Commentary
Endopolyploidy, Genome Size, and Flow Cytometry THE
characterization of organisms by their genome size, or more generally, by the DNA content of their nuclei, has attracted considerable interest in recent years. Genome size has increasingly been treated as an important character in its own right with the potential to influence phenotype (1). Nuclear DNA content is also commonly estimated for reasons to which the genome size itself is secondary, such as when somatic tissue DNA content is used as an indicator of an organism’s cytotype (2) or when the distributions of DNA replication levels (“C-levels”) across tissues is of primary interest (3). Regardless of the motivation, all such applications depend on accurately attributing a particular DNA content to a particular chromosomal complement, most often the baseline somatic chromosome set (2C DNA content), the set contained within a meiotically reduced gamete (1C, “holoploid” DNA content) or the base chromosome number for the species (1Cx, “monoploid” DNA content) (4). Flow cytometry has become the predominant method for estimating nuclear DNA content in plants (5). The DNA content of a set of nuclei is estimated based on the relative fluorescence of these nuclei compared to those from a standard of known DNA content, after staining with a DNA-selective fluorochrome. The C-level of these nuclei is identified (e.g., 2C, 4C), and related measures, such as 1C and 1Cx, can then be determined based on simple division (1Cx requiring knowledge of the organisms cytotype). The second step, identifying the C-level of the nuclei, is clearly critical to the process, but in common practice, may appear so straightforward as to receive little attention: in a typical plant genome size or ploidy determination study, somatic tissue (usually leaf) is tested and the set of nuclei with the lowest relative fluorescence is identified as having 2C DNA content. For many plants, the fluorescence output for somatic nuclei will contain only one prominent nuclei peak, readily identified as 2C, but multiple prominent peaks may appear in endopolyploid species. Endopolyploidy is the presence of cells of more than one C-level or ploidy within a tissue or
Received 17 June 2015; Accepted 22 June 2015 *Correspondence to: Paul Kron, Department of Integrative Biology, 50 Stone Road East, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. E-mail: [email protected]
Cytometry Part A 87A: 887 889, 2015
organism, the result of DNA replication without cell division (3). This is a common phenomenon in plants, and the functional significance of endopolyploidy is an interesting topic in itself, with implications for physiology, ecology, and development (3). However, as demonstrated by Travnıcˇek et al. (in this issue, page 958), it can also be a source of technical problems and errors when estimating genome size (6). In endopolyploid species, the number of C-levels present and the proportions of nuclei in each are tissue- and environment-specific, and 2C nuclei do not necessarily predominate in somatic tissue (3,6). As Travnıcˇek et al.’s extensive study of the Orchidaceae makes clear, 2C nuclei may be entirely absent in some tissues for some species. In fact, in at least half of the orchid species Travnıcˇek et al. tested, there was at least one tissue type for which testing with a na€ıve, “lowest peak 5 2C” approach would result in the incorrect calculation of 1C. They also note that even when present, 2C peaks may be small enough to be overlooked, a problem that can be made worse when data quality is suboptimal, for example, when nuclei counts are low and/or debris levels are high (Fig. 1) (7). Clearly, when a set of measured nuclei in somatic tissue is misidentified as 2C, either because the true 2C peak is absent or overlooked, the result will be an incorrect genome size estimate, ploidy assignment, or endoreduplication index. Travnıcˇek et al. demonstrate a solution: the testing of reproductive tissues (ovaries or pollinaria in the case of orchids) to conclusively identify expected 1C and/ or 2C peak positions on fluorescence histograms. This approach works because these tissues can be relied upon to have either conspicuous numbers of 1C nuclei (pollinaria) or 2C nuclei (ovaries). Pollinaria have 1C nuclei because of the presence of 1N vegetative nuclei in the pollen. Ovaries always contain significant numbers of 2C nuclei because of the presence of megasporangia as well as surrounding somatic tissue, but lack 1C nuclei because unpollinated orchid flowers have pre-meiotic ovaries. For each tissue,
Published online 17 July 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22718 C 2015 International Society for Advancement of Cytometry V
Commentary
Figure 1. Fluorescence histograms (fluorescence area for propidium iodide stained nuclei) for somatic nuclei from a Galium species and an internal DNA content standard (Raphanus sativus). Both species are endopolyploid. 2C, 4C, and 8C peaks are apparent for Galium, and 2C and 4C for the standard (S2C, S4C). A: Mature leaf tissue with relatively few 2C nuclei and high debris, with 2C scarcely noticeable within the debris curve; (B) Young stem tissue with a prominent 2C peak.
the lowest fluorescence peak therefore can be confidently identified with respect to its C-level, and the relative fluorescence of these peaks can be used either to directly estimate genome size, or to guide the interpretation of histograms derived from other tissues. The recommendation to use reproductive tissues to identify 1C or 2C peaks comes with some caveats. Travnıcˇek et al. note that seasonality is an important restriction (i.e., the plant must be in flower), and they also observed difficulties with getting consistently good results with pollinaria. Also, under unusual circumstances, reproductive tissues may 888
contain nuclei with unexpected C-levels. Travnıcˇek et al. point out that in autogamous orchids, pollination could occur within buds, triggering meiosis and resulting in ovaries containing 1C nuclei (although this did not occur in two such orchids they tested). More broadly, it is possible in rare cases for plants to produce only unreduced pollen, that is, with no 1C nuclei (e.g., Ref. 8). Nevertheless, ovaries and pollinaria were used successfully to identify the 2C peak in all 48 orchid species tested, and this general approach should work for the great majority of endopolyploid plants. Travnıcˇek et al. have emphasized the Orchidaceae in this empirical study, but they note that the issues they raise apply to other groups as well. There are several plant families known to be predominantly endopolyploid, including economically important ones such as the Brassicaceae, Cucurbitaceae, Fabaceae, and Solanaceae (3). Extending Travnıcˇek et al.’s methodology beyond the Orchidaceae would not be difficult in many cases, because it is possible to acquire good quality pollen nuclei histograms across a wide range of angiosperm families (9). Mosses are also commonly endopolyploid (10), and while the green, haploid gametophyte tissue is the most accessible tissue for genome size estimation, diploid sporophyte tissue may be used to confirm C-level identification (Fig. 2). Nor is this issue restricted to plants, as endopolyploidy is not uncommon among animals (11). It would be an interesting exercise to search for outlier values of 1C and 1Cx in plant and animal families with high endopolyploidy rates, and to reexamine both somatic and reproductive tissue in outlier species with this source of error in mind. Travnıcˇek et al. also draw attention to the unusual phenomenon of progressively partial endoreplication (PPE) (12), in which only part of the genome is duplicated, so that DNA contents of endoreduplicated nuclei do not increase by factors of two. Species with PPE appear to be prone to the absence of 2C peaks in some tissues, but the authors also point out another possible source of error in working with PPE species. It is sometimes the case that when multiple peaks appear in a nuclei fluorescence histogram, some peaks are dismissed as probable contaminants because their fluorescence measures (and associated DNA contents) do not correspond to expected values relative to other peaks, that is, their DNA contents do not differ by factors of two (e.g., see Ref. 7). In species with PPE, this could result in the disregarding of true nuclei peaks. Although PPE appears to be rare, this is a potentially important observation. Travnıcˇek et al. (in this issue, page 958) have drawn attention to an important consideration in genome size studies using flow cytometry. Cautionary comments about the dangers of endopolyploidy for genome size estimation have been raised before (2,7), as has the idea of testing gamete nuclei to verify C-levels in endopolyploid plants (9) and animals (11). Travnıcˇek et al. move beyond these observations to provide a strong empirical study demonstrating both the potential for misidentifying peaks in endopolyploid species and the value of using reproductive tissue to avoid this. Endopolyploidy, Genome Size, and Flow Cytometry
Commentary
Figure 2. Propidium iodide stained nuclei from a moss’s haploid gametophyte (A,B) and diploid sporophyte (C,D) tissue (Plagiomnium cuspidatum). A,C: Show relative fluorescence area (585 nm) on a linear scale and (B,D) show relative fluorescence height (670 nm) on a log scale. Sporophyte tissue includes immature (pre-meiotic) capsules. Histograms have been gated for debris on a fluorescence area vs. side scatter plot.
Paul Kron* Department of Integrative Biology University of Guelph Guelph, Ontario, Canada
LITERATURE CITED 1. Greilhuber J, Leitch IJ. Genome size and the phenotype. In: Leitch IJ, Greilhuber J, Dolezel J, Wendel JF, editors. Plant Genome Diversity, Vol. 2: Physical Structure, Behaviour and Evolution of Plant Genomes. Vienna: Springer-Verlag; 2013. pp 323–344. 2. Suda J, Kron P, Husband BC, Travnıcˇek P. Flow cytometry and ploidy: Applications in plant systematics, ecology and evolutionary biology. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley–VCH Verlag GmbH & Co.; 2007. pp 103–130. 3. Barow M, Jovtchev G. Endopolyploidy in plants and its analysis by flow cytometry. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley–VCH Verlag GmbH & Co.; 2007. pp 349–372.
Cytometry Part A 87A: 887 889, 2015
4. Greilhuber J, Dolezel J, Lysak MA, Bennett MD. The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents. Ann Bot 2005;95:255–260. 5. Leitch IJ, Bennett MD. Genome size and its uses: The impact of flow cytometry. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometry with Plant Cells. Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley-VCH; 2007. pp 153–176. 6. Galbraith DW, Harkins KR, Knapp S. Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiol 1991;96:985–989. 7. Pellicer J, Leitch IJ. The application of flow cytometry for estimating genome size and ploidy level in plants. In: Besse P, editor. Molecular Plant Taxonomy: Methods and Protocols. New York: Springer Science 1 Business Media; 2014. pp 279–307. 8. Pichot C, El Ma^ataoui M. Unreduced diploid nuclei in Cupressus dupreziana A. Camus pollen. Theor Appl Genet 2000;101:574–579. 9. Kron P, Husband BC. Using flow cytometry to estimate pollen DNA content: Improved methodology and applications. Ann Bot 2012;110:1067–1078. 10. Bainard JD, Newmaster SG. Endopolyploidy in bryophytes:widespread in mosses and absent in liverworts. J Bot 2010:Article ID 316356. 11. Gregory TR. The C-value enigma in plants and animals: A review of parallels and an appeal for partnership. Ann Bot 2005;95:133–146. 12. Bory S, Catrice O, Brown S, Leitch IJ, Gigant R, Chiroleu F, Grisoni M, Duval MF, Besse P. Natural polyploidy in Vanilla planifolia (Orchidaceae). Genome 2008; 51:816–826.
889
Endopolyploidy, Genome Size, and Flow Cytometry THE
characterization of organisms by their genome size, or more generally, by the DNA content of their nuclei, has attracted considerable interest in recent years. Genome size has increasingly been treated as an important character in its own right with the potential to influence phenotype (1). Nuclear DNA content is also commonly estimated for reasons to which the genome size itself is secondary, such as when somatic tissue DNA content is used as an indicator of an organism’s cytotype (2) or when the distributions of DNA replication levels (“C-levels”) across tissues is of primary interest (3). Regardless of the motivation, all such applications depend on accurately attributing a particular DNA content to a particular chromosomal complement, most often the baseline somatic chromosome set (2C DNA content), the set contained within a meiotically reduced gamete (1C, “holoploid” DNA content) or the base chromosome number for the species (1Cx, “monoploid” DNA content) (4). Flow cytometry has become the predominant method for estimating nuclear DNA content in plants (5). The DNA content of a set of nuclei is estimated based on the relative fluorescence of these nuclei compared to those from a standard of known DNA content, after staining with a DNA-selective fluorochrome. The C-level of these nuclei is identified (e.g., 2C, 4C), and related measures, such as 1C and 1Cx, can then be determined based on simple division (1Cx requiring knowledge of the organisms cytotype). The second step, identifying the C-level of the nuclei, is clearly critical to the process, but in common practice, may appear so straightforward as to receive little attention: in a typical plant genome size or ploidy determination study, somatic tissue (usually leaf) is tested and the set of nuclei with the lowest relative fluorescence is identified as having 2C DNA content. For many plants, the fluorescence output for somatic nuclei will contain only one prominent nuclei peak, readily identified as 2C, but multiple prominent peaks may appear in endopolyploid species. Endopolyploidy is the presence of cells of more than one C-level or ploidy within a tissue or
Received 17 June 2015; Accepted 22 June 2015 *Correspondence to: Paul Kron, Department of Integrative Biology, 50 Stone Road East, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. E-mail: [email protected]
Cytometry Part A 87A: 887 889, 2015
organism, the result of DNA replication without cell division (3). This is a common phenomenon in plants, and the functional significance of endopolyploidy is an interesting topic in itself, with implications for physiology, ecology, and development (3). However, as demonstrated by Travnıcˇek et al. (in this issue, page 958), it can also be a source of technical problems and errors when estimating genome size (6). In endopolyploid species, the number of C-levels present and the proportions of nuclei in each are tissue- and environment-specific, and 2C nuclei do not necessarily predominate in somatic tissue (3,6). As Travnıcˇek et al.’s extensive study of the Orchidaceae makes clear, 2C nuclei may be entirely absent in some tissues for some species. In fact, in at least half of the orchid species Travnıcˇek et al. tested, there was at least one tissue type for which testing with a na€ıve, “lowest peak 5 2C” approach would result in the incorrect calculation of 1C. They also note that even when present, 2C peaks may be small enough to be overlooked, a problem that can be made worse when data quality is suboptimal, for example, when nuclei counts are low and/or debris levels are high (Fig. 1) (7). Clearly, when a set of measured nuclei in somatic tissue is misidentified as 2C, either because the true 2C peak is absent or overlooked, the result will be an incorrect genome size estimate, ploidy assignment, or endoreduplication index. Travnıcˇek et al. demonstrate a solution: the testing of reproductive tissues (ovaries or pollinaria in the case of orchids) to conclusively identify expected 1C and/ or 2C peak positions on fluorescence histograms. This approach works because these tissues can be relied upon to have either conspicuous numbers of 1C nuclei (pollinaria) or 2C nuclei (ovaries). Pollinaria have 1C nuclei because of the presence of 1N vegetative nuclei in the pollen. Ovaries always contain significant numbers of 2C nuclei because of the presence of megasporangia as well as surrounding somatic tissue, but lack 1C nuclei because unpollinated orchid flowers have pre-meiotic ovaries. For each tissue,
Published online 17 July 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22718 C 2015 International Society for Advancement of Cytometry V
Commentary
Figure 1. Fluorescence histograms (fluorescence area for propidium iodide stained nuclei) for somatic nuclei from a Galium species and an internal DNA content standard (Raphanus sativus). Both species are endopolyploid. 2C, 4C, and 8C peaks are apparent for Galium, and 2C and 4C for the standard (S2C, S4C). A: Mature leaf tissue with relatively few 2C nuclei and high debris, with 2C scarcely noticeable within the debris curve; (B) Young stem tissue with a prominent 2C peak.
the lowest fluorescence peak therefore can be confidently identified with respect to its C-level, and the relative fluorescence of these peaks can be used either to directly estimate genome size, or to guide the interpretation of histograms derived from other tissues. The recommendation to use reproductive tissues to identify 1C or 2C peaks comes with some caveats. Travnıcˇek et al. note that seasonality is an important restriction (i.e., the plant must be in flower), and they also observed difficulties with getting consistently good results with pollinaria. Also, under unusual circumstances, reproductive tissues may 888
contain nuclei with unexpected C-levels. Travnıcˇek et al. point out that in autogamous orchids, pollination could occur within buds, triggering meiosis and resulting in ovaries containing 1C nuclei (although this did not occur in two such orchids they tested). More broadly, it is possible in rare cases for plants to produce only unreduced pollen, that is, with no 1C nuclei (e.g., Ref. 8). Nevertheless, ovaries and pollinaria were used successfully to identify the 2C peak in all 48 orchid species tested, and this general approach should work for the great majority of endopolyploid plants. Travnıcˇek et al. have emphasized the Orchidaceae in this empirical study, but they note that the issues they raise apply to other groups as well. There are several plant families known to be predominantly endopolyploid, including economically important ones such as the Brassicaceae, Cucurbitaceae, Fabaceae, and Solanaceae (3). Extending Travnıcˇek et al.’s methodology beyond the Orchidaceae would not be difficult in many cases, because it is possible to acquire good quality pollen nuclei histograms across a wide range of angiosperm families (9). Mosses are also commonly endopolyploid (10), and while the green, haploid gametophyte tissue is the most accessible tissue for genome size estimation, diploid sporophyte tissue may be used to confirm C-level identification (Fig. 2). Nor is this issue restricted to plants, as endopolyploidy is not uncommon among animals (11). It would be an interesting exercise to search for outlier values of 1C and 1Cx in plant and animal families with high endopolyploidy rates, and to reexamine both somatic and reproductive tissue in outlier species with this source of error in mind. Travnıcˇek et al. also draw attention to the unusual phenomenon of progressively partial endoreplication (PPE) (12), in which only part of the genome is duplicated, so that DNA contents of endoreduplicated nuclei do not increase by factors of two. Species with PPE appear to be prone to the absence of 2C peaks in some tissues, but the authors also point out another possible source of error in working with PPE species. It is sometimes the case that when multiple peaks appear in a nuclei fluorescence histogram, some peaks are dismissed as probable contaminants because their fluorescence measures (and associated DNA contents) do not correspond to expected values relative to other peaks, that is, their DNA contents do not differ by factors of two (e.g., see Ref. 7). In species with PPE, this could result in the disregarding of true nuclei peaks. Although PPE appears to be rare, this is a potentially important observation. Travnıcˇek et al. (in this issue, page 958) have drawn attention to an important consideration in genome size studies using flow cytometry. Cautionary comments about the dangers of endopolyploidy for genome size estimation have been raised before (2,7), as has the idea of testing gamete nuclei to verify C-levels in endopolyploid plants (9) and animals (11). Travnıcˇek et al. move beyond these observations to provide a strong empirical study demonstrating both the potential for misidentifying peaks in endopolyploid species and the value of using reproductive tissue to avoid this. Endopolyploidy, Genome Size, and Flow Cytometry
Commentary
Figure 2. Propidium iodide stained nuclei from a moss’s haploid gametophyte (A,B) and diploid sporophyte (C,D) tissue (Plagiomnium cuspidatum). A,C: Show relative fluorescence area (585 nm) on a linear scale and (B,D) show relative fluorescence height (670 nm) on a log scale. Sporophyte tissue includes immature (pre-meiotic) capsules. Histograms have been gated for debris on a fluorescence area vs. side scatter plot.
Paul Kron* Department of Integrative Biology University of Guelph Guelph, Ontario, Canada
LITERATURE CITED 1. Greilhuber J, Leitch IJ. Genome size and the phenotype. In: Leitch IJ, Greilhuber J, Dolezel J, Wendel JF, editors. Plant Genome Diversity, Vol. 2: Physical Structure, Behaviour and Evolution of Plant Genomes. Vienna: Springer-Verlag; 2013. pp 323–344. 2. Suda J, Kron P, Husband BC, Travnıcˇek P. Flow cytometry and ploidy: Applications in plant systematics, ecology and evolutionary biology. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley–VCH Verlag GmbH & Co.; 2007. pp 103–130. 3. Barow M, Jovtchev G. Endopolyploidy in plants and its analysis by flow cytometry. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley–VCH Verlag GmbH & Co.; 2007. pp 349–372.
Cytometry Part A 87A: 887 889, 2015
4. Greilhuber J, Dolezel J, Lysak MA, Bennett MD. The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents. Ann Bot 2005;95:255–260. 5. Leitch IJ, Bennett MD. Genome size and its uses: The impact of flow cytometry. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometry with Plant Cells. Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley-VCH; 2007. pp 153–176. 6. Galbraith DW, Harkins KR, Knapp S. Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiol 1991;96:985–989. 7. Pellicer J, Leitch IJ. The application of flow cytometry for estimating genome size and ploidy level in plants. In: Besse P, editor. Molecular Plant Taxonomy: Methods and Protocols. New York: Springer Science 1 Business Media; 2014. pp 279–307. 8. Pichot C, El Ma^ataoui M. Unreduced diploid nuclei in Cupressus dupreziana A. Camus pollen. Theor Appl Genet 2000;101:574–579. 9. Kron P, Husband BC. Using flow cytometry to estimate pollen DNA content: Improved methodology and applications. Ann Bot 2012;110:1067–1078. 10. Bainard JD, Newmaster SG. Endopolyploidy in bryophytes:widespread in mosses and absent in liverworts. J Bot 2010:Article ID 316356. 11. Gregory TR. The C-value enigma in plants and animals: A review of parallels and an appeal for partnership. Ann Bot 2005;95:133–146. 12. Bory S, Catrice O, Brown S, Leitch IJ, Gigant R, Chiroleu F, Grisoni M, Duval MF, Besse P. Natural polyploidy in Vanilla planifolia (Orchidaceae). Genome 2008; 51:816–826.
889
