TSitologiya i Genetika 2022, vol. 56, no. 6, 19-30
Cytology and Genetics 2022, vol. 56, no. 6, 494–503, doi: https://www.doi.org/10.3103/S0095452722060111

Organization of the 5S rDNA intergenic spacer and its use in molecular taxonomy of the genus Aconitum L.

Tynkevich Y.O., Novikov A.V., Chorney I.I., Volkov R.А.

  1. Yuriy Fedkovych Chernivtsi National University Kotsiubynsky str. 2, 58012 Chernivtsi, Ukraine
  2. State Museum of Natural History, National Academy of Sciences of Ukraine, Teatralna str. 18, 79008 Lviv, Ukraine

SUMMARY. The genus Aconitum L. includes a large number of toxic and pharmaceutical important plants. One of the centers of diversity of this genus is located on the territory of the Eastern Carpathians. In this region there are many representatives of the genus with unclear taxonomic status, in particular, members of the complex A. anthora s. l. The taxonomic position of this complex within the genus also remains controversial, as the regions of the chloroplast and nuclear genomes previously used for phylogenetic analysis appeared to be insufficiently variable. Therefore, the search for an optimal molecular marker with a high level of polymorphism within the genus Aconitum remains a relevant task. The 5S rDNA IGS (intergenic spacer) is an evolutionarily variable region of the nuclear genome, which was previously successfully used for phylogeny reconstruction in many groups of angiosperms. In this work, using methods of molecular genetics and bioinformatics, we obtained 5S rDNA IGS sequences for representatives of the A. anthora complex and phylogenetically distant species of the genus Aconitum. Analysis of IGS sequences showed that this region is relatively long in species of the genus, 574–619 bp. The IGS variability is due to numerous nucleotide substitutions, while short oligonucleotide indels occur only at the 5′ end of the spacer. Four conserved regions were found in the IGS of Aconitum, two of which correspond to the external promoter and terminator elements of RNA polymerase III, while the function of the other two regions remains unknown. First of them shows homology to the 5S rRNA coding region, while the second one demonstrates high similarity to the sequences from the genomes of representatives of taxonomically distant families of monocots and dicots, suggesting horizontal gene transfer. The phylogenetic analysis applying the 5S rDNA IGS supports the inter-pretation of A. anthora s. l. as a separate subgenus within the genus Aconitum.

Keywords: 5S rDNA intergenic spacer, horizontal gene transfer (HGT), molecular evolution and taxonomy, Aconitum, Ranunculaceae

TSitologiya i Genetika
2022, vol. 56, no. 6, 19-30

Current Issue
Cytology and Genetics
2022, vol. 56, no. 6, 494–503,
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References

Anisimova, M. and Gascuel, O., Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative, Syst. Biol., 2006, vol. 55, no. 4, pp. 539–552. https://doi.rg/10.1080/10635150600755453

Bartha, L., Mandáková, T., et al., Intact ribosomal DNA arrays of Potentilla-origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer, New Phytol., 2021, vol. 235, no. 3, pp. 1246–1259. https://doi.org/10.1111/nph.18171

Blöch, C., Weiss-Schneeweiss, H., et al., Molecular phylogenetic analyses of nuclear and plastid DNA sequences support dysploid and polyploid chromosome number changes and reticulate evolution in the diversification of Melampodium (Millerieae, Asteraceae), Mol. Phylogenet. Evol., 2009, vol. 53, no. 1, pp. 220–233. https://doi.org/10.1016/j.ympev.2009.02.021

Boron, P., Wróblewska, A., et al., Phylogeny of Aconitum subgenus Aconitum in Europe, Acta Soc. Bot. Pol., 2020, vol. 89, no. 3. https://doi.org/10.5586/asbp.8933

Bruni, I., De Mattia, F., et al., Identification of poisonous plants by DNA barcoding approach, Int. J. Legal Med., 2010, vol. 124, no. 6, pp. 595–603. https://doi.org/10.1007/s00414-010-0447-3

Cardoni, S., Piredda, R., et al., 5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species, Plant J., 2022, vol. 109, no. 4, pp. 909–926. https://doi.org/10.1111/tpj.15601

Carles, M., Cheung, M.K.L., et al., A DNA microarray for the authentication of toxic traditional Chinese medicinal plants, Planta Med., 2005, vol. 71, no. 6, pp. 580–584. https://doi.org/10.1055/s-2005-864166

Chen, G., Stepanenko, A., et al., Mosaic arrangement of the 5S rDNA in the aquatic plant Landoltia punctata (Lemnaceae), Front. Plant Sci., 2021, vol. 12, p. 678689. https://doi.org/10.3389/fpls.2021.678689

Cronn, R.C., Zhao, X., et al., Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons, J. Mol. Evol., 1996, vol. 42, no. 6, pp. 685–705. https://doi.org/10.1007/BF02338802

De Souza, T.B., Gaeta, M.L., et al., IGS sequences in Cestrum present AT-and GC-rich conserved domains, with strong regulatory potential for 5S rDNA, Mol. Biol. Rep., 2020, vol. 47, pp. 55–66. https://doi.org/10.1007/s11033-019-05104-y

Didukh, Y.P., Chervona knyha Ukrainy. Roslynnyi svit (Red Data Book of Ukraine. Plant Kingdom), Kyiv: Globalconsulting, 2009.

Douet, J. and Tourmente, S., Transcription of the 5S rRNA heterochromatic genes is epigenetically controlled in Arabidopsis thaliana and Xenopus laevis, Heredity, 2007, vol. 99, pp. 5–13. https://doi.org/10.1038/sj.hdy.6800964

Fehrer, J., Slavíkova, R., et al., Molecular evolution and organization of ribosomal DNA in the hawkweed tribe Hieraciinae (Cichorieae, Asteraceae), Front. Plant Sci., 2021, vol. 12, p. 647375. https://doi.org/10.3389/fpls.2021.647375

Fulnec̆ek, J., Lim, K.Y., et al., Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species, Heredity, 2002, vol. 88, no. 1, pp. 19–25. https://doi.org/10.1038/sj.hdy.6800001

Garcia, S., Wendel, J.F., et al., The utility of graph clustering of 5S ribosomal DNA homoeologs in plant allopolyploids, homoploid hybrids, and cryptic introgressants, Front. Plant Sci., 2020, vol. 11, p. 41.

Garrido-Cardenas, J.A., Mesa-Valle, C., et al., Trends in plant research using molecular markers, Planta, 2018, vol. 247, no. 3, pp. 543–557. https://doi.org/10.1007/s00425-017-2829-y

He, J., Wong, K.L., et al., Identification of the medicinal plants in Aconitum L. by DNA barcoding technique, Planta Med., 2010, vol. 76, no. 14, pp. 1622–1628. https://doi.org/10.1055/s-0029-1240967

Huff, M., Seaman, J., et al., A high-quality reference genome for Fraxinus pennsylvanica for ash species restoration and research, Mol. Ecol. Resour., 2022, vol. 22, no. 4, pp. 1284–1302. https://doi.org/10.1111/1755-0998.13545

Ishchenko, O.O., Panchuk, I.I., et al., Molecular organization of 5S ribosomal DNÀ of Deschampsia antarctica, Cytol. Genet., 2018, vol. 52, no. 6, pp. 416–421. https://doi.org/10.3103/S0095452718060105

Ishchenko, O.O., Bednarska, I.O., et al., Application of 5S ribosomal DNA for molecular taxonomy of subtribe Loliinae (Poaceae), Cytol. Genet., 2021, vol. 55, no. 1, pp. 13–22. https://doi.org/10.3103/S0095452721010096

Jabbour, F. and Renner, S.S., A phylogeny of Delphinieae (Ranunculaceae) shows that Aconitum is nested within Delphinium and that Late Miocene transitions to long life cycles in the Himalayas and Southwest China coincide with bursts in diversification, Mol. Phylogenet. Evol., 2012, vol. 62, no. 3, pp. 928–942. https://doi.org/10.1016/j.ympev.2011.12.005

Katoh, K., Rozewicki, J., et al., MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization, Briefings Bioinf., 2019, vol. 20, no. 4, pp. 1160–1166. https://doi.org/10.1093/bib/bbx108

Kim, Y., Yi, J.S., et al., The complete chloroplast genome of Aconitum coreanum (H. Lév.) Rapaics (Ranunculaceae), Mitochondrial DNA, Part B, 2019, vol. 4, no. 2, pp. 3404–3406. https://doi.org/10.1080/23802359.2019.1674213

Krak, K., Caklová, P., et al., Horizontally Acquired nrDNAs persist in low amounts in host Hordeum genomes and evolve independently of native nrDNA, Front. Plant Sci., 2021, vol. 12, p. 672879. https://doi.org/10.3389/fpls.2021.672879

Kumar, S., Stecher, G., et al., MEGA X: molecular evolutionary genetics analysis across computing platforms, Mol. Biol. Evol., 2018, vol. 35, no. 6, pp. 1547–1549. https://doi.org/10.1093/molbev/msy096

Kumari, K., Bhargava, S., et al., Molecular depiction of thirteen Indian toxic plants with ITS markers, Arab J. Forensic Sci. Forensic Med., 2020, vol. 2, no. 2, pp. 159–166. https://doi.org/10.26735/YGUY5295

Liangqian, L. and Kadota, Y., Aconitum L. Flora of China, Beijing: Science Press, 2001, pp. 149–222, vol. 6.

Luo, Y., Zhang, F.M., et al., Phylogeny of Aconitum subgenus Aconitum (Ranunculaceae) inferred from ITS sequences, Plant Syst. Evol., 2005, vol. 252, nos. 1–2, pp. 11–25. https://doi.org/10.1007/s00606-004-0257-5

Matyásek, R., Tate, J.A., et al., Concerted evolution of rDNA in recently formed Tragopogon allotetraploids is typically associated with an inverse correlation between gene copy number and expression, Genet, 2007, vol. 176, no. 4, pp. 2509–2519. https://doi.org/10.1534/genetics.107.072751

Mitka, J., Sutkowska, A., et al., Reticulate evolution of high-alpine Aconitum (Ranunculaceae) in the Eastern Carpathians (Central Europe), Acta Biol. Cracov., Ser. Bot., 2007, vol. 49, no. 2, pp. 15–26.

Mlinarec, J., Satovic, Z., et al., Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin, Ann. Bot., 2012, vol. 110, no. 3, pp. 703–712. https://doi.org/10.1111/boj.12452

Mlinarec, J., Franjevic, D., et al., Diverse evolutionary pathways shaped 5S rDNA of species of tribe Anemoneae (Ranunculaceae) and reveal phylogenetic signal, Bot. J. Linn. Soc., 2016, vol. 182, no. 1, pp. 80–99. https://doi.org/10.1111/boj.12452

Navrotska, D., Andreev, I., et al., Assessment of the molecular cytogenetic, morphometric and biochemical parameters of Deschampsia antarctica from its southern range limit in maritime Antarctic, Polish Polar Res., 2018, vol. 39, no. 4, pp. 525–548. https://doi.org/10.24425/118759

Novikoff, A.V. and Mitka, J., Taxonomy and ecology of the genus Aconitum L. in the Ukrainian Carpathians, Wulfenia, 2011, vol. 18, pp. 37–61.

Paštová, L., Belyayev, A., et al., Molecular cytogenetic characterisation of Elytrigia × mucronata, a natural hybrid of E. intermedia and E. repens (Triticeae, Poaceae), BMC Plant Biol., 2019, vol. 19, no. 1, p. 230. https://doi.org/10.1186/s12870-019-1806-y

Peška, V., Mandaková, T., et al., Comparative dissection of three giant genomes: Allium cepa, Allium sativum, and Allium ursinum, Int. J. Mol. Sci., 2019, vol. 20, no. 3, p. 733. https://doi.org/10.3390/ijms20030733

Porebski, S., Bailey, L.G., et al., Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components, Plant Mol. Biol. Rep., 1997, vol. 15, no. 1, pp. 8–15. https://doi.org/10.1007/BF02772108

Richard, P. and Manley, J.L., Transcription termination by nuclear RNA polymerases, Genes Dev., 2009, vol. 23, pp. 1247–1269. https://doi.org/10.1101/gad.1792809

Saini, A. and Jawali, N., Molecular evolution of 5S rDNA region in Vigna subgenus Ceratotropis and its phylogenetic implications, Plant Syst. Evol., 2009, vol. 280, no. 3, p. 187. https://doi.org/10.1007/s00606-009-0178-4

Simon, L., Rabanal, F.A., et al., Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana, Nucleic Acids Res., 2018, vol. 46, no. 6, pp. 3019–3033. https://doi.org/10.1093/nar/gky163

Stepanenko, A., Chen, G., et al., 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. https://doi.org/10.3389/fpls.2022.819750

Tynkevich, Y.O. and Volkov, R.A., Structural organization of 5S ribosomal DNA in Rosa rugosa, Cytol. Genet., 2014, vol. 48, no. 1, pp. 1–6. https://doi.org/10.3103/S0095452714010095

Tynkevich, Y.O. and Volkov, R.A., 5S ribosomal DNA of distantly related Quercus species: molecular organization and taxonomic application, Cytol. Genet., 2019, vol. 53, no. 6, pp. 459–466. https://doi.org/10.3103/S0095452719060100

Tynkevich, Y.O., Biliai, D.V., et al., Utility of the trnH-psbA region for DNA barcoding of Aconitum anthora L. and related taxa, Factors Exp. Evol. Org., 2022a, vol. 31, pp. 134–141.https://doi.org/10.7124/FEEO.v31.1450

Book

Tynkevich, Y.O., Shelyfist, A.Y., et al., 5S Ribosomal DNA of genus Solanum: molecular organization, evolution, and taxonomy, Front. Plant Sci., 2022b, vol. 13, p. 852406. https://doi.org/10.3389/fpls.2022.852406

Utelli, A.B., Roy, B.A., et al., Molecular and morphological analyses of European Aconitum species (Ranunculaceae), Plant Syst. Evol., 2000, vol. 224, no. 34, pp. 195–212. https://doi.org/10.1007/BF00986343

Volkov, R.A., Bachmair, A., et al., 25S-18S rDNA intergenic spacer of Nicotiana sylvestris (Solanaceae): Primary and secondary structure analysis, Plant Syst. Evol., 1999a, vol. 218, no. 1, pp. 89–97. https://doi.org/10.1007/BF01087037

Volkov, R.A., Borisjuk, N.V., et al., Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum, Mol. Biol. Evol., 1999b, vol. 16, no. 3, pp. 311–320. https://doi.org/10.1093/oxfordjournals.molbev.a026112

Volkov, R.A., Panchuk, I.I., et al., Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna, BMC Plant Biol., 2017, vol. 17, p. 21. https://doi.org/10.1186/s12870-017-0978-6

Vozárová, R., Herklotz, V., et al., 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. https://doi.org/10.3389/fpls.2021.643548

Wang, W., Liu, Y., et al., Gymnaconitum, a new genus of Ranunculaceae endemic to the Qinghai-Tibetan Plateau, Taxon, 2013, vol. 62, no. 4, pp. 713–722. https://doi.org/10.12705/624.10

Wendel, J.F., Schnabel, A., et al., Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium), PNAS, 1995, vol. 92, no. 1, pp. 280–284. https://doi.org/10.1073/pnas.92.1.280

Ye, J., McGinnis, S., et al., BLAST: improvements for better sequence analysis, Nucleic Acids Res., 2006, vol. 34, no. 2, pp. 6–9. https://doi.org/10.1093/nar/gkl164

Zhai, W., Duan, X., et al., Chloroplast genomic data provide new and robust insights into the phylogeny and evolution of the Ranunculaceae, Mol. Phylogenet. Evol., 2019, vol. 135, pp. 12–21. https://doi.org/10.1016/j.ympev.2019.02.024

Ziman, S.M. and Bulakh, O.V., Genus Aconitum L. (Ranunculaceae Juss.) within the flora of the Ukrainian Carpathians: comparative-morphological and taxonomic study, Sci. Herald Chernivtsi Univ., Biol. (Biol. Syst.), 2011, vol. 3, no. 2, pp. 142–149.