See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/270104064 Estimation of fungal genome size: Comparison of image cytometry and photometric cytometry Article in Folia Cryptogamica Estonica · January 2006 CITATIONS READS 6 159 2 authors: Bellis Kullman Wladimir Teterin Estonian University of Life Sciences University of Rostock 45 PUBLICATIONS 491 CITATIONS 7 PUBLICATIONS 10 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: 8-2/T9040PKMO "Mükoloogilised kogud (31.03.2009−31.12.2018)", Kadri Pärtel, Eesti Maaülikool, Põllumajandus- ja keskkonnainstituut. View project SF0170057s09 "Plant protection for sustainable crop production (1.01.2009−31.12.2014)", Marika Mänd, Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences. View project All content following this page was uploaded by Bellis Kullman on 28 December 2014. The user has requested enhancement of the downloaded file. Folia Cryptog. Estonica, Fasc. 42: 43-56 (2006) Estimation of fungal geome size: comparison of image cytometry and photometric cytometry Bellis Kullman1 & Wladimir Teterin2 1 Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Riia Street 181, 51014 Tartu, Estonia. E-mail: [email protected] . 2 Institute of Medical Microbiology, Virology and Hygiene, Rostock University, Schillingallee 70, D-18057 Rostock, Germany. Abstract: Besides photometric cytometry (PC), fl uorescence microscopy combined with computerised image analysis, i.e. image cytometry (IC), offers an alternative tool for assessing genome size. These techniques allow direct visualization of hyphae and simultaneous measurement of nuclear fl uorescence intensity. We developed a simple method for quantitative evaluation of nuclear DNA in fungi using DAPI-IC. The intensity of signals from individual nuclei was quantitatively measured in digitized images. In agreement with the results of parallel PC experiments, this simple IC performed on fruitbodies or on pure culture preparations enables to detect the amount of nuclear DNA in fungal cells. This result validates IC as an alternative to PC in such experiments. Kokkuvõte: Seene genoomi suuruse määramine: pildianalüüsi ja fotomeetriise tsütomeetria võrdlus. Genoomi suuruse määramise meetodiks klassikalise tsütofotomeetria (PC) kõrval on fluorestsents-mikroskoopia kombineerituna kompuuter-pildianalüüsiga (IC). See meetod võimaldab mõõta hüüfi tuumade fl uorestsentsi intensiivsust in situ. Töös esitatakse lihtne meetod tuuma DNA-sisalduse kvantitatiivseks määramiseks DAPI-IC. Digitaliseeritud pildil mõõdeti igast tuumast tulevate signaalide intensiivsust kvantitatiivselt. Paralleelsete PC eksperimentide tulemused kinnitasid, et see lihtne IC meetod võimaldab viljakeha või puhaskultuuri preparaatidest kindlaks teha tuuma DNA-sisaldust ja ta on kasutatav alternatiivse meetodina. INTRODUCTION al., 2002) to 795 Mbp (Scutellospora castanea, The study of genome size variation is important Hijri & Sanders, 2005) (see Kullman et al., 2005, from a number of practical and theoretical http://www.zbi.ee/fungal-genomesize/). perspectives. Nuclear DNA content in an In providing quantitative data of nuclear unreplicated haploid chromosome complement DNA for the purpose of fungal taxonomy, (1C-value) is a key diversity character with microspectrophotometry and microfl uorometry many uses (Bennett & Leitch, 1998, 2005, (cytofluorometry) have played an important for terminology see Greilhuber et al., 2005). role (see for a review Kullman, 2000). These Processes inducing quantum or doubling cytometric methods of determination of genome series variation in genome size are common. size consist in the analysis of nuclear DNA These abrupt shifts have significant effects molecules in situ afterafter qquantitativeuantitative sstaining,taining, aandnd on phenotypic attributes at both cellular and comparative evaluation of the data of different organism levels and may play an important species. By means of these methods it is possible role in evolution (Gregory & Hebert, 1999). to measure relative DNA amount in the nucleus: The questions of the C-value enigma transcend the ratio of the result of the measurement of taxonomic boundaries, and increased the studied organism to that of the standard communication is therefore urged among those organism is calculated. who study genome size evolution, whether in Fluorometry relies on quantitative staining plants, animals or other organisms (Gregory, of DNA with fl uorochromes. The amount of 2005 a). Databases of plant and animal genome DNA is estimated from the amount of emitted size are growing rapidly (Gregory, 2005b; light measured with a microscope-photometer Bennett & Leitch, 2004). To date, comparatively or with a fl ow cytometer* (FC; Dolezhel, 1997; few data are available for the genome size of Dolezhel et al., 1998; Kullman, 2000; Saar & fungi. The 1C-values in fungi range from 6.5 Kullman, 2000). A classical fl uorometric method Mbp (Pneumocystis carinii f. sp. muris, Birren et which has been used mostly in mycology (see 44 Folia Cryptog. Estonica review Kullman, 2000) involves measurement Important steps in measurement of of intensity with instruments combining a nuclear DNA content in fungi are standardized microscope and a photometer. The photometer procedures for DNA staining and measurement, can now be replaced with an image analysis as well as the choice of the standard species. system which grabs images from the microscope The aim of the present study was to compare PC via a digital camera and calculates intensity or and IC on the basis of the nuclei stained with optical density from the grey values of the pixels the fl uorochrome. in the nucleus (for a review see Vidal, 1997; Hardie et al., 2002). The classical method is MATERIAL AND METHODS referred to as photometric cytometry (PC) in contrast to computer-based image cytometry Experimental design (IC) (Vilhar et al., 2001). Nuclear DNA content in 5 fungal species was Comparison has shown that using Feulgen measured in two laboratories with IC and PC, staining, FC and IC as well as PC and IC, respectively, using DAPI staining. Also, two IC, provide a similar effi cacy of DNA quantifi cation the software Image-Pro Plus 4.5 and the software (Borgiani et al., 1994; Vilhar et al., 2001). In Image J 1.34f, were used to study the same measurement of plant and animal genome size, slides of 8 specimens (from 6 species). the Feulgen image analysis has met an ever Two calibration standards, Trichophaea wider application. This method is also used in hemisphaerioides (TFC 97-71) 23.3 Mbp plant pathology (Volgmayr & Greilhuber, 1998; (Kullman, 2000) and the species Neottiella Rincones et al., 2003). rutilans 530530 MbpMbp (Kullman,(Kullman, 2002a),2002a), andand thethe Employing PC for measurement of fungal prophase/telophase method were applied for nuclear DNA, mostly fl uorochrome DAPI has fungal cytometry. The genome size of the other been used for estimation of the ploidy level specimens was estimated in relation to the (Bresinsky et al., 1987a, b; Wittmann-Meixner, standards. 1989; Wittmann-Meixner & Bresinsky, 1989; Measured fluorescence intensity is Whittaker et al., 1991; Wittmann-Meixner et proportional to DNA content in the nucleus. al., 1989; Weber & Bresinsky, 1992; Weber, When measuring nuclei in the haplophase, 1992; Bresinsky & Wittmann-Bresinsky, 1995; a distribution curve is obtained whose Bresinsky et al., 1999; Kullman, 2000, 2002 a, first maximum, indicating the nuclei with b). DAPI is a small water-soluble fl uorescent unreplicated chromosomes in the G0/G1 molecule with extremely high avidity and phase of the cell cycle, corresponds to genome specifi city for DNA, preferentially binding to size. The resulting fluorescence histograms the A-T rich regions of DNA. A comparative can be analysed for calculating the difference study of the DAPI dye and the intercalating in nuclear DNA content between the specimens. dyes revealed a higher fl uorescence intensity By including the internal standard, relative DNA and resolution of the former (Otto & Tsou, 1985). content is converted to the absolute amount. DAPI staining is less affected by the state of The genome size of an unknown specimen is chromatin condensation compared with staining obtained by dividing the mean relative DNA with other fluorochromes (Shapiro, 1995). content of the unknown G0/G1 population Comparing the Feulgen image analysis and DAPI of nuclei by the mean of the standard G0/G1 image analysis, Volkova (2005) reported higher population of nuclei and by multiplying the image resolution in the case of DAPI staining. result by the genome size of the standard. DAPI is also widely used in current plant FC for DNA content is expressed in megabase pairs ploidy analysis. of nucleotides (Mbp) (NB 1 pg = 978 Mbp (see IC has only rarely been used in mycology Dolezhel et al., 2003). (Rincones et al., 2003; Volkova, 2005). Moreover, When the genome size of T. hemisphaerioides, published data provide no evidence that IC with obtained in Kullman (2000), is 23.3 Mpb and the DAPI staining yields results comparable to the mean value of the same species, measured in results obtained with PC. Most PC studies arbitrary units (a.u.) by Weber (1992), is 54.4, report only relative DNA values, while usually then 1 a.u.= 0.43 Mbp. In this case also the no attempt has been made to present the value genome size of the other species