Quantification of DNA Content in Freshwater Microalgae Using Flow Cytometry: a Modified Protocol for Selected Green Microalgae

Fottea 11(2): 317–328, 2011 317

Quantification of DNA content in freshwater microalgae using flow cytometry: a modified protocol for selected green microalgae

Petra Ma z a l o v á , Petra Ša r h a n o v á , Vl a d a n On d ř e j & Aloisie Po u l í č k ov á

Department of Botany, Faculty of Science, Palacký University Olomouc, Šlechtitelů 11, CZ–783 71 Olomouc, Czech Republic; e–mail: [email protected]

Abstract: The study of genome size variation in microalgae lags behind that of comparable research in higher plants and seaweeds. This situation is essentially caused by: (1) difficulties in obtaining sufficient biomass for experiments; (2) problems with protoplast isolation due to cell–wall heterogeneity and complexity; and (3) the absence of suitable standards for routine measurements. We propose a multi–step protocol that leads to the quantification of DNA content in desmids using flow cytometry. We present detailed culture conditions, the minimal biomass necessary for three repetitive measurements, a method to isolate protoplasts and selection of suitable standards. Our protocol, which is mainly based on studies with higher plants and commercially available enzyme mixtures, is useful in Streptophyta, especially members of the Zygnematophyceae, because of their close phylogenetic relationship to higher plants, in particular the similarity of their cell wall organization. Moreover, the suggested protocol also works for some Chlorophyta (Chloroidium ellipsoideum, Tetraselmis subcordiformis) and Heterokontophyta (Tribonema vulgare). We suggest and characterize a new standard for flow cytometry of microalgae (Micrasterias pinnatifida). Modification of the enzyme mixture is probably necessary for microalgae whose cell walls are surrounded by a mucilaginous envelope (Planktosphaeria), those that contain alganan (Chlorella), monads with a pellicle or chlamys (Euglena, Chlamydomonas). While we did not anticipate any success with diatoms (Pinnularia), because of their silica frustules, the enzyme mixture also failed for some other green microalgae (Xanthidium, Kentrosphaera, Stigeoclonium, Trentepohlia and Pseudendoclonium).

Keywords: DNA content, flow cytometry, desmids, microalgae, standards,Micrasterias pinnatifida

Introduction to apply existing genome size estimation methods to this group of microalgae in particular. Although data relating to genome size are extre- Several methods have been used to mely important in the assessment of phylogenetic quantify nuclear DNA in algae, primarily relationships in plants, there remain enormous Feulgen microdensitometry and similar gaps in the current genome size database. DNA microspectrophotometric methods (Ha r d i e et al. content have so far been determined for 7058 2002; Ka p r a u n 2007). Although flow cytometry species, comprising 6287 angiosperms, 204 (FC) is routinely used in higher plants, it has gymnosperms, 82 pteridophytes, 232 bryophytes rarely been used for microalgae (Le Ga l l et al. and 253 algae (Be n n e t t & Le i t c h 2010). Genome 1993; Si mo n et al. 1994; Ve l d h u i s et al. 1997). size estimates have been studied by Ka p r a u n Moreover these studies estimated nuclear DNA (2005, 2007), who reported values for almost content from whole cells, but recently it turned 400 species of red, green and brown macroscopic out that only estimates based on isolated nuclei algae. Unicellular freshwater microalgae have are accurate enough for general comparisons rarely been studied (Ka p r a u n 2007), but genome (Ka p r a u n 2007). size data can be used for testing the role of In contrast to higher plants and seaweeds, polyploidy in algal evolution and speciation studies on microalgae require isolation of single (Ma n n & Po u l í č k ov á 2010), particularly in cells and maintainence of unialgal cultures microalgal groups with sexual reproduction to obtain a reasonable amount of biomass for (Zygnematophyceae, Bacillariophyceae). Our experiments. As a result, research on microalgae research interests include cryptic diversity and is time consuming. In addition, we have found reproductive isolation in desmids and so we tried that methods for nuclei separation used in higher 318 Ma z a l o v á et al.: Flow cytometry in microalgae plants (chopping tissue with a razor blade; Materials and methods Ga l b r a i t h et al. 1983), do not work in our model microalgae, the desmids. Consequently, we tried Algal strains, media, culture conditions, cropping of grinding the material in a mortar, but this did not cells and LM observations. The algal material used in yield any positive results. Techniques for cell these experiments was obtained from clonal unialgal cultures. Algal strains were either our isolates or those wall removal using cell wall–degrading enzymes obtained from culture collections (Table 1). Our strains have previously been used in higher plants for (699, 723, 745, Pin1Cra) were isolated using standard intact plant cells and in the preparation of somatic methods (An d e r s e n 2005) from Irish or Scottish lakes. hybrids after protoplast fusion (Do l e ž e l et al. They are held in the culture collection of the Department 2007). This technique has also been used for of Botany, Palacký University in Olomouc. Cultures some seaweeds (Mi l l n e r et al. 1979; Bu t l e r et were maintained at 16 °C with 12:12 light:dark cycle, al. 1990). However, algae are a heterogeneous under cool–white fluorescent lights at an irradiation of group and exhibit a variety and complexity of 20 μmol.m–2.s–1. cell wall organization which contrasts with the Two basic liquid culture media were used: an relative homogeneity characteristic of higher oligotrophic medium for desmids used in the CAUP collection (OGM; Če r n á & Ne u s t u p a 2010) and plants. As in higher plants, the algal cell wall can Bold‘s Basal medium for other microalgae (BBM; sometimes be interpreted as a two phase system: Bold 1949). Euglena was cultured in soil extract a crystalline phase (the skeleton) embedded in a medium (An d e r s e n 2005) diluted 1:1 with distilled more amorphous phase (the matrix). However, water, Tetraselmis in ASW medium (McLa c h l a n 1964) the algal cell wall differs from land plants in the and Pinnularia in diatom medium WC with silicate abundance of matrix compounds compared to (Gu i l l a r d & Lo r e n z e n 1972). Storage cultures were the skeletal components (Bu t l e r et al. 1990). In kept in 50 mm Petri dishes. Subsequently, 2 weeks higher plants the three primary components of the before planned flow cytometric measurements, a rich cell wall are cellulose, hemicellulose and pectins, inoculum of each strain (ca 1 ml) was transfered to fresh and protoplasts can be isolated from virtually any medium in 100 mm Petri dishes and kept at a higher –2 –1 plant species using a combination of cellulases, irradiation (40 μmol.m .s ) with 16:8 light:dark cycle. Approximately 10 days after subculturing, the bottom hemicellulases and pectinases (Bu t l e r et al. 1990). of the Petri dish was conspicuously green and such an Because of the phylogenetic position of desmids amount of cells in exponential growth phase was used close to higher plants (Streptophyta), we decided for cytometric measurements. For three replacated to start with commercial preparations of enzymes DNA measurements of each algal strain, we used three available for the routine isolation of higher plant Petri dishes of material. Desmids were usually growing protoplasts. Propidium iodide was used as a on the bottom of the Petri dish. Therefore superfluous fluorescent dye which intercalates quantitatively medium was gently poured off and cells were carefully to the double–stranded DNA, therefore absolute scraped from the bottom using a bacteriological DNA amount can be quantified. squeegee and transfered into 15 ml centrifuge tubes and Commonly used higher plant standards centrifuged (1500 rpm for 5 min). Superfluous medium was again gently removed and the sedimented cells (Do l e ž e l et al. 1992), Pisum sativum, Lycopersicon were used for subsequent treatment. LM observations esculentum, Raphanus sativus and Zea mays have were done under a light Zeiss AxioImager microscope been tested and used for characterization of new and Zeiss Axiovert inverted microscope. desmid standard. Enzyme treatment. Conspicuously green cultures in the exponential phase of growth (10–15 days after In this study we aimed to develop a protocol inoculation) were concentrated by centrifugation so that for the quantification of nuclear DNA by flow the tip of a conical 15 ml tube was filled with biomass. cytometry suitable primarily for desmids. As the Then, 2 ml of the enzymatic mixture of 2% Cellulase method consists of several steps, we aimed to find Onozuka R–10 (Duchefa Biochemie, Netherlands), 1) an easy and fast culturing method to obtain a 0.5% Macerozyme R–10 (Duchefa Biochemie, Netherlands) dissolved in modified rinsing solution minimal biomass necessary for the experiments, 2) PGly (Table 2) was added (De b e a u j o n & Br a n c h a r d an easy way to isolate protoplasts and 3) a suitable 1992). Suspensions were kept for 16 h in the dark at standard for measurement of DNA content and/or 27 °C. After 16 h, suspensions were centrifuged (700 define a new microalgal standard. We also tried rpm for 5 min), the supernatant was poured off and to verify, by selective screening, whether our replaced by 1 ml of PGly to rinse the pellet. Suspensions protocol is useful for other microalgae. were recentrifuged (700 rpm for 5 min) and PGly was Fottea 11(2): 317–328, 2011 319

Fig. 1. Histograms of relative nuclear DNA content of Micrasterias pinnatifida (RALFS) and various standards: (1) Pisum sativum, (2) Zea mays, (3) Lycopersi- con esculentum, (4) Rapha- nus sativus. (G1) G1 phase of the standards, (G2) G2 phase of the standards, (P1) haploid stage of the sample, (P2) diploid stage of the sample. removed. The obtained biomass of cells was placed on the flow cytometer up to 3000 particles. The absolute ice before lysis buffer (flow cytometry) was added to nuclear DNA amount was calculated from a linear ratio protect the cells from regenerating cell walls and to between G1 peak position of the standard and the peak enhance release of the nuclei from the treated cells. of the sample according to Do l e ž e l & Ba r t o š (2005): (sample G1 peak mean / standard G1 peak mean) * Flow cytometry and DNA amount estimation. The standard 2C DNA content. absolute nuclear DNA amount of the studied algae All samples were measured at least twice, was estimated by FC using a ML CyFlow instrument usually three times. Some authors had shown that (Partec GmbH, Münster, Germany). Each sample was greater ploidy levels in one algal culture can occur analyzed separately to choose a proper standard of (Ha m a d a 1987; Ha i g 2010). Although we did not study different genome size, but still relatively close enough the number of chromosomes or ploidy level, in those to the sample to avoid nonlinearity of the instrument. cases where we obtained two peaks, we labelled the A second measurement of the studied samples was first lower ploidy peak as 1C and the second main peak done together with the internal standard. Raphanus as 2C in accordance with Gr e i l h u b e r et al. (2005). sativus cv. Saxa (2C = 1.11 pg, Do l e ž e l et al. 1992), Lycopersicon esculentum cv. Stupicke (2C = 1.96 pg, New microalgal standard identification. The vigo- Do l e ž e l et al. 1992), Zea mays, CE–777, (2C = 5.46 pg, rously growing desmid strain available in the Hamburg Su d a personal communication) or Pisum sativum cv. culture collection (Sammlung von Conjugaten– Ctirad (2C = 8.76 pg, Su d a personal communication) were used as standards. Leaf tissues of standards were Kulturen), Micrasterias pinnatifida (Kü t z i n g ) ex chopped with a razor blade in 1 ml of LB01 lysis buffer Ra l f s (SVCK 411; http://www.biologie.uni–hamburg. (15 mM TRIS; 2 mM EDTA; 0.5 mM spermine–4HCl; de/b–online/d44_1/44_1.htm) has been characterized 80 mM KCl; 20 mM NaCl; 0.1 % Triton X–100; as a first microalgal standard. 15mM 2–mercaptoethanol.; pH 8.0; Do l e ž e l et al. Micasterias pinnatifida strain was measured 1989). Then the algal solution was added to the seventeen times with different standards following the chopped standard, mixed and the suspension of nuclei method described above. As internal standards, Pisum was filtered through nylon mesh (42 μm) into a tube sativum was measured ten times, Zea mays four times, containing another 300 μl of lysis buffer. After 1 hour Lycopersycon esculentum twice and Raphanus sativus incubation on ice, the suspension was decanted to a new once. To cross–check genome sizes, standards were tube to remove the sediment and 50 μl of propidium also analysed in combination: a) P. sativum, Z. mays iodid (PI) was added. Measurements were done on and R. sativus and b) P. sativum and L. esculentum. 320 Ma z a l o v á et al.: Flow cytometry in microalgae

Table 1. Basic characteristics of the strains under study. Sources of the strains: (ASW) Algensammlung Wien, University of Viena, nowadays deposit in the Culture Collection of Algae at the University of Cologne (CCAC); (CAUP) Culture Collection of Algae, Charles University in Prague, Czech Republic; (M) Research Culture Collection Melkonian, University of Cologne, Germany; (NIES) Microbial Culture Collection, National Institute for Environmental Studies, Japan; (SAG) Sammlung von Algenkulturen Göttingen, University of Göttingen, Germany; (SVCK) Sammlung von Conjugaten–Kulturen (http://www.biologie.uni-hamburg.de/b-online/d44_1/44_1.htm). The strains not held by above mentioned collections are keeping in culture collection of the Department of Botany, Palacký University in Olomouc (Czech Republic).

Species Strain Origin/locality Medium

Zygnematophyceae Cosmarium contractum Ki r c h - M 3039 Doksy (CZ) OGM n e r

Euastrum verrucosum Ra l f s 699 Lough an Fhraoigh OGM (Connemara, IRL) Micrasterias pinnatifidaR a l f s SVCK 411 Laguna de Mucubaji, OGM Merida (YV) Micrasterias rotata Ra l f s SVCK 212 near Potsdam (D) OGM Micrasterias truncata var. NIES 783 Centenial Park (Sydney, OGM pusilla G.S. We s t AUS) Pleurotaenium ehrenbergii 745 vicinity of Upper Lake OGM (Ra l f s ) De Ba r y (Kerry, IRL) Staurastrum arctiscon (Ra l f s ) 723 Lough Eirk (Kerry, OGM Lu n d e l l IRL) Staurastrum orbiculare Ra l f s M 2217 vicinity of Vladivostok OGM (RU) Staurodesmus dickiei (Ra l f s ) ASW 07056 fen Rotmoos near Horn- OGM Li l l i e r o t h spitz (AUS) Triploceras gracile Ba i l e y SAG 24.82 Rotary Pond at Falmou- OGM th (MA, USA) Triploceras gracile Ba i l e y SVCK 366 Sumatra OGM

Xanthidium octocorne Ra l f s M3057 Pískovny Cep (CZ) OGM Zygnema circumcarinatum CAUP K meadow ditch near BBM Cz u r d a 402a Doksy (CZ) Zygnema cylindricum Tr a n - CAUP K 403 Doksy (CZ) BBM s e a u Klebsormidiophyceae Klebsormidium flaccidum CAUP J 302 Adršpach (CZ) BBM (Kü t z i n g ) Si l v a , Ma t t o x & Bl a c k w e l l Trebouxiophyceae Chloroidium ellipsoideum CAUP H on a tree bark, Adršpach BBM (Ge r n e c k ) Da r i e n k o et al. 1949 (CZ) Chlorella vulgaris Be y e r i n c k CAUP H Žebrákovský creek, BBM 1993 Czech-Moravian High- lands (CZ) Chlorophyceae Stigeoclonium sp. CAUP J 603 Žebrákovský creek, BBM river basin of Sázava, Czech-Moravian High- lands (CZ) Chlamydomonas geitleri Et t l CAUP G 224 pond near Opatov (CZ) BBM Desmodesmus communis (E. CAUP H 522 unknown BBM He g e w a l d ) E. He g e w a l d Planktosphaeria gelatinosa CAUP H soil from garden, Wo- BBM G.M.Sm i t h 1401 ods Hole (MA, USA) Fottea 11(2): 317–328, 2011 321

Table 1 Cont.

Ulvophyceae Kentrosphaera sp. CAUP H soil, top of the Boreč BBM 5308 Hill (České Středohoří Mts, CZ) Pseudendoclonium basiliense SAG 466-2 bog water, Bot. Gard. BBM Vi s c h e r Univ. Basel (CH) Trentepohlia sp. CAUP J 1601 bark, Singapore BBM Prasinophyceae Tetraselmis subcordiformis CAUP M 201 Sandy Hook (NJ, USA) ASW (Wi l l e ) Bu t c h e r Bacillariophyceae Pinnularia cf. gibba Eh r e n - Pin1Cra Loch of Craiglush (UK) WC b e r g

Euglenophyceae Euglena gracilis Kl e bs CAUP E 201 peaty pool nearby Třti- soil ce (CZ) extract medium Xanthophyceae Tribonema vulgare Pa s c h e r CAUP D 501 Palach Pond near Led- BBM nice (CZ)

Results and discussion such conditions we obtained from each Petri dish sufficient cell suspension for at least one Range of tested microalgae measurement. In the case of Micrasterias rotata, This paper proposes an easy protocol for the a greater degree of biomass cover of the Petri quantification of nuclear DNA by flow cytometry, dish was necessary to yield a sufficient amount suitable primarily for Zygnematophyceae, but of cell suspension. In contrast, the suspensions of also useful for some other microalgae, depending small genome size species including Tetraselmis in particular on cell wall organization. We subcordiformis and Tribonema vulgare were tested 28 microalgal cultures and succeeded highly concentrated and one microliter of the with Streptophyta representatives (Desmidiales, suspension was sufficient for the flow cytometric Zygnematales and Klebsormidiales; Table analysis. The species with larger genome sizes 3). In addition, 39 desmid cultures of the tend to have bigger cells (Gr e go r y 2001; Le i t c h genus Micrasterias sensu lato have been & Be n n e t t 2007) and therefore the cover of the successfully analyzed in another study (Šk a l o u d Petri dish seems to be more intense. Consequently unpublished). Although the protocol (particularly the real amount of cells rather than the intensity of protoplast isolation) failed in Bacillariophyceae, green colour is crucial for sufficient concentration Euglenophyta and most of the tested Chlorophyta, of cells in the FC–solution. Culturing in Petri it was successful for Chloroidium ellipsoideum dishes is cheap, does not require a lot of space (Oocystaceae), Tetraselmis subcordiformis and the health and density of the culture can (Prasinophyceae) and Tribonema vulgare easily be checked under an inverted microscope (Xanthophyceae, Heterokontophyta). (Po u l í č k ov á & Ma n n 2006).

Minimum biomass requirement and culture Protoplast isolation conditions Isolated protoplasts are living plant cells from In contrast to higher plants and seaweeds, biomass which the walls have been removed. They provide for microalgal experiments originates from clonal a large, relatively homogeneous suspension of unialgal cultures. From our experience, most of wall–less, single plant cells for physiological and the tested species grow well in liquid media. We biochemical research. Protoplasts surrounded mostly used culture media for green microalgae only by a plasma membrane are a convenient and desmids (Bold‘s Basal medium–BBM; Bo l d starting material for obtaining high yields 1949 and oligotrophic medium for desmids as used of delicate cell organelles such as nuclei or in the CAUP collection–OGM; Če r n á & Ne u s t u p a microbodies. Techniques for the removal of the 2010) and an elevated irradiance level compared cell wall using cell wall–degrading enzymes were to maintenance cultures (40 μmol.m–2.s–1). Under developed in the 1960s and are routinely used in 322 Ma z a l o v á et al.: Flow cytometry in microalgae

Table 2. Composition of the modified solution PGly (original: Micrasterias rotata, Micrasterias truncata De b e a u j o n & Br a n c h a r d 1992). With continuous stirring Pleurotaenium ehrenbergii, Staurastrum arcti- dissolve all components in distilled water. Bring the total volume to 1000 ml. Adjust to pH 5.8 with 1M HCl and sterilize scon, Staurastrum orbiculare, Staurodesmus by filtration. *MES 2–(N-morpholino)ethanesulfonic acid dickiei, Triploceras gracile), some other microalgae within Streptophyta (Klebsormidium flaccidum, –1 Zygnema circumcarinatum and Zygnema Component g.l H2O cylindricum), a few Chlorophyta (Chloroidium PGly KH2PO4 0.0272 ellipsoideum, Tetraselmis subcordiformis) and KNO 0.101 3 Heterocontophyta (Tribonema vulgare). How- CaCl (anhydrous) 1.1176 2 ever, in contrast to higher plant samples, the algal

MgSO4.7H2O 0.246 cell walls have not been digested completely glycine 11.15 by the enzymes in some cases. Sometimes the glucose 18.016 desmid cell wall was eliminated only from one (younger) semicell, or protoplast left the cell MES* 0.5857 wall by the split in the isthmus area. However, manitol 65.58 even these samples were often useful for flow cytometric measurements. This is the reason why young cultures (in the exponential growth higher plants (Ta k e b e & Ot s u k i 1969; Co c k i n g phase) are recommended, because the cell wall 1960; Do l e ž e l et al. 2007) for biotechnological is not fully developed. The method failed in the purposes. In principle, there is no reason why cases of Chlorella vulgaris and Desmodesmus such techniques cannot be successfully applied communis. Their cell walls contain alganan to algae. Methods for preparation of protoplasts and Planktosphaeria gelatinosa has a robust of marine macroalgae have been developed only mucilaginous envelope. As we expected, we did recently (Bu t l e r et al. 1990; In o u e et al. 2010; Wa- not successed in the diatom Pinnularia cf. gibba k a b a y a s h i et al. 1999) and have never been used on account of its silica frustule, nor monads with for microalgae. The algal cell wall differs from protein–containing periplast [Euglena gracilis, land plants in its heterogeneity and complexity. Chlamydomonas geitleri] (Gr a h a m et al. 2008). Commercial mixtures of enzymes routinely used The enzyme mixture also failed for Xanthidium in higher plants mostly consist of cellulases, octocorne, Kentrosphaera sp., Stigeoclonium hemicellulases and pectinases. Cellulose is very sp., Trentepohlia sp. and Pseudendoclonium abundant in green algae, in which it accounts for basiliense. Specific enzyme mixtures will be up to 70 % of the cell wall (Pr e s t o n 1974). On the necessary for these microalgae. other hand, the cellulose content of most brown and red algae is low, being less than 10 % and Flow cytometry and DNA amount estimation some algae do not contain any cellulose (Bu t l e r FC is the optimal method for nuclear genome et al. 1990). Our recent research focuses primarily size estimation. Compared to traditional methods on desmids, which belong to Streptophyta, thus like Feulgen microdensitometry, scanning a high similarity of their cell wall organization microspectrophotometry or DNA image cytometry with higher plants can be expected. There exist which were used in the past, the FC enables the actually differences in cell wall composition measurement of large amounts of cells in a short within different algal taxa (St a c e 1991), but the period of time (Do l e ž e l & Ba r t o š 2005). Usability concentrations of the main cell wall polymers of FC for estimation of DNA amount from whole of land plants are similar to that of Streptophyta cells or protoplasts refers to cytoplasmatic (Ed e r et al. 2008, An d e r so n & Ki n g 1961). autofluorescens and nonspecific background We modified the enzyme mixture used for which lead to overestimation of results (Ka p r a u n the genera Cucumis and Cucurbita (Ga j d ov á et 2007; Ve l d h u i s et al. 1997). Therefore release al. 2007; On d ř e j et al. 2009) and marine green of the nuclei from the protoplast by lysis buffer macroalgae (Re d d y et al. 2006; Upp a l a p a t i & enables precise estimation of the nuclear genome Fu j i t a 2002; Ch e n & Sh i h 2000). After this size. One of the most important steps in genome treatment we were able to measure the amount size estimation is the selection of an appropriate of DNA in desmids (Cosmarium contractum, standard. It should have a genome size close to the Euastrum verrucosum, Micrasterias pinnatifida, peaks of the target material, but not overlap with Fottea 11(2): 317–328, 2011 323

the cells of interest (Pr i c e & Jo h n s t o n 1996). If the estimated genome size. genome sizes are too dissimilar, non–linearity of At present two standard fluorescent dyes the instrument can arise. We chose different plant are routinely used for DNA staining for FC standards for different samples to avoid overlap- measurements: 4’,6–diamidino–2–phenylindole ping or nonlinearity of the instrument (Table 3). In (DAPI) binding to AT–rich regions (Ba r c e l l o n a three cases (Micrasterias pinnatifida, Cosmarium et al. 1986) and propidium iodide (PI) intercalating contractum and Tetraselmis subcordiformis) more to the whole DNA (Cr i ssm a n & St e i n k a mp standards were used to choose the optimal one. 1973). The applicability of DAPI staining in Results did not lead to significant differences in absolute DNA amount measurements clashes 324 Ma z a l o v á et al.: Flow cytometry in microalgae with unbalanced proportion of different bases to 46.8 pg. Our results are broadly comparable in the genome. Ba r ow & Me i s t e r (2002) have with recorded ranges, however as we did not shown that the proportion of AT:GC–rich regions determine ploidy level of the studied cultures, we in higher plants is not equal between families nor used terminology of 2C value only in cases with species. The intraspecific differences were also two peaks (desmids, Zygnematophyceae). In the documented by Šm a r d a et al. (2008) on fescues case of primitive unicellular algae (Chloroidium which range from 42.53% to 46.41%. Similar ellipsoideum) the DNA amount (Table 3) should variation with a range from 42.9% to 46.7% was represent the haploid vegetative stage, while detected for several algae species by Le Ga l l et the situation in algae with sexual reproduction al. (1993). The most marked discrepancy caused (desmids, diatoms) can be more complicated. by the use of specifically binding dyes was shown We detected in 5 of 17 algal cultures by Do l e ž e l et al. (1992). They found that the (Cosmarium contractum, Micrasterias pinnatifida, difference was caused not only by the differences M. truncata var. pusilla, Staurastrum arctiscon in overall AT/GC ratios for studied material and Zygnema circumcarinatum) two peaks = and standard, but also by the species–specific ploidy levels where the first peak (most likely differences in binding of these fluorochromes to 1C level) was notably smaller than the second DNA. Hence, use of PI for absolute genome size (2C). This phenomenon was reviewed for several estimation is unambiguous. algae species by Ha i g (2010). They explain that Our protocol succeeded in 17 cases from 28 occurence of both haploid and diploid cells is cultures used in this study. The estimated absolute caused by the meiosis and syngamy both followed nuclear DNA amount in these 17 microalgal by mitosis leading to vegetative growth of the two cultures varied from 0.28 pg to 28.2 pg (Table 3). ploidy levels in culture. The two smallest genomes belong to the families Interesting differences were found in the Klebsormidophyceae (Klebsormidium flaccidum case of two strains of Triploceras gracile. The strain – 0.28 pg) and Xanthophyceae (Tribonema isolated from Sumatra with 28.2 pg has a DNA vulgare – 0.41 pg). Variability in the family content three times higher than the strain isolated Zygnematophyceae ranged from 2.05 to 28.2 pg, from USA (9.39 pg). The same phenomenon was which probably reflects an increase in ploidy level detected by Ka sp r i k (1973), who counted numbers during diversification and evolution of the species. of chromosomes of several Micrasterias species. This phenomenon was previously described by He detected different numbers of chromosomes in Wa n g et al. (1986) for polyploid series of Spirogyra each population of M. americana corresponding maxima (Zygnemataceae) in both culture and wild to three ploidy levels: 2x, 3x and 4x. populations. The original culture developed into The coefficient of variation (CV) refers three ploidy levels: 2x, 3x and 4x in accordance to the quality of the relative fluorescent peaks. to findings in natural populations. In contrast, In most cases CVs below 3% are acceptable as Hos h a w et al. (1985) observed a spontaneous CVs below 5% are considered to be sufficient for decrease in ploidy level in clonal cultures of “problematic” species (Doležel & Bartoš 2005). Spyrogira singularis. Ka p r a u n (2007) recorded a DAPI staining shows lower CVs than those for range of 2C nuclear DNA content for Chlorophyta PI as the binding of DNA is not influenced by from 0.01 to 5.8 pg and for Streptophyta from chromatine structure (Sa n t i s t e b a n et al. 1992). 0.2 to 6.4 pg excluding the highly polyploid Coefficients of variation in our study varied from Desmidiales which have geonome sizes of up 2.35% (Micrasterias pinnatifida, selected as a

Table 4. Values of the absolute nuclear DNA amount (pg) of standards used in our study, from different sources.

Do l e ž e l et al. Do l e ž e l et al. Ly s á k & Do l e - Jo h n s t o n et al. Su d a personal (1992) (1998) ž e l (1998) (1999) communication Raphanus sativus 1.11 1.41 1.12 Lycopersicon 1.96 1.96 esculentum Zea mays 5.72 5.82 5.43 5.73 5.46 Pisum sativum 9.07 9.09 9.39 8.76 Fottea 11(2): 317–328, 2011 325 new standard for microalgae) to 5.38%, with only and distribute to other laboratories and stable one exception (Tetraselmis subcordiformis; 14.76 genome size due to vegetative propagation. %), which was excluded from the analysis. Gating In addition it seems to be free of compounds of the peaks (understand as trimming of the peak interfering with PI staining. with high background) was necessary in the case In the majority of the 17 measurements of of Micrasterias rotata and Chloroidium ellipsoi- Micrasterias pinnatifida, two peaks of nuclear deum, to obtain explicit results. Modification of DNA were detected. In four measurements the the protoplast isolation enzymatic mixture for first peak was not detected, although CV for these species could lead to better results. the second peak was low, indicating that the In the case of the filamentous species absence was not caused by debris in the sample. Zygnema circumcarinatum and Z. cylindricum, To identify the genome size of M. pinnatifida, chopping of the sample with a razor blade after all four plant standards were used as the primary enzyme treatment was necessary for nuclei reference standards (Fig. 1). However, the use of isolation. We tested the hypothesis that chopping Lycopersicon and Raphanus as internal standards without enzymatic treatment is sufficient for refers to alternating of 1C and 2C peaks (Fig. cytometric analyses for Z. cylindricum as it is 1) and increase the CV and inaccuracy of the possible for most of the higher plants. Coefficient measurement. Therefore we decided to exclude of variation in that case was 21.65% in comparison these standards from absolute genome size with the chopped sample treated with enzymes – estimation. The genome size of new algal standard CV = 5.15%. Nevertheless, analysis of unchopped was counted from 14 analyses with Pisum and and enzymatically treated sample did not give any Zea, whose means of CVs were 3.63% for alga, results. 2.36% for Pisum and 3.59% for Zea. In all cases peak of 2C–value of alga was narrow and with New microalgal standard M. pinnatifida maximum CV = 4.61%. The determined size of As we have shown in our study, the genome Micrasterias pinnatifida from Hamburg culture size of microalgae may differ largely from one collection is 3.4 pg ± 0.07. to another species or population. Selection of a To validate the DNA amount of internal suitable standard is necessary for precise analyses. standards (Table 4) we measured them in In the last few decades, different standards for combination: a) Pisum sativum, Zea mays and FC analyses were used e.g. human leucocytes Raphanus sativus or b) Pisum sativum and (Ly s á k et al. 2000), chicken red blood cells Lycopersicon esculentum. Cross calculation gave (CRBCs, Ga l b r a i t h et al. 1983; Ve l d h u i s et al. means of genome sizes: 8.77 pg for P. sativum, 1997), salmon erythrocytes (Iv e r s e n & La e r u m 5.48 pg for Z. mays, 1.95 pg for L. esculentum 1987) or different plant species (various authors). and 1.09 pg for R. sativus. The standard deviation As was proven by many authors (Do l e ž e l et al. of known and counted genome sizes did not exce- 1992; Jo h n s t o n et al. 1999; Ga l b r a i t h et al. 1983; ed 0.01 and we consider use of known genome Ti e r s c h et al. 1989), human and chicken cells do sizes as legitimate. not seem to be a convenient standard for genome size estimation due to intraspecific and sex– Acknowledgements The authors are grateful to Kath Evans (University of related variation (De Vi t a et al. 1994; Me ffo r d Edinburgh) for English corrections. The research was et al. 1997). For absolute genome size estimation, supported by projects GACR 206/09/0906, 206/08/0389 use of biologically similar material with identical and IGA PrF_2011_003. We are grateful to P. Škaloud sample preparation is recommended (Do l e ž e l (Charles University of Prague) for inspirative com- & Ba r t o š 2005). Therefore we suggest a new ments and for providing us with cultures. algal standard, Micrasterias pinnatifida for FC estimations, which can be the starting point for FC–standards development suitable for material References of isolated protoplasts. The species match most of An d e r s e n , R. A. (ed.) (2005): Algal culturing technique. the requirements for such standards: unproblematic – 596 pp., Academic Press, London. preparation leading to narrow peaks with low An d e r so n , D.M.W. & Ki n g , N. J. (1961): Polysaccha- CV, simultaneous preparation of standard and rides of the Characeae III. The carbohydrate protoplast samples prior to cytometric analyses, content of Chara australis. – Biochimica et available in sufficient quantities, easy to cultivate Biophysica Acta 52: 449–454. 326 Ma z a l o v á et al.: Flow cytometry in microalgae

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