L. varieties. Factors of experimental evolution of organisms. 2015. Vol. 17. P. 82–86 [in Ukrainian]
Pirko Ya.V., Postovoitova A.S., Rabokon A.M., Kalafat L.O., Privаlikhin S.M., Bilonozhko Yu.O., Pirko N.M., Blume Ya.B. Study of intron length polymorphism of the α -tubulin genes as a method of analysis of the genetic differentiation in plants. Ukr. Bot. J. 2018. Vol. 75 (6). P. 576–584. [in Ukrainian] doi: 10.15407/ukrbotj75.06.576.
Pirko Ya.V., Buy D.D., Postovoitova A.S., Rabokon A.M., Kalafat L.O., Blume Ya.B. New ILP method based on γ--tubulin genes intron length polymorphism. Dopov. Nac. akad. nauk Ukr. 2018. № 12. P. 87–92. [in Ukrainian] doi: 10.15407/dopovidi2018.12.087.
Postovoitova A. S., Pirko Ya.V., Blume Ya.B. Polymorphism of actin gene introns as an instrument for genotyping of the representatives from Solanaceae family. Science Bulletin of NUBiP. Biological systems: theory and innovation. [S.l.]. № 287. P. 70–78. [in Ukrainian]
Postovoitova A.S., Yotka O.Yu., Pirko Ya.V., Blume Ya.B. The intron length polymorphism analysis of the actin genes in representatives of the genus LINUM L. Plant Biology and Biotechnology: Third Conference for Young Scientists (Kyiv, May 16–18 2017). Kyiv, 2017. P. 40. [in Ukrainian]
Benbouza H., Jean-Marie J., Jean-Pierre B. Optimization of a reliable, fast, cheap and sensitive silver staining method to detect SSR markers in polyacrylamide gels. Biotechnol Agron Soc Environ. 2006. Vol. 10 (2). P. 77–81.
Pydiura N., Pirko Ya., Galinousky D., Postovoitova A., Yemets A., Kilchevsky A., Blume Ya. Genome-wide identification, phylogenetic classification, and exon-intron structure characterisation of the tubulin and actin genes in flax (Linum usitatis-simum). Cell Biol. Intl. 2018. doi: 10.1002/cbin.11001