Abstract
Flow cytometry delivers a key value to detect differences between species and hybrids and to determine ploidy. In addition, DNA weight per nucleus appears to be related to various properties, such as the size of the cell and the nucleus, the duration of mitosis and meiosis and the generation time. The variation in DNA weight is also useful to analyze biodiversity, genome evolution and relationships between related taxa and recognizing ploidy-chimeras. Moreover, it is essential to know the size of a genome before one starts to determine the base sequences of DNA of a plant. To be able to measure the amount of DNA per nucleus, a standard that is genetically stable with constant genome size, easy to use and commonly available is recommended for flow cytometry. In this study, standards from selected perennial plants instead of annuals are recommended as these are more stable than individual seedlings and don’t need greenhouse facilities. Four different methods are used here to determine genome sizes or 2C-value (the amount of DNA per nucleus) for the standard values of perennial plants: 1. Eight selected standards are measured separately against four primary standards 2. Starting with the lowest two standards, each result is used cascade-wise as a standard for the next plant. 3/4. Combining six to nine standard plants in a single chopping event: 3. Calculating all standards against a single standard and 4. Calculate all plants cascade-wise. The last combination 3/4 was also used to compare the results from buffers of Bharathan, Galbraith, Otto and Doležel. The aim of this study was to present selected perennial plants to be useful in flow cytometry. Selected perennial plants have several advantages like permanent availability, surviving for weeks without watering and the absence of variation as is possible in seedlings of annuals. The obtained 2C-values of perennial plants, ranging from 2.7 to 70.5 pg, are presented as a valuable tool in the determination of genome sizes. Agave americana’Aureomarginata’ is presented as the preferred reference standard.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815. https://doi.org/10.1038/35048692
Bagwell CB, Baker D, Whetstoe S, Munson M, Hitchcox S, Ault K, Lovett EJ (1989) A simple and rapid method for determining the linearity of a flow cytometer amplification system. Cytometry 10:689–694. https://doi.org/10.1002/cyto.990100604
Baranyi M, Greilhuber J (1996) Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum. Theor Appl Genet 92:297–307. https://doi.org/10.1007/bf00223672
Bennett MD, Leitch IJ, Price HJ, Johnston S (2003) Comparisons with Caenorhapditis (∼100Mb) and Drosophila (∼175Mb) using Flow Cytometry show genome size in Arabidopsis to be ∼157 Mb and thus 25% larger that the Arabidopsis Genome Initiative estimate of ∼125 Mb. Ann Bot (Oxford) 91:547–557. https://doi.org/10.1093/aob/mcg057
Bharathan G, Lambert G, Galbraith DW (1994) Nuclear DNA contents of monocotyledons and related taxa. Amer J Bot 81:381–386. https://doi.org/10.1002/j.1537-219719994.tb15475.x
de Groot JJ, Tojibaev KS (2019) Tulipa zonneveldii (Liliaceae), A new species from the eastern Chatkal Mountains. Kyrgyzstan. Int Rock Gard 93:20–26
de Groot JJ, Zonneveld BJM (2020) Two new tulip species from the Altai Mountains. Kazakhstan. Int Rock Gard 95:18–23
de Laat AMM, Göhde W, Vogelzang MJDC (1987) Determination of ploidy of single plants and plant populations by flow cytometry. Pl Breed 99:303–307. https://doi.org/10.1111/j.1439-0523.1987.tb01186.x
Doležel J, Bartoš J (2005) Plant DNA Flow Cytometry and Estimation of Nuclear Genome Size. Ann Bot (Oxford) 95:99–110. https://doi.org/10.1093/aob/mci005
Doležel J, Bartoš J, Voglmayer H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry 51:127–128. https://doi.org/10.1002/cyto.a.10013
Doležel J, Binarová P, Lucretti S (1989) Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Pl 31:113–120. https://doi.org/10.1007/BF02907241
Doležel J, Greilhuber J (2010) Nuclear genome size: are we getting closer? Cytometry 77:635–642. https://doi.org/10.1002/cyto.a.20915
Doležel J, Greilhuber J, Suda J (2007) Estimation of Nuclear DNA content in plants using flow cytometry. Nat Protoc 2–9:2233–2244. https://doi.org/10.1038/nprot.2007.310
Doležel J, Sgorbati S, Lucretti S (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Pl 85:625–631. https://doi.org/10.1111/j.1399-3054.1992.tb04764.x
Doležel J, Čížková J, Šimková J, Bartoš J (2018) One Major Challenge of sequencing Large Plant genomes is to know how big they really are. Int J Molec Sci 19:3554. https://doi.org/10.3390/ijms19113554
Doležel J, Greilhuber J, Lucretti S, Meister A, Lysák MA, Nardi L, Obermayer R (1998) Plant genome size estimation by flow cytometry: inter-laboratory comparison. Ann Bot (Oxford) 82 (Suppl A): 17–26. https://doi.org/10.1006/anbo.19998.0794
Drenckhahn D, Baumgartner W, Zonneveld BJM (2017) Different genome sizes of Western and Eastern Ficaria verna lineages shed light on steps of Ficaria evolution. Forum Geobot 7:27–33. https://doi.org/10.3264/FG.2017.0xx
Loureiro J, Rodriguez E, Santos C, Doležel J, Suda J (2008) FLOWer: A plant DNA flow cytometry database (release 1.0, May 2008). Available at: https://botany.natur.cuni.cz/flower/index.php
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051. https://doi.org/10.1126/science.220.4601.1049
Garnatje T, Canela MÁ, Garcia S, Hidalgo O, Pellicer J, Sánchez-Jiménez I, Siljak-Yakovlev S, Vitales D, Vallès J. (2011) GSAD: a genome size database in the Asteraceae. Cytometry 79A:401–404. https://doi.org/10.1002/cyto.a.21056
Gerard M , Michez D, Fournier D, Maebek K, Smagghe G, Biesmeijer JC , De Meulemeester T. (2015) Discrimination of haploid and diploid males of Bombus terrestris (Hymenoptera; Apidae) based on wing shape. Int Apidology 46:644–653
Gregory TR (2005) Animal Genome Size Database. Available at: http://www.genomesize.com
Greilhuber J (2005) Intraspecific variation in genome size in angiosperms: identifying its existence. Ann Bot (Oxford) 95:91–98. https://doi.org/10.1093/aob/mci004
Greilhuber J (2008) Cytochemistry and C-values: The less-well-known world of nuclear DNA amounts. Ann Bot (Oxford) 101:791–804. https://doi.org/10.1093/aob/mcn035
Greilhuber J, Doležel J, Lysa´k M, Bennett MD (2005) The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents. Ann Bot (Oxford) 95:255–260. https://doi.org/10.1093/aob/mci019
Hanson L, Brown RL, Boyd A, Johnson MAT, Bennett MD (2003) First nuclear DNA C-values for 28 Angiosperm genera. Ann Bot (Oxford) 91:31–38. https://doi.org/10.1093/aob/mcg005
Hulgenhof E, Weidhase RA, Schlegel R, Tewes A (1988) Flow cytometric determination of DNA content in isolated nuclei of cereals. Genome 30:565–569. https://doi.org/10.1139/g88-095
IHGSC (International Human Genome Sequencing Consortium) (2001) Initial sequencing and analysis of the human genome. Nature 409: 860–921. https://doi.org/10.1038/35057062
Johnston JS, Bennett MD, Rayburn AL, Galbraith DW, Price HJ (1999) Reference standards for determination of DNA content of plant nuclei. Amer J Bot 86:609–613. https://doi.org/10.2307/2656569
Leitch IJ, Johnston E, Pellicer J, Hidalgo O, Bennett MD (2019) Angiosperm DNA C-values database. Available at: https://cvalues.science.kew.org/
Matzk F, Meister A, Schubert I (2000) An efficient screen for reproductive pathways using mature seeds of monocots and dicots. Pl J 21:97–108. https://doi.org/10.1046/j.1365-313x.2000.00647.x
Otto FJ, Oldiges H, Göhde W, Jain VK (1981) Flow cytometric measurement of nuclear DNA content variations as a potential in vivo mutagenicity test. Cytometry 2: 189–191. https://doi.org/10.1002/cyto.990020311
Praça-Fontes MM, Carvalho CR, Clarindo WR, Cruz CD (2011) Revisiting the DNA C-values of the genome size-standards used in plant flow cytometry to choose the “best primary standards.” Pl Cell Rep 30:1183–1191. https://doi.org/10.1007/s00299-011-1026-x
Rice et al (2015) The Chromosome Counts Database (CCDB) - a community resource of plant chromosome numbers. New Phytol 206:19–26. https://doi.org/10.1111/nph.2015.206-4
Roux N, Toloza A, Radecki Z, Zapata-Arias FJ, Doležel J (2003) Rapid detection of aneuploidy in Musa using flow cytometry. Pl Cell Rep 21:483–490. https://doi.org/10.1007/s00299-002-0512-6
Suda J, Leitch IJ (2010) The quest for suitable reference standards in genome size research. Cytometry 77:717–720. https://doi.org/10.1002/cyto.a.20907
Tiersch TR, Chandler R, Wachtel SSM, Ellia S (1989) Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10:706–710. https://doi.org/10.1002/cyto.990100606
Vindeløv LL, Christensen IJ, Nissen NI (1983) Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. Cytometry 3:328–331. https://doi.org/10.1002/cyto.990030504
Zonneveld BJM (2001) Nuclear DNA content of all species of Helleborus (Ranunculaceae) discriminate between species and sectional divisions. Pl Syst Evol 229:125–130. https://doi.org/10.1007/s006060170022
Zonneveld BJM (2002) Genome size analysis of selected species of Aloe (Aloaceae) reveals the most primitive species and results in some new combinations. Bradleya 20:5–11. https://doi.org/10.25223/brad.n20.2002.a3
Zonneveld BJM (2003) Systematic value of nuclear DNA content in Agave L. and some related genera of the Agavaceae. Bradleya 21:121–125. https://doi.org/10.25223/brad.n21.2003.a17
Zonneveld BJM (2004) The systematic value of nuclear DNA content in Clivia. Herbertia 57:41–47
Zonneveld BJM (2004) De waarde van de hoeveelheid DNA per kern voor de naamgeving in Senecio 4: de overige succulente senecio’s. Succulenta 84:162–168
Zonneveld BJM (2004) De genoomgrootte in Hydrangea. In: van Gelderen CJ, van Gelderen DM. Het grote Hortensia boek. Terra Press, pp 245−251
Zonneveld BJM (2008) Systematic value of nuclear DNA content for all species of Narcissus L. (Amaryllidaceae). Pl Syst Evol 275:109–132. https://doi.org/10.1007/s00606-008-0015-1
Zonneveld BJM (2009) Systematic value of nuclear genome size for ‘“all”’ species of Tulipa L. (Liliaceae). Pl Syst Evol 281:217–245. https://doi.org/10.1007/s00606-008-0203-7
Zonneveld BJM (2012) Genome sizes of all 19 species of Araucaria are correlated with their geographical distribution. Pl Syst Evol 298:1249–1255. https://doi.org/10.1007/s06-012-0631-710.1007/s006
Zonneveld BJM (2012) Genome sizes for all genera of the Cycadales, compared with earlier cladistic analysis. Pl Biol 14–3:253–256. https://doi.org/10.1111/plb.2012.14
Zonneveld BJM (2013) Conifer genome sizes of 172 species covering 64 out of 67 genera, range from 8 to 72 picogram. Nordic J Bot 30:490–502. https://doi.org/10.1111/j.1756-1051.2012.01516.x
Zonneveld BJM (2019) DNA weights per nucleus (genome size) of more than 2350 species of the Flora of The Netherlands, of which 1370 are new to science, including the pattern of their DNA peaks. Forum Geobot 8: 24–78 (184 including supplement). https://doi.org/10.3264/FG-2019-1022
Zonneveld BJM, Duncan G (2003) Systematic value of nuclear DNA content in all species of Agapanthus. Pl Syst Evol 242:115–123. https://doi.org/10.1007/s00606-003-0038.6
Zonneveld BJM, Duncan G (2006) Genome sizes for the species of Nerine Herb. (Amaryllidaceae) and its evident correlation with growth cycle, leaf width and other morphological characters. Pl Syst Evol 257:251–260. https://doi.org/10.1007/s00606-005-0381-x
Zonneveld BJM, Grimshaw JM, Davis AP (2003) The systematic value of nuclear DNA content in Galanthus. Pl Syst Evol 241:89–102. https://doi.org/10.1007/s00606-003-0016-z
Zonneveld BJM, van Iren F (2001) Genome size and pollen viability as taxonomic criteria: application to the genus Hosta. Pl Biol 3:176–185. https://doi.org/10.1055/s-2001-12900
Zonneveld BJM, van Jaarsveld EJ (2005) Taxonomic implications of genome size for all species of the genus Gasteria Duval. (Aloaceae). Pl Syst Evol 251:217–227. https://doi.org/10.1007/soo606-004-0244-x
Zonneveld BJM, Leitch IJ, Bennett MD (2005) First nuclear DNA amounts in more than 300 Angiosperms. Ann Bot (Oxford) 96:229–244. https://doi.org/10.1093/aob/mci170
Zonneveld BJM, Lindstrom AJ (2016) Genome sizes for the genus Zamia L. (Cycadales: Zamiaceae) correspond with three biogeographic regions. Nordic J Bot 34:744–751. https://doi.org/10.1111/njb.01094,ISSN1756-1051
Zonneveld BJM, Pollock WI (2012) Flow cytometric analysis of somaclonal variation in lineages of Hosta sports detects polyploidy and aneuploidy chimeras. Pl Biol 30:490–512
Zonneveld BJM, Fritz GPJ (2010) Three species recognized in Chortolirion Berger (Asphodelaceae: Alooideae). Bradleya 28: 27−36. https://doi.org/10.25223/brad.n28.2010.a5
Zonneveld BJM, Pollock W (2018) Sporting rules in Hosta: A primer. The American Hosta Society, Oakbrook Terrace
Acknowledgements
This work is made possible through the contributions of Bertie−Joan van Heuven and Rob Langelaan by maintaining the device and watering my standard plants. H. Duistermaat is thanked for her efforts for making the laboratory facilities accessible in Corona times. I also like to thank the editors and the reviewers for their valuable suggestions.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of Interest
The author declares that there is no conflict of interest.
Additional information
Handling editor: Jorg Fuchs.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Information on Electronic Supplementary Material
Information on Electronic Supplementary Material
Online Resource 1. Comparing buffers, using 6 standard plants chopped together, arranged according the number of the experiment and calculated against Agave americana =15.9 pg in combination and against the lowest value in experiment in cascade.
Online Resource 2. Comparing buffers, using 6 standard plants chopped together, calculated as in Online resource 1, but arranged according to buffer type. Also Standard deviations and Averages are calculated.
Rights and permissions
About this article
Cite this article
Zonneveld, B.J.M. Selected perennial plants do provide convenient standards for the determination of genome sizes with flow cytometry. Plant Syst Evol 307, 28 (2021). https://doi.org/10.1007/s00606-021-01747-2
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00606-021-01747-2