The use of the 5S rDNA IGS region for DNA barcoding and molecular taxonomy of two Ukrainian species of the genus Tulipa L.
Abstract
Taxonomic identification of closely related representatives of the genus Tulipa L. is complicated by the convergent similarity of morphological characters, interspecific hybridization, polyploidy and asexual reproduction. Among the tulips in the flora of Ukraine, the most problematic is the status of taxa of the complex species T. sylvestris L. Aim. To examine the organization and evolution of the nuclear 5S rDNA IGS region for four closely related Tulipa taxa and to evaluate the possibility of its use in the molecular taxonomy of this plant group. Methods. PCR amplification, cloning and sequencing of 5S rDNA repeat units, bioinformatic and phylogenetic analysis. Results. In this work, 5S rDNA repeats were sequenced for the species T. graniticola and T. ophiophylla, which are considered to be synonyms of T. sylvestris L. by many authors. We also assembled the main ribotypes from full-genome libraries of short Illumina reads for T. patens, and T. saxatilis, the species related to T. sylvestris. A comparative analysis of the structure of 5S rDNA sequences and a phylogenetic analysis based on the IGS region were carried out. Conclusions. A high level of 5S rDNA IGS variability has been demonstrated for representatives of the subgenus Eriostemones of the genus Tulipa. The use of 5S rDNA IGS for phylogenetic analysis enabled the clear delimitation of the closely related species T. saxatilis, T. patens, and the complex species T. sylvestris, as well as the delineation of the taxa with the controversial status, T. graniticola and T. ophiophylla. Therefore, the use of this region of the nuclear genome is a promising approach to the molecular taxonomy of the genus Tulipa.
References
Andreev I. O., Mel’nyk V. M., Parnikoza I. Y., Kunakh V. A. Molecular organization and intragenomic variability of intergenic spacer of 5S rRNA genes in Colobanthus quitensis. Cytol. Genet. 2023. Vol. 57(5). P. 399-405. doi: 10.3103/S0095452723050018.
Anisimova M., Gascuel O. Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst. Biol. 2006. Vol. 55. P. 539-552. doi: 10.1080/10635150600755453.
Baker W. J., Bailey P., Barber V., Barker A., et al. A comprehensive phylogenomic platform for exploring the angiosperm tree of life. Systematic biology. 2022. Vol. 71(2). P. 301-319. doi: 10.1093/sysbio/syab035.
Besendorfer V., Krajačić-Sokol I., Jelenić S., Puizina J., Mlinarec J., Sviben T., Papeš D. Two classes of 5S rDNA unit arrays of the silver fir, Abies alba Mill. structure, localization and evolution. Theor. Appl. Genet. 2005. Vol. 110. P. 730-741. doi: 10.1007/s00122-004-1899-y
Botschantzeva Z. Tulips: taxonomy, morphology, cytology, phytogeography and physiology. Crc Press. 1982.
Chen X., Helen V. M. Tulipa Linnaeus, Flora of China, Samuel B. J., Wu Z. Y., and Raven P. H., Eds. Science Press, Beijing, China, 2000.
Christenhusz M. J., Govaerts R., David J. C., Hall T. et al. Tiptoe through the tulips–cultural history, molecular phylogenetics and classification of Tulipa (Liliaceae). Bot. J. Linn. Soc. 2013. Vol. 172(3). P. 280-328. doi: 10.1111/boj.12061.
Ciganda M., Williams N. Eukaryotic 5S rRNA biogenesis. Wiley Interdiscip. Rev.: RNA. 2011. Vol. 2(4). P. 523-533. doi: 10.1002/wrna.74.
Cloix C., Yukawa Y., Tutois S., Sugiura M., Tourmente S. In vitro analysis of the sequences required for transcription of the Arabidopsis thaliana 5S rRNA genes. Plant J. 2003. Vol. 35(2). P. 251-261. doi: 10.1046/j.1365-313X.2003.01793.x.
De Souza T. B., Gaeta M. L., Martins C., Vanzela A. L. L. IGS sequences in Cestrum present AT-and GC-rich conserved domains, with strong regulatory potential for 5S rDNA. Mol. Biol. Rep. 2020. Vol. 47. P. 55-66. doi: 10.1007/s11033-019-05104-y.
Douet J., Tourmente S. Transcription of the 5S rRNA heterochromatic genes is epigenetically controlled in Arabidopsis thaliana and Xenopus laevis. Hered. 2007. Vol. 99. P. 5-13. doi: 10.1038/sj.hdy.6800964.
Garcia S., Wendel J. F., Borowska-Zuchowska N., Ainouche M., Kuderova A., Kovarik A. The utility of graph clustering of 5S ribosomal DNA homoeologs in plant allopolyploids, homoploid hybrids, and cryptic introgressants, Front. Plant Sci. 2020. Vol. 11(41). doi: 10.3389/fpls.2020.00041.
Grabiele M., Aguilera P. M., Ducasse D. A., Debat H. J. Molecular characterization of the 5S rDNA non-transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Rodriguésia. 2021. Vol. 72. doi: 10.1590/2175-7860202172071.
Hajdari A., Pulaj B., Schmiderer C., Mala X., et al. A phylogenetic analysis of the wild Tulipa species (Liliaceae) of Kosovo based on plastid and nuclear DNA sequence. Adv. Genet. 2021. Vol. 2(3). e202100016. doi: 10.1002/ggn2.202100016.
Ishchenko O. O., Bednarska I. O., Panchuk І. І. Application of 5S ribosomal DNA for molecular taxonomy of subtribe Loliinae (Poaceae). Cytol. Genet. 2021. Vol. 55. P.10-18. doi: 10.3103/S0095452721010096.
Katoh K., Rozewicki J., Yamada K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings Bioinf. 2017. Vol. 20(4). P. 1160–1166. doi: 10.1093/bib/bbx108.
Kritskaya T. A., Kashin A. S., Perezhogin Y. V., Murtazaliev R. A., Anatov D. M., Friesen N. Genetic diversity of Tulipa suaveolens (Liliaceae) and its evolutionary relationship with early cultivars of T. gesneriana. Plant Syst. Evol. 2020. Vol. 306. P. 1-15. doi: 10.1007/s00606-020-01667-7.
Kumar S., Stecher G., Knyaz C., Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018. Vol. 35(635). P. 1547-1549. doi: 10.1093/molbev/msy096.
Liu G., Lan Y., Qu L., Zhao Y., Xin H., Xi M. Analyzing the genetic relationships in Tulipa based on karyotypes and 5S rDNA sequences. Sci. Hortic. 2022. Vol. 302. P. 111178. doi: 10.1016/j.scienta.2022.111178.
Marasek-Ciolakowska A., Ramanna M. S., Arens P., Van Tuyl J. M. Breeding and cytogenetics in the genus Tulipa. Floricult. Ornam. Biotechnol. 2012. Vol. 6. P. 90-97.
Mizuochi H., Marasek A., Okazaki K. Molecular cloning of Tulipa fosteriana rDNA and subsequent FISH analysis yields cytogenetic organization of 5S rDNA and 45S rDNA in T. gesneriana and T. fosteriana. Euphytica. 2007. Vol. 155. P. 235-248. doi: 10.1007/s10681-006-9325-y.
Nikitina E. V., Karimov F. I., Savina N. V., Kubrak S. V., Kilchevsky A. V. Inventory of some Tulipa species from Uzbekistan using DNA barcoding. In BIO Web of Conferences. 2021. Vol. 38. P. 00086. EDP Sciences. doi: 10.1051/bioconf/20213800086.
Okonechnikov K., Golosova O., Fursov M., Ugene Team. Unipro UGENE: a unified bioinformatics toolkit. Bioinf. 2012. Vol. 28(8). P. 1166-1167. doi: 10.1093/bioinformatics/bts091.
Onyshchenko V. A., Mosyakin S. L., Korotchenko I. A., Danylyk I. M. et al. IUCN Red List categories of vascular plant species of Ukrainian flora / ed. by V. A. Onyshchenko. Kyiv: FOP Huliaeva V. M. 2022. 198 p. [In Ukrainian]
Panchuk I. I., Volkov R. A. A practical course in molecular genetics. Chernivtsi: Ruta. 2007. 120 p. [In Ukrainian]
Peregrym M. M. Representation of bulb and bulbotuberiferous species of the natural flora of Ukraine in protected plant lists of different levels. Ukr. Botan. Journ. 2012. Vol. 69(6). P. 832-846 [In Ukrainian]
Porebski S., Bailey L. G., Baum B. R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep. 1997. Vol. 15(1). P. 8-15. doi: 10.1007/BF02772108.
Pourkhaloee A., Khosh-Khui M., Arens P., Salehi H. et al. Molecular analysis of genetic diversity, population structure, and phylogeny of wild and cultivated tulips (Tulipa L.) by genic microsatellites. Hort. Environ. Biotech. 2018. Vol. 59(6) P. 875-888. doi: 10.1007/s13580-018-0055-6.
POWO. Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew. Published on the Internet; http://www.plantsoftheworldonline.org (accessed December 05 2023).
Rusak O. O., Petrashchuk V. I., Panchuk I. I., Volkov R. A. Molecular organization of 5S rDNA in two Ukrainian populations of Sycamore (Acer pseudoplatanus). Visn. Ukr. Tov. Genet. Selek.. 2016. Vol. 14(2). P. 216-220. [In Ukrainian] doi: 10.7124/visnyk.utgis.14.2.691.
Saini A., Jawali N. Molecular evolution of 5S rDNA region in Vigna subgenus Ceratotropis and its phylogenetic implications. Plant Syst. Evol. 2009. Vol. 280. P. 187-206. doi: 10.1007/s00606-009-0178-4.
Stepanenko A., Chen G., Hoang P. T., Fuchs J., Schubert I., Borisjuk N., The ribosomal DNA loci of the ancient monocot Pistia stratiotes L. (Araceae) contain different variants of the 35S and 5S ribosomal RNA gene units. Front. Plant Sci. 2022. Vol. 13. P. 819750. doi: 10.3389/fpls.2022.819750.
Turktas M., Metin Ö. K., Baştuğ B., Ertuğrul F. et al. Molecular phylogenetic analysis of Tulipa (Liliaceae) based on noncoding plastid and nuclear DNA sequences with an emphasis on Turkey. Bot. J. Linn. Soc. 2013. Vol. 172(3). P. 270-279. doi: 10.1111/j.0024-4074.2004.00194.x.
Tynkevich Y. O., Bushyla K. D., Volkov R. A. Organization of the 5S rDNA intergenic spacer of Quercus rubra L. and its relationship to the Ukrainian Quercus species. Factors Experimental Evol. Organisms. 2020. Vol. 26. P. 125-131 [In Ukrainian] doi: 10.7124/FEEO.v26.1254.
Tynkevich Y. O., Kozub L. V., Volkov R. A. Organization and polymorphysm of 5S rDNA intergenic spacer of blackthorn (Prunus spinosa L.). Visn. Ukr. Tov. Genet. Selek.. 2021. Vol. 19(1-2). P. 40-46 [In Ukrainian] doi: 10.7124/visnyk.utgis.19.1-2.1439.
Tynkevich Y. O., Moysiyenko I. I., Volkov R. A. The use of the intergenic spacer region psbA-trnH of the chloroplast genome for the analysis of the taxonomic position and genetic polymorphism of the Ukrainian populations of Tulipa quercetorum Klokov et Zoz. Visn. Ukr. Tov. Genet. Selek.. 2022а. Vol. 20(1-2). P. 8-15. [In Ukrainian] doi: 10.7124/visnyk.utgis.20.1-2.1508.
Tynkevich Y. O., Shelyfist A. Y., Kozub L. V., Hemleben V., Panchuk I. I., Volkov R. A. 5S ribosomal DNA of genus Solanum: molecular organization, evolution, and taxonomy. Front. Plant Sci. 2022b. Vol. 13. P. 852406. doi: 10.3389/fpls.2022.852406.
Tynkevich Y. O., Valin M. O., Moysiyenko I. I., Panchuk I. I., Volkov R. A. 5S ribosomal DNA in the family Plumbaginaceae. Cytol. Genet. 2023. Vol. 57(6). P. 524-537. doi: 10.3103/S0095452723060099.
Tynkevich Y. O., Volkov R. A. 5S ribosomal DNA of distantly related Quercus species: molecular organization and taxonomic application. Cytol. Genet. 2019. Vol. 53. P. 459-466. doi: 10.3103/S0095452719060100.
Van Eijk, J. P., Van Raamsdonk, L. W. D., Eikelboom, W., Bino, R. J. Interspecific crosses between Tulipa gesneriana cultivars and wild Tulipa species: a survey. Sexual Plant Reproduction. 1991 Vol. 4, P. 1-5.
Vozárová R., Herklotz V., Kovařík A., Tynkevich Y. O., Volkov R. A., Ritz C. M., Lunerová J. Ancient origin of two 5S rDNA families dominating in the genus Rosa and their behavior in the Canina-type meiosis. Front. Plant Sci. 2021. Vol. 12. P. 643548. doi: 10.3389/fpls.2021.643548
Wang W., Zhang X., Garcia S., Leitch A.R., Kovařík A. Intragenomic rDNA variation - the product of concerted evolution, mutation, or something in between? Hered. 2023. P. 1-10. doi: 10.1038/s41437-023-00634-5.
WFO World Flora Online, 2023, Available from: http://www.worldfloraonline.org/ (accessed 17 November 2023).
Wilson B. Tulipa: the taxonomy and evolutionary history of the genus and its impact on conservation priorities in Central Asia. Doctoral dissertation, University of Cambridge. 2023. 258 p. doi: 10.17863/CAM.94432.