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Plant Genome Size Estimation by Flow Cytometry

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Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genome size in plants. In contrast to general expectations, results obtained in different laboratories showed some striking discrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care was taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C=approx. 0.3–30 pg DNA). Each of the four laboratories involved in this study used a different buffer and/or procedure for nuclei isolation. Two laboratories used arc lamp-based instruments while the other two used laser-based instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but statistically significant, differences were found between data obtained with individual instruments. Differences between the same type of instruments were negligible, while larger differences were observed between lamp- and laser-based instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flow cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, the study confirmed an urgent need for an agreement on standards. Given the small but systematic differences between different types of flow cytometers, analysis of very small differences in genome size should be made in the same laboratory and using the same instrument.
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  Annals of Botany  82  (Supplement A): 17–26, 1998Article No. bo980730 Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison* J. DOLEZ       EL †‡ , J. GREILHUBER § , S. LUCRETTI  , A. MEISTER ¶ , M. A. LYSA     K † **, L. NARDI  ,and R. OBERMAYER §† De Montfort Uni   ersity Norman Borlaug Centre for Plant Science, Institute of Experimental Botany, Olomouc,Czech Republic,  § Institute of Botany, Uni   ersity of Vienna, Vienna, Austria,   ENEA, Casaccia Research Centre,INN Department, Plant Biotechnology Di   ision, Rome, Italy,  ¶ Institute of Plant Genetics and Crop PlantResearch, Gatersleben, Germany and **Department of Botany, Palacky   Uni   ersity, Olomouc, Czech Republic Received: 4 November 1997 Returned for revision: 9 April 1998 Accepted: 8 May 1998 Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genomesize in plants. In contrast to general expectations, results obtained in different laboratories showed some strikingdiscrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care wastaken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C  approx. 0  3–30 pg DNA). Each of the four laboratories involved in this study used a different buffer and  orprocedurefornucleiisolation.Twolaboratoriesusedarclamp-basedinstrumentswhiletheothertwousedlaser-basedinstruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well withthose obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (bindingpreferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, butstatistically significant, differences were found between data obtained with individual instruments. Differencesbetween the same type of instruments were negligible, while larger differences were observed between lamp- and laser-based instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained bylamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flowcytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, thestudy confirmed an urgent need for an agreement on standards. Given the small but systematic differences betweendifferent types of flow cytometers, analysis of very small differences in genome size should be made in the samelaboratory and using the same instrument.   1998 Annals of Botany Company Key words:  DAPI, Feulgen densitometry, flow cytometry, plant genome size, nuclear DNA content, propidiumiodide, standardization. INTRODUCTIONMost of the genetic information of an organism is coded byDNA localized in cell nuclei. However, it has been notedthat most or all eukaryotic organisms contain more DNAthan is needed for coding and regulatory sequences, andthat the quantity of DNA in a nucleus (genome size) is notcorrelated with organismic complexity. This inconsistencyhas been called the ‘C-value paradox’ (Thomas, 1971). Ithas been suggested that the DNA amount itself hasphenotypic effects via its influence on cell size and mitoticcycle time; these effects have been termed ‘nucleotypic’, theterm ‘nucleotype’ denoting the physico-mechanical proper-ties of the nucleus (Bennett, 1972). The best knownnucleotypic effect is that of genome size on life-cycle time inherbaceous angiosperms, where annuals, and in particularephemerals,havesmallergenomesthanperennials(Bennett, * Some results of this study were presented in a poster session dur-ing the Angiosperm Genome Size Workshop and Discussion Meeting,Jodrell Laboratory, Royal Botanic Gardens, Kew, 1997. ‡  For correspondence at: Institute of Experimental Botany, Lab-oratory of Molecular Cytogenetics and Cytometry, Sokolovska     6, CZ-77200 Olomouc, Czech Republic. E-mail: dolezel  risc.upol.cz 1972). Although the genome size of only about 1% of theworldflorahasbeeninvestigatedsofar(BennettandLeitch,1997), the general correlation of genome size with cell sizeand cell cycle time indicates a pivotal role of genome size inmany aspects of plant evolution and adaptation (Bennett,1972, 1987; Price and Bachmann, 1976; Grime andMowforth, 1982; Jasienski and Bazzaz, 1995; Bharathan,1996).Within angiosperms, genome size varies between speciesat least up to about 800-fold ( Fritillaria assyriaca , 1C  127  4 pg,   s .  Arabidopsis thaliana , 1C  0  165 pg; Bennettand Smith, 1976; Bennett and Leitch, 1997). However,estimates for the same species are sometimes surprisinglydivergent (see Bennett and Smith, 1976, 1991; Bennett,Smith and Heslop-Harrison, 1991; Bennett and Leitch,1995, 1997). The existence and extent of this intraspecificvariation in genome size is a particularly difficult andcontroversial field and is currently receiving much attention(Greilhuber and Ebert, 1994; Bennett and Leitch, 1995;Baranyi and Greilhuber, 1996; Greilhuber and Obermayer,1997). Naturally, the extent of variation ought to be muchsmaller within a species than between species, if it exists atall.Nevertheless,remarkableintraspecificvariationhasbeen0305-7364  98  0A0017  10 $30.00  0   1998 Annals of Botany Company  18  Dolez   el   et al.—  Estimation of Plant Genome Size using Flow Cytometry T   1.  Excitation light source and optical filter set up of flow cytometers in indi   idual laboratories Excitation wavelengths or filters Emission filtersLaboratory Light source Propidium iodide DAPI Propidium iodide DAPIL1 Argon ion laser 514 nm 351  1–363  8 nm 630 nm BP 400 nm LP5W 500 mW 100 mWL2 Mercury arc lamp 535 nm BP — 590 nm LP — HBO 100W  2L3 Mercury arc lamp 535 nm BP UG1 590 nm LP 435 nm LPHBO 100W  2L4 Argon ion laser 514 nm — 630 nm BP — 5W 200 mWBP, Band pass; LP, long pass. reported many times from the late sixties to the present day(Miksche, 1968; Laurie and Bennett, 1985; Rayburn  et al  .,1985, 1997; Graham, Nickell and Rayburn, 1994; to quoteonly the earliest and some more recent references). Certainresults even seem to support the concepts of a ‘plasticgenome’, i.e. quantitative changes in genomic DNA of anorganism in response to environmental or developmentalstimuli (e.g. Evans, Durrant and Rees, 1966; Price andJohnston, 1996).Incontrasttointerspecificcomparisonswhengenomesizedifferences are not unexpected, technical variation becomesa much more obvious problem when analysing intraspecificvariation. Clearly, rapid but at the same time reliablemethods for genome size estimation are needed. At present,thetwomostwidelyusedmethodsareFeulgendensitometryand flow cytometry. The former has been by far the mostfrequently used method and is still providing the highestnumber of estimates (Bennett and Leitch, 1997). Flowcytometry is more convenient and rapid, and hence isbecoming increasingly popular. However, the simplicity of flow cytometry may be deceiving and may lead to thegeneration of flawed data. For instance, it has been shownthat fluorochromes which bind preferentially at AT- or GC-rich regions of DNA are not suitable for genome sizeestimation in plants (Michaelson  et al  ., 1991, Dolez      el,Sgorbati and Lucretti, 1992, Godelle  et al  ., 1993). However,some authors prefer to use DAPI as the fluorochrome forgenome size estimation and find propidium iodide (PI) lessreliable (e.g. Rayburn, Auger and McMurphy, 1992), afinding that contradicts the results mentioned above. Inaddition, attention should be paid also to the properfluorochrome concentration (Dolez      el, 1991).The choice and correct use of reference standards isanother critical factor which has been largely neglected;practically each major laboratory uses different standards(animal or plant). As a result, the quality of genome sizedata obtained using flow cytometry compared with Feulgendensitometrydoesnotgenerallyseemtohavebeenimproved(Bennett and Leitch, 1995). Estimates obtained by flowcytometry in the same species may vary up to 100% as inmaize (see Bennett and Leitch, 1997: Table 1). This isespecially concerning given that the technique has thepotential to detect differences in genome size as small as 1%(Lysa    k, Dolez      elova and Dolez      el, 1997).The aim of this work was to evaluate the extent of variation and to test the reproducibility of DNA flowcytometryforgenomesizeestimation.Weanalysedratiosof DNA content between nine plant species, ranging over 100-fold in DNA content. Based on previous experience, allspecies used in this study can be used as reference standardsfor estimation of nuclear genome size using flow cytometry.Furthermore, they are seed propagated and all of them maybe obtained as pure lines or cultivars.Four laboratories participated in this experiment. Twolaboratories used laser flow cytometers while the other twoused arc lamp flow cytometers. Because the aim of theexperimentwastoprovidethemostrealisticpictureofinter-laboratory variation, each of the laboratories used its ownprocedure for nuclei isolation and staining. In addition, wecompared data obtained by flow cytometry using twodifferent types of DNA fluorochromes with data obtainedon the same material by Feulgen densitometry.MATERIALS AND METHODS Plant material  The following plant species were used:  Allium cepa  ‘Alice’, Vicia faba  ‘Inovec’,  Secale cereale  ‘Dankovske’,  Hordeum  ulgare  ‘Ditta’,  Pisum sati   um  ‘Ctirad’,  Zea mays  line CE-777,  Glycine max  ‘Polanka’,  Raphanus sati   us  ‘Saxa’ and Arabidopsis thaliana  ‘Columbia’. With the exception of   A . thaliana , which was donated by Dr E. Chytilova     (MasarykUniversity, Brno) all seeds were received as certified seedlots from breeders responsible for maintenance breeding of respective cultivars. Seeds were sown in pots and plantswere grown in a glasshouse. Young healthy leaves of youngplantlets (2–4 weeks old) were used for sample preparation. Experimental design Four laboratories participated in the project (Table 1).Two laboratories used laser-based flow cytometers with a jet-in-air configuration (designated L1 and L4), and twolaboratories (L2 and L3) used mercury arc lamp-based flowcytometers with enclosed-stream design. All four labora-tories estimated relative DNA content using propidiumiodide (a DNA intercalator), two laboratories (one with a  Dolez   el   et al.—  Estimation of Plant Genome Size using Flow Cytometry  19 T   2.  Procedures used to prepare the samples for flow cytometric analyses Method of nucleiStainingLaboratory isolation Isolation buffer DAPI Propidium iodideL1 Chopping Mg  +  buffer 1  µ g ml −   50  µ g ml −  (Galbraith  et al  ., 1983)   50  µ g ml −   RNaseL2 Chopping LB01 buffer — 50  µ g ml −  (Dolez      el  et al  ., 1989)   50  µ g ml −   RNaseL3 Chopping Two step procedure 4  µ g ml −   50  µ g ml −  (Baranyi and Greilhuber, 1996)   150  µ g ml −   RNaseL4 Chopping LB01 buffer — 50  µ g ml −  (Dolez      el  et al  ., 1989)   50  µ g ml −   RNase laser-andonewithanarclamp-basedinstrument)estimatedDNA content using DAPI (binding preferentially to AT-rich regions of DNA). In addition, one laboratory (L3)estimated relative DNA content using Feulgen densito-metry.The ratio of nuclear DNA content was estimated for thefollowing pairs of species: (1)  V  .  faba : A .  cepa ; (2)  S  . cereale : V  .  faba ; (3)  H  .   ulgare : S  .  cereale ; (4)  P  .  sati   um : H  .  ulgare ; (5)  Z  .  mays : P  .  sati   um ; (6)  G .  max : Z  .  mays ; (7)  R . sati   us : G .  max ; (8)  A .  thaliana : R .  sati   us . For certaincomparisons, relative DNA content was calculated usingthe ratio of DNA content and assigning the DNA contentof   A .  cepa  to 100 arbitrary units. Ratios of DNA contentand relative DNA contents (in arbitrary units) obtainedafter flow cytometry and after Feulgen densitometry werecompared. Flow cytometric analysis Each laboratory used their preferred method for genomesize estimation (type of buffer, nuclei isolation and stainingprocedure; Table 2). Nuclei were isolated by choppingyoung leaf tissue from both plants of each species pairsimultaneouslyinisolation buffer.The sampleswerestainedeither with propidium iodide (PI) which intercalates intodouble-stranded DNA or 4,6  -diamidino-2-phenylindole(DAPI) which binds at AT-rich regions of DNA. Stainingwith PI involved treatment with RNase. Prior to analysis,each instrument was checked for linearity and aligned toachieve the highest resolution. In each sample, at least10000 nuclei were analysed and the ratio of G   peak meanswas calculated (see Fig. 1). Ten samples were analysed fromeach species pair, only two samples per day to avoid errorsdue to random instrument drift. Feulgen densitometry Root and shoot tips were fixed either in neutralformaldehyde or 3:1 fixative and stored in ethanol. Afterhydrolysis in 5    HCl at 20  C, samples were stained inFeulgen reagent, washed in SO  -water and squashed.Integrated extinction was determined with a scanningdensitometer at 570 nm using a square diaphragm (0  5  µ m)and step size of 0  5 or 1  0  µ m. Early prophase or telophasenuclei were measured in plants with large genomes ( Z  .  mays 20050B    N  u  m   b  e  r  o   f  n  u  c   l  e   i 400100250Relative nuclear DNA content G. max 1500300200100  Z. mays 0 A 800 G. max 600400200  Z. mays F  . 1. Histograms of relative DNA content obtained after analysis of nuclei isolated from young leaf tissues of   Glycine max  and  Zea mays .The nuclei were isolated and stained simultaneously with propidiumiodide (A) or DAPI (B). The ratio of DNA content was determined bydividing the G   peak mean of the smaller genome ( G .  max ) by the G  peak mean of the larger genome ( Z  .  mays ). Note that due topreferential binding of DAPI to AT-rich regions of DNA, the ratio of DNA content observed after DAPI staining (0  736) differs by 68%from that observed after staining with PI (0  438). to  A .  cepa ), metaphase or anaphase nuclei were measured inplants with small genomes ( A .  thaliana  to  G .  max ). Statistical analysis Data were analysed using the NCSS 6.0 (StatisticalSolutions, Ireland) and STATISTICA 4.01 (StatSoft Inc.,USA) statistical software.  20  Dolez   el   et al.—  Estimation of Plant Genome Size using Flow Cytometry RESULTSIn this work, each laboratory used a different procedure forsample preparation and analysed the samples using adifferent instrument. Nevertheless, analyses resulted in highresolution histograms of nuclear DNA content in alllaboratories (Fig. 1). Overall means of coefficients of variation of G   peaks determined in individual laboratoriesranged from 2  17 to 3  38% and from 2  77 to 4  15% for PI-and DAPI-stained samples, respectively (data not shown).Measurements were highly reproducible, resulting in verylow intra-laboratory variation in ratios of relative DNAcontent with very low standard deviations (Table 4). Comparison of flow cytometry and Feulgen densitometry Ratios of nuclear DNA content estimated for selectedpairs of species using Feulgen densitometry are listed inTable 3. Analysis of variance (ANOVA) demonstrated thatdifferences between the ratios obtained after fixation with3:1 fixative and neutral formaldehyde were not significant( P   0  05).Similarly,differencesbetweentheratiosobtainedwith roots and shoot were not significant ( P   0  05). Thusdata obtained with both fixatives and tissues were pooledand mean values were used in subsequent analyses. Ratiosof nuclear DNA content obtained with flow cytometry afterpropidium iodide staining are given in Table 4.T   3.  Ratios of nuclear DNA content estimated for pairs of species using Feulgen densitometry Ratio of relative DNA content (mean  s.d.)Tissue  fixation* V  .  faba  A .  cepaS  .  cereale  V  .  fabaH  .   ulgare  S  .  cerealeP  .  sati   um  H  .   ulgareZ  .  mays  P  .  sati   umG .  max  Z  .  maysR .  sati   us  G .  maxA .  thaliana  R .  sati   us Root  3:1 0  790  0  001 0  608  0  001 0  635  0  001 0  876  0  001 0  605  0  008 0  439  0  006 0  416  0  004 0  282  0  009Root  F 0  737  0  004 0  569  0  005 0  659  0  007 0  936  0  016 0  577  0  025 0  451  0  004 0  521  0  001 0  298  0  011Shoot  3:1 0  762  0  042 0  599  0  009 0  626  0  008 0  886  0  009 0  582  0  013 0  464  0  006 0  452  0  007 0  312  0  004Shoot  F 0  757  0  003 0  564  0  013 0  659  0  008 0  920  0  004 0  594  0  011 0  457  0  001 0  469  0  007 0  311  0  020Mean ratio 0  762  0  022 0  585  0  022 0  645  0  017 0  905  0  028 0  590  0  013 0  453  0  011 0  465  0  044 0  301  0  014* 3:1, Methanol:acetic acid; F, neutral formaldehyde. †  Eachmean represents a mean of two replications. In each replication,shoots or roots from three different seedlings were analysed. From eachseedling, ten nuclei were measured and the ratio of DNA content was calculated. T   4.  Ratios of nuclear DNA content estimated for pairs of species using flow cytometry after propidium iodide staining Ratio of relative DNA content (mean  s.d.)Laboratory V  .  faba  A .  cepaS  .  cereale  V  .  fabaH  .   ulgare  S  .  cerealeP  .  sati   um  H  .   ulgareZ  .  mays  P  .  sati   umG .  max  Z  .  maysR .  sati   us  G .  maxA .  thaliana  R .  sati   us L1 (laser) 0  778  0  007 0  613  0  006 0  647  0  004 0  874  0  009 0  639  0  021 0  469  0  031 0  506  0  006 0  310  0  003L2 (lamp) 0  776  0  010 0  595  0  005 0  638  0  005 0  863  0  007 0  609  0  008 0  441  0  007 0  462  0  008 0  300  0  002L3 (lamp) 0  752  0  016 0  586  0  008 0  632  0  007 0  879  0  004 0  586  0  003 0  438  0  005 0  465  0  009 0  313  0  011L4 (laser) 0  792  0  028 0  606  0  017 0  661  0  005 0  869  0  008 0  658  0  019 0  519  0  004 0  464  0  003 0  302  0  001Mean ratio 0  774  0  017 0  600  0  012 0  645  0  013 0  870  0  007 0  623  0  032 0  467  0  038 0  474  0  021 0  306  0  006Largest difference (%) 5  1 4  4 4  4 1  8 10  9 15  6 8  7 4  2Difference betweenlaser cytometers (%)1  8 1  1 2  1 0  6 2  9 9  6 8  3 2  6Difference betweenlamp cytometers (%)3  1 1  5 0  9 1  8 3  8 0  7 0  6 4  2 Linear regression analysis indicated a strong and stat-istically highly significant correlation between the ratios of DNA content estimated by Feulgen densitometry and byflow cytometry of PI-stained samples ( r  0  999,  n  8, P   0  0001; Fig. 2A). On the other hand, ratios estimatedbyflowcytometryofDAPI-stainedsamplesweredifferenttothose obtained after PI staining for most of species pairs(Table 7); the difference reached 58% in the case of  G .  max  Z  .  mays  (see also Fig. 1). Correlation betweenratios of DNA content estimated by flow cytometryof DAPI-stained samples and by Feulgen densitometry(Fig.2B)was notsignificantat P   0  025( r  0  769, n  8).In these calculations, we analysed overall means calculatedfor DNA content ratios obtained in all four or twolaboratories for PI-stained and DAPI-stained samples,respectively. The same results were obtained when theanalysis was performed for each laboratory separately (datanot shown).Ratios of DNA content determined by Feulgen densito-metry (Table 3) and by flow cytometry after PI staining(Table 4) were used to calculate relative DNA content of individual species. In these calculations, the relative DNAcontent of   A .  cepa  was arbitrarily set to 100 arbitraryunits (A.U.). Linear regression analysis between the relativeDNA content estimated by Feulgen densitometry andrelative DNA content estimated by flow cytometry of PI-stained samples (Fig. 3) showed a strong and
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