Intragenomic polymorphism of the 35S rDNA internal transcribed spacers (ITS) in three species and three interspecific hybrids of Pulsatilla (Ranunculaceae)
UDC 582.675.1+575.174.015.3
Keywords:
interspecific hybridization, ITS1, nothospecies, polymorphic sites, sequencing
Abstract
Morphological criteria do not always allow to determine the hybrid origin of samples. The study of intragenomic polymorphism of either protein-coding or rRNA-coding genes provides a more accurate assessment. ITS regions of 35S rDNA are often used as one of such markers. In the presented work, we performed a comparative analysis of ITS1 in three hybrids (nothospecies) of the genus Pulsatilla: P. × intermedia, P. × spuria and P. × hackelii and their parental species P. patens, P. vernalis, P. pratensis from Leningrad Region (NW Russia). The ITS1-5.8S rDNA-ITS2 region was sequenced by Sanger’s sequencing method. On the chromatograms of the ITS sequences of the parental species there were on average, 1.7 (P. pratensis) to 3.3 (P. vernalis) polymorphic sites (PSs) – positions where two different nucleotides were detected. All three studied nothospecies exhibited more PSs – from 4.7 (P. × intermedia) to 9.5 (P. × hackelii). Study of intragenomic polymorphism of the transcribed spacer ITS1 by locus-specific NGS confirmed an obviously assumption that polymorphic sites detected by Sanger sequencing actually reflect the presence in the genomes of different rDNA variants (ribotypes) that were received by the plant from its ancestors, if the proportion of the unusual ribotype is about 20 % or more. Rarer ribotype variants can be detected only by NGS. Among the major ribotypes (ZOTU) of the studied nothospecies there were found any specific ZOTU, which were not detected in the parental species. The intragenomic polymorphism of the ITS1 region revealed by either Sanger sequencing or NGS in parental species may indicate earlier events of homoploid hybridization accompanying speciation within the genus.
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References
Aichele D., Schwegler H. W. 1957. Die Taxonomie der Gattung Pulsatilla. Feddes Repert. 60: 1–230.
Andreasen K., Baldwin B. G. 2003. Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae). Mol. Phylogenet. Evol. 29(3): 563–581.
Baumberger von H. 1971. Chromosomenzahlbestimmungen and Karyotypanelysen bei den Gattungen Anemone, Hepatica and Pulsatilla. Ber. Schweiz. Bot. Ges. 80: 17–95.
Bolger A. M., Lohse M., Usadel B. 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30(15): 2114–2120.
Chase M. W., Hills H. H. 1991. Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40(2): 215–220.
Dawe J. C., Murray D. F. 1979. In IOPB chromosome number reports LXIII. Taxon 28: 265–268.
Доронина А. Ю. Новые местонахождения редких видов сосудистых растений на Карельском перешейке (Ленинградская область и Санкт-Петербург) // Вестник СПбГУ. Серия 3. Биология, 2006. Т. 3. С. 34–40.
Edgar R. C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460–2461. DOI: 10.1093/bioinformatics/btq461
Edgar R. C. 2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10: 996–998. DOI: 10.1038/nmeth.2604
Edgar R. C. 2016. UNOISE2: Improved error-correction for Illumina 16S and ITS amplicon reads. BioRxiv. DOI: 10.1101/081257
Feliner G. N., Rosselló J. A. 2007. Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Mol. Phylogenet. Evol. 44(2): 911–919.
Hegi G., Weber H. 1975. Illustrierte Flora von Mittel-Europa. Bd. 3. Teil 3. Berlin: P. Parey. 356 s. [In German].
Huson D. H., Bryant D. 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 2: 254–267.
Hyvärinen E., Juslen A., Kemppainen E., Uddström A., Liukko U.-M. 2019. Suomen lajien uhanalaisuus ß Punainen kirja. In: The 2019 red list of Finnish species. Helsiniki: Ympäristöministeriö & Suomen impäristökeskus. 704 pp.
Гельтман Д. В. Pulsatilla pratensis; Pulsatilla vernalis // Красная книга Российской Федерации (растения и грибы). М.: Тов-во науч. изд. КМК, 2008. С. 483–485.
Гельтман Д. В. Pulsatilla patens; P. pratensis; P. vernalis // Красная книга Ленинградской области. Объекты растительного мира. СПб.: Марафон, 2018. С. 229–230, 762–764.
Grant V. 1981. Plant speciation. New York; Columbia university press. 564 pp.
Карташова Н. Н., Малахова Л. А., Козлова А. А., Дуброва Н. А. Числа хромосом у ряда полезных растений из природных популяций флоры Приобья // Биология и биофизика: Материалы итоговой науч. конф. (по законченным в 1973 г. темам). Томск: Изд-во Том. ун-та, 1974. C. 47–53.
Kotseruba V., Pistrick K., Blattner F. R., Kumke K., Weiss O., Rutten T., Fuchs J., Endo T., Nasuda S., Ghukasyan A., Houben A. 2010. The evolution of the hexaploid grass Zingeria kochii (Mez) Tzvel. (2n = 12) was accompanied by complex hybridization and uniparental loss of ribosomal DNA. Mol. Phylogenet. Evol. 56(1): 146–155.
Kovarik A., Dadejova M., Lim Y. K., Chase M. W., Clarkson J. J., Knapp S., Leitch A. R. 2008. Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics. Ann. Bot. 101(6): 815–823.
Красников А. А. 1991. Хромосомные числа некоторых видов сосудистых растений из Новосибирской области // Бот. журн., 1991. Т. 76, № 3. С. 476–479.
Krejová N. Hybridizace mezi Pulsatilla pratensis a P. patens? Skutenost nebo mýtus? Diplomová práce. Praha: Univerzita Karlova, Přírodovědecká fakulta, Katedra botaniky, 2014. 99 pp.
Kricsfalusy V. V. 2015. Taxonomy and phylogeny of Anemone patens L. sensu lato (Ranunculaceae): A critical review. Thaiszia J. Bot. 25: 153–164.
Kumar S., Stecher G., Li M., Knyaz C., Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 35: 1547–1549.
Liu J. S., Schardl C. L. 1994. A conserved sequence in internal transcribed spacer 1 of plant nuclear rRNA genes. Plant Mol. Biol. 26(2): 775–778.
Lopez-Alvarez D., López-Herranz M. L., Betekhtin A., Catalán P. 2012. A DNA barcoding method to discriminate between the model plant Brachypodium distachyon and its close relatives B. stacei and B. hybridum (Poaceae). PLoS ONE 7(12): 51058.
Lunerová J., Renny-Byfield S., Matyášek R., Leitch A., Kovařík A. 2017. Concerted evolution rapidly eliminates sequence variation in rDNA coding regions but not in intergenic spacers in Nicotiana tabacum allotetraploid. Plant Syst. Evol. 303: 1043–1060.
Matyášek R., Renny-Byfiel S., Fulneče J., Maca J., Grandbastien M. A., Nichols R., Leitch A., Kovařík A. 2012. Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids. BMC genomics 13(1): 1–12.
Noyes R. D. 2006. Intraspecific nuclear ribosomal DNA divergence and reticulation in sexual diploid Erigeron strigosus (Asteraceae). Amer. J. Bot. 93(3): 470–479.
Okonechnikov K., Golosova O., Fursov M., the UGENE team. 2012. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 28: 1166–1167. DOI: 10.1093/bioinformatics/bts091
Пунина E. O., Гриф В. Г. Кариосистематическое исследование видов и естественных гибридов рода Pulsatilla (Ranunculaceae) в Ленинградской области // Бот. журн., 1984. Т. 69, № 12. С. 1673–1678.
Punina E. O., Machs E. M., Krapivskaya E. E., Kim E. S., Mordak E. V., Myakoshina Y. A., Rodionov A. V. 2012. Interspecific hybridization in the genus Paeonia (Paeoniaceae): polymorphic sites in transcribed spacers of the 45S rRNA genes as indicators of natural and artificial peony hybrids. Russ. J. Genet. 48(7): 684–697.
Punina E. O., Machs E. M., Krapivskaya E. E., Rodionov A. V. 2017. Polymorphic sites in transcribed spacers of 35S rRNA genes as an indicator of origin of the Paeonia cultivars. Russ. J. Genetics. 53(2): 202–212.
Qiu T., Liu Z., Liu B. 2020. The effects of hybridization and genome doubling in plant evolution via allopolyploidy. Mol Biol. Rep. 47: 5549–5558. DOI: 10.1007/s11033-020-05597-y
Ridgway K. P., Duck J. M., Young J. P. W. 2003. Identification of roots from grass swards using PCR-RFLP and FFLP of the plastid trnL (UAA) intron. BMC Ecol. 3: 3–8. DOI: 10.1186/1472-6785-3-8
Rodionov A. V., Dobryakova K. S., Punina E. O. 2018. Polymorphic sites in ITS1-5.8 S rDNA-ITS2 region in hybridogenic genus × Elyhordeum and putative interspecific hybrids Elymus (Poaceae: Triticeae). Russ. J. Genet. 54(9): 1025–1039.
Rodionov A. V., Gnutikov A. A., Kotsinyan A. R., Kotseruba V. V., Nosov N. N., Punina E. O., Rayko M. P., Tyupa N. B., Kim E. S. 2017. ITS1-5.8 S rDNA-ITS2 sequence in 35S rRNA genes as marker for reconstruction of phylogeny of grasses (Poaceae family). Biol. Bull. Rev. 7(2): 85–102.
Rodionov A. V., Machs E. M., Mikhailova Y. V., Krainova L. M., Loskutov I. G., Amosova A. V., Muravenko O. V., Gnutikov A. A. 2020. Phenomenon of multiple mutations in the 35S rRNA genes of the C subgenome of polyploid Avena L. Rus. J. Genet. 56: 657–666.
Ronikier M., Costa A., Aguilar J. F., Feliner G. N., Küpfer P., Mirek Z. 2008. Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): Chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia. J. Biogeogr. 35: 1650–1664.
Sang T., Crawford D. J., Stuessy T. F. 1995. Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proc. Natl. Acad. Sci. USA 92(15): 6813–6817.
Soltis P. S., Soltis D. E. 2009. The role of hybridization in plant speciation. Annu. Rev. Plant Biol. 60: 561–588.
Sopova M., Sekovski Z. 1982. Chromosome atlas of some Macedonian angiosperms. III. God. Zborn. Biol. Fak. Univ. Kiril Metod. 35: 145–161.
Sramkó G., Laczkó L., Volkova P. A., Bateman R. M., Mlinarec J. 2019. Evolutionary history of the Pasque-flowers (Pulsatilla, Ranunculaceae): Molecular phylogenetics, systematics and rDNA evolution. Mol. Phylogenet. Evol. 135: 45–61. DOI: 10.1016/j.ympev.2019.02.015
Sun Y.-L., Park W.-G., Oh H.-K., Hong S.-K. 2014. Genetic diversity and hybridization of Pulsatilla tongkangensis based on the nrDNA ITS region sequence. Biologia 69: 24–31. DOI: 10.2478/s11756-013-0284-1
Szczecińska M., Łazarski G., Bilska K., Sawicki J. 2017. The complete plastid genome and nuclear genome markers provide molecular evidence for the hybrid origin of Pulsatilla × hackelii Pohl. Turk. J. Bot. 41(4): 329–337. DOI: 10.3906/bot-1610-28
Szczecińska M., Sawicki J. 2015. Genomic resources of three Pulsatilla species reveal evolutionary hotspots, species-specific sites and variable plastid structure in the family Ranunculaceae. Int. J. Mol. Sci. 16: 22258–22279.
Szczecińska M., Sramkó G., Wołosz K., Sawicki J. 2016. Genetic diversity and population structure of the rare and endangered plant species Pulsatilla patens (L.) Mill. in East Central Europe. PLoS ONE 11(3): e0151730. DOI: 10.1371/journal.pone.0151730
Цвелёв Н. Н. Pulsatilla L. // Флора Восточной Европы. Т. 10. СПб.: Мир и семья, 2001. С. 85–94.
Uotila P. 1980. Pulsatilla patens x vernalis Suomessa. Memoranda Soc. Fauna Fl. Fenn. 56: 111–117.
Valuyskikh O. E., Teteryuk L. V., Pylina Y. I., Sushentsov O. E., Martynenko N. A., Shadrin D. M. 2020. Phylogenetic relationships and status of taxa of Pulsatilla uralensis and P. patens s. str. (Ranunculaceae) in north-eastern European Russia. PhytoKeys 162: 113–130. DOI: 10.3897/phytokeys.162.53361
White T. J., Bruns T., Lee S., Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M. A. Innis, D. H. Gelfand, J. I. Sninsky, T. J. White (eds.). PCR Protocols: Guide to Methods and Applications. San Diego: Academic Press. Pp. 315–322.
Winterfeld G., Schneider J., Röser M. 2009. Allopolyploid origin of Mediterranean species in Helictotrichon (Poaceae) and its consequences for karyotype repatterning and homogenisation of rDNA repeat units. Syst. Biodivers. 7(3): 277–295.
Andreasen K., Baldwin B. G. 2003. Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae). Mol. Phylogenet. Evol. 29(3): 563–581.
Baumberger von H. 1971. Chromosomenzahlbestimmungen and Karyotypanelysen bei den Gattungen Anemone, Hepatica and Pulsatilla. Ber. Schweiz. Bot. Ges. 80: 17–95.
Bolger A. M., Lohse M., Usadel B. 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30(15): 2114–2120.
Chase M. W., Hills H. H. 1991. Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40(2): 215–220.
Dawe J. C., Murray D. F. 1979. In IOPB chromosome number reports LXIII. Taxon 28: 265–268.
Доронина А. Ю. Новые местонахождения редких видов сосудистых растений на Карельском перешейке (Ленинградская область и Санкт-Петербург) // Вестник СПбГУ. Серия 3. Биология, 2006. Т. 3. С. 34–40.
Edgar R. C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460–2461. DOI: 10.1093/bioinformatics/btq461
Edgar R. C. 2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10: 996–998. DOI: 10.1038/nmeth.2604
Edgar R. C. 2016. UNOISE2: Improved error-correction for Illumina 16S and ITS amplicon reads. BioRxiv. DOI: 10.1101/081257
Feliner G. N., Rosselló J. A. 2007. Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Mol. Phylogenet. Evol. 44(2): 911–919.
Hegi G., Weber H. 1975. Illustrierte Flora von Mittel-Europa. Bd. 3. Teil 3. Berlin: P. Parey. 356 s. [In German].
Huson D. H., Bryant D. 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 2: 254–267.
Hyvärinen E., Juslen A., Kemppainen E., Uddström A., Liukko U.-M. 2019. Suomen lajien uhanalaisuus ß Punainen kirja. In: The 2019 red list of Finnish species. Helsiniki: Ympäristöministeriö & Suomen impäristökeskus. 704 pp.
Гельтман Д. В. Pulsatilla pratensis; Pulsatilla vernalis // Красная книга Российской Федерации (растения и грибы). М.: Тов-во науч. изд. КМК, 2008. С. 483–485.
Гельтман Д. В. Pulsatilla patens; P. pratensis; P. vernalis // Красная книга Ленинградской области. Объекты растительного мира. СПб.: Марафон, 2018. С. 229–230, 762–764.
Grant V. 1981. Plant speciation. New York; Columbia university press. 564 pp.
Карташова Н. Н., Малахова Л. А., Козлова А. А., Дуброва Н. А. Числа хромосом у ряда полезных растений из природных популяций флоры Приобья // Биология и биофизика: Материалы итоговой науч. конф. (по законченным в 1973 г. темам). Томск: Изд-во Том. ун-та, 1974. C. 47–53.
Kotseruba V., Pistrick K., Blattner F. R., Kumke K., Weiss O., Rutten T., Fuchs J., Endo T., Nasuda S., Ghukasyan A., Houben A. 2010. The evolution of the hexaploid grass Zingeria kochii (Mez) Tzvel. (2n = 12) was accompanied by complex hybridization and uniparental loss of ribosomal DNA. Mol. Phylogenet. Evol. 56(1): 146–155.
Kovarik A., Dadejova M., Lim Y. K., Chase M. W., Clarkson J. J., Knapp S., Leitch A. R. 2008. Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics. Ann. Bot. 101(6): 815–823.
Красников А. А. 1991. Хромосомные числа некоторых видов сосудистых растений из Новосибирской области // Бот. журн., 1991. Т. 76, № 3. С. 476–479.
Krejová N. Hybridizace mezi Pulsatilla pratensis a P. patens? Skutenost nebo mýtus? Diplomová práce. Praha: Univerzita Karlova, Přírodovědecká fakulta, Katedra botaniky, 2014. 99 pp.
Kricsfalusy V. V. 2015. Taxonomy and phylogeny of Anemone patens L. sensu lato (Ranunculaceae): A critical review. Thaiszia J. Bot. 25: 153–164.
Kumar S., Stecher G., Li M., Knyaz C., Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 35: 1547–1549.
Liu J. S., Schardl C. L. 1994. A conserved sequence in internal transcribed spacer 1 of plant nuclear rRNA genes. Plant Mol. Biol. 26(2): 775–778.
Lopez-Alvarez D., López-Herranz M. L., Betekhtin A., Catalán P. 2012. A DNA barcoding method to discriminate between the model plant Brachypodium distachyon and its close relatives B. stacei and B. hybridum (Poaceae). PLoS ONE 7(12): 51058.
Lunerová J., Renny-Byfield S., Matyášek R., Leitch A., Kovařík A. 2017. Concerted evolution rapidly eliminates sequence variation in rDNA coding regions but not in intergenic spacers in Nicotiana tabacum allotetraploid. Plant Syst. Evol. 303: 1043–1060.
Matyášek R., Renny-Byfiel S., Fulneče J., Maca J., Grandbastien M. A., Nichols R., Leitch A., Kovařík A. 2012. Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids. BMC genomics 13(1): 1–12.
Noyes R. D. 2006. Intraspecific nuclear ribosomal DNA divergence and reticulation in sexual diploid Erigeron strigosus (Asteraceae). Amer. J. Bot. 93(3): 470–479.
Okonechnikov K., Golosova O., Fursov M., the UGENE team. 2012. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 28: 1166–1167. DOI: 10.1093/bioinformatics/bts091
Пунина E. O., Гриф В. Г. Кариосистематическое исследование видов и естественных гибридов рода Pulsatilla (Ranunculaceae) в Ленинградской области // Бот. журн., 1984. Т. 69, № 12. С. 1673–1678.
Punina E. O., Machs E. M., Krapivskaya E. E., Kim E. S., Mordak E. V., Myakoshina Y. A., Rodionov A. V. 2012. Interspecific hybridization in the genus Paeonia (Paeoniaceae): polymorphic sites in transcribed spacers of the 45S rRNA genes as indicators of natural and artificial peony hybrids. Russ. J. Genet. 48(7): 684–697.
Punina E. O., Machs E. M., Krapivskaya E. E., Rodionov A. V. 2017. Polymorphic sites in transcribed spacers of 35S rRNA genes as an indicator of origin of the Paeonia cultivars. Russ. J. Genetics. 53(2): 202–212.
Qiu T., Liu Z., Liu B. 2020. The effects of hybridization and genome doubling in plant evolution via allopolyploidy. Mol Biol. Rep. 47: 5549–5558. DOI: 10.1007/s11033-020-05597-y
Ridgway K. P., Duck J. M., Young J. P. W. 2003. Identification of roots from grass swards using PCR-RFLP and FFLP of the plastid trnL (UAA) intron. BMC Ecol. 3: 3–8. DOI: 10.1186/1472-6785-3-8
Rodionov A. V., Dobryakova K. S., Punina E. O. 2018. Polymorphic sites in ITS1-5.8 S rDNA-ITS2 region in hybridogenic genus × Elyhordeum and putative interspecific hybrids Elymus (Poaceae: Triticeae). Russ. J. Genet. 54(9): 1025–1039.
Rodionov A. V., Gnutikov A. A., Kotsinyan A. R., Kotseruba V. V., Nosov N. N., Punina E. O., Rayko M. P., Tyupa N. B., Kim E. S. 2017. ITS1-5.8 S rDNA-ITS2 sequence in 35S rRNA genes as marker for reconstruction of phylogeny of grasses (Poaceae family). Biol. Bull. Rev. 7(2): 85–102.
Rodionov A. V., Machs E. M., Mikhailova Y. V., Krainova L. M., Loskutov I. G., Amosova A. V., Muravenko O. V., Gnutikov A. A. 2020. Phenomenon of multiple mutations in the 35S rRNA genes of the C subgenome of polyploid Avena L. Rus. J. Genet. 56: 657–666.
Ronikier M., Costa A., Aguilar J. F., Feliner G. N., Küpfer P., Mirek Z. 2008. Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): Chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia. J. Biogeogr. 35: 1650–1664.
Sang T., Crawford D. J., Stuessy T. F. 1995. Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proc. Natl. Acad. Sci. USA 92(15): 6813–6817.
Soltis P. S., Soltis D. E. 2009. The role of hybridization in plant speciation. Annu. Rev. Plant Biol. 60: 561–588.
Sopova M., Sekovski Z. 1982. Chromosome atlas of some Macedonian angiosperms. III. God. Zborn. Biol. Fak. Univ. Kiril Metod. 35: 145–161.
Sramkó G., Laczkó L., Volkova P. A., Bateman R. M., Mlinarec J. 2019. Evolutionary history of the Pasque-flowers (Pulsatilla, Ranunculaceae): Molecular phylogenetics, systematics and rDNA evolution. Mol. Phylogenet. Evol. 135: 45–61. DOI: 10.1016/j.ympev.2019.02.015
Sun Y.-L., Park W.-G., Oh H.-K., Hong S.-K. 2014. Genetic diversity and hybridization of Pulsatilla tongkangensis based on the nrDNA ITS region sequence. Biologia 69: 24–31. DOI: 10.2478/s11756-013-0284-1
Szczecińska M., Łazarski G., Bilska K., Sawicki J. 2017. The complete plastid genome and nuclear genome markers provide molecular evidence for the hybrid origin of Pulsatilla × hackelii Pohl. Turk. J. Bot. 41(4): 329–337. DOI: 10.3906/bot-1610-28
Szczecińska M., Sawicki J. 2015. Genomic resources of three Pulsatilla species reveal evolutionary hotspots, species-specific sites and variable plastid structure in the family Ranunculaceae. Int. J. Mol. Sci. 16: 22258–22279.
Szczecińska M., Sramkó G., Wołosz K., Sawicki J. 2016. Genetic diversity and population structure of the rare and endangered plant species Pulsatilla patens (L.) Mill. in East Central Europe. PLoS ONE 11(3): e0151730. DOI: 10.1371/journal.pone.0151730
Цвелёв Н. Н. Pulsatilla L. // Флора Восточной Европы. Т. 10. СПб.: Мир и семья, 2001. С. 85–94.
Uotila P. 1980. Pulsatilla patens x vernalis Suomessa. Memoranda Soc. Fauna Fl. Fenn. 56: 111–117.
Valuyskikh O. E., Teteryuk L. V., Pylina Y. I., Sushentsov O. E., Martynenko N. A., Shadrin D. M. 2020. Phylogenetic relationships and status of taxa of Pulsatilla uralensis and P. patens s. str. (Ranunculaceae) in north-eastern European Russia. PhytoKeys 162: 113–130. DOI: 10.3897/phytokeys.162.53361
White T. J., Bruns T., Lee S., Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M. A. Innis, D. H. Gelfand, J. I. Sninsky, T. J. White (eds.). PCR Protocols: Guide to Methods and Applications. San Diego: Academic Press. Pp. 315–322.
Winterfeld G., Schneider J., Röser M. 2009. Allopolyploid origin of Mediterranean species in Helictotrichon (Poaceae) and its consequences for karyotype repatterning and homogenisation of rDNA repeat units. Syst. Biodivers. 7(3): 277–295.
How to Cite
Punina E. O., Mikhailova Y. V., Shneyer V. S., Krapivskaya E. E., Machs† E. M., Rodionov A. V. Intragenomic polymorphism of the 35S rDNA internal transcribed spacers (ITS) in three species and three interspecific hybrids of Pulsatilla (Ranunculaceae) // Turczaninowia, 1. Vol. 27, № 4. P. 67–85 DOI: 10.14258/turczaninowia.27.4.8. URL: http://turczaninowia.asu.ru/article/view/16626.
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- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
