Estimation of fungal genome size by FC

Bellis Kullman

Department of Mycology
Institute of Agricultural and Environmental Sciences,
Estonian University of Life Sciences

1C-value, fungal genome size in Mbp

 Fungal genome size
Genome size is an important biodiversity character the study of which has both practical
and theoretical uses in biology.
Variation in fungal genome sizes ranges:
Parasitological findings
from the brain abscess.
from 2.5 Mb in Encephalitozoon cuniculi Ditrich O et al. J. Clin. Microbiol.
2011;49:2769-2771
10 μm 10 μm

to 3050 Mbp in Neottiella sp. highly polyploid genera of ascomycetes

Genome size (total DNA content of the unreplicated haploid nuclear genome, 1C-value)
Fungal Genome Size Database. Kullman et al., 2005
http://www.zbi.ee/fungal-genomesize
FUNGAL C-values (1641 entries, 122414 views) 2014

The genome size of over 1800 fungi is measured with different methods.
Tavares et al. 2014. Front Plant Sci. 5: 422

ca 90% of available values remain between 10 Mb and 60 Mb

0.01 pg – 0.06 pg

1C-value, genome size in pg

 Flow Cytometry (FC or FCM)

Flow Cytometry (cyto=cell) (metry=measurement)

Measurements performed
On individual cells
In a liquid stream. http://www.flowcytometri.dk/literature/Leslie-FCBasic.pdf

The first fluorescence-based flow cytometer was developed in 1968 by Wolfgang

Göhde from the University of Münster, and was commercialized one year later by
Partec in Germany.

It soon became an essential method in medicine, veterinary science, food

microbiology and water analysis.

However, it has remained unknown and unused in mycology.

 Number of publications in Web of Science (from All Databases)

• Searched for „flow cytometry“ and fungi – ca 460 (of these, „genome size“ 17)
• Searched for „flow cytometry“ and plant – ca 6 000
• Searched for „flow cytometry“ and animal – ca 65 000
 Web of science (from All Databases)
Searched for „flow cytometry“ and fungi – 460
 My background

I have studied problems connected with measurement of DNA content with FC in order
to investigate the presence of polyploidy in filamentous fungi (hyphal-forming fungi).
I started this research in 1996 at Tartu University, Estonia, not knowing that this is
almost impossible by FC.
This method was and really is highly appropriate for single-celled fungi, e.g. Yeast cells.
However, I was lucky to get in contact with the best scientists in their respective fields
in the course my scholarships (1998, 2002, 2007 DAAD and 2000 DFG)
Prof Andreas Bresinsky from the University of Regensburg
Prof Wolfgang Göhde from the University of Münster
Dr Wladimir Teterin from the University of Rostock
 My background
Prof Bresensky applied a unique, Zeiss- built fluorescence microscope equipped with
a microspectrophotometer for measurement of relative nuclear DNA content.

Prof Göhde was interested in implementing new methods to study fungal genome
size by his Ploidy Analyser PAS

Dr Teterin equipped the fluorescence microscope with a CCD camera and Image
analysis program (ImageProPlus)
It was possible to begin with image-cytometry (IC), which is sometimes
indispensable in the case of fungi.

So, it was possible to use simultaneously three different methods.

Now I know something about why measurement of fungal genome by FC is so
complicated.
 FC for measurement of fungi: application, advantages and pitfalls

The main advantage of FC is that it is a fast and robust technique.

It is really very easy to measure with FC but the question is what in fact you are
measuring.
So, I will not speak much about how to manage a flow cytometer
(this can be found in manuals) but about the problem caused by the specificity of
fungi, which must be known in order to understand what you are really measuring.

It is Important that you have obtained intact nuclei or intact cells.

In latter case they must be without autofluorescence (AF).
Both nuclei and cells can be measured one by one.
Conceptual figure of a flow cytometer

FC is a technique for the measurement and counting of

small particles in a fluid stream.

FC comprises three systems: fluidics, optics and electronics.

Every single particle is excited by a light source and displayed
on a graph.

FC detects: forward scatter (FSC), sideward scatter (SSC)

and fluorescent wavelengths (FL1-3)

FSC depends on the size of the cell.

SSC depends on the external granularity and shape of the cell.

PMTs - photomultiplier tubes.

D’HONDT et al. 2011.

 As a result you can obtain relative measurements
of all detected parameters - Multivariate Analysis

SSC

FSC

Gating on the dot plot of FSC/SSC

http://www.slideshare.net/viviansareno/flow-basics2ppt2?related=2 modified
 Multivariate Analysis
of spores of P. ostreatus FLIII/FSC

Gating on the dot plot of FLIII/FSC

Size of particel / events

R1 – gate of spores
R2 – gate of released nuclei
R3 – gate of fluorescent beads (diameter 5.2 m)

Due to the known size of the beads, the

nuclei can be positioned and their absolute
size in microns determined
Fluorescence intensity

FLIII represents interaction of fluorocrome with particele

Univariate Analysis - Size Histogram Analysis
of spores of P. ostreatus

Size of particele / events in a.u.

Nuclei are represented by the first peak (RN1)

spores, by the second peak (RN2).
 Polybead fluorescent microspheres

• Fluorescent beads (diameter 5.2 m) are applicable in the serial

measurement of nuclear DNA content.
• Measurement accuracy can be enhanced by adding beads to the
samples to be compared.
• In an ideal case, they remain in the same channel and can be used
also for measurement of the size of nuclei and cells.
• FC is useful in the measurement of cell and nuclear size with the aim
to study the relationship between cell size and cell DNA content.
 Fluorochromes used to analyse DNA content
• PI - propidium iodide, which intercalates into double-stranded DNA

• DAPI - 4,6’-diamidino-2-phenylindole, which binds at AT-rich regions

The use of base preferring fluorochromes can lead to errors!

Maximum excitation of DAPI, bound to DNA, is at 359 nm,

and its maximum emission is at 461 nm
Maximum excitation of PI, bound to DNA, is at 536 nm and
its maximum emission is at 617 nm
• PI has often been used to test nonviable cells
• DAPI stains also viable cells as well
 DNA measurement by flow cytometry (FC)

• DNA measurement by FC is based on the quantitative binding of a

DNA specific dye (fluorochrome) to DNA
• FC allows measurement of the fluorescence intensity of the DNA-
fluorochrome complex separately for each cell (spore)
• Fluorescence intensity is proportional to the amount of fluorescent
substance and hence to DNA content
 FC data analysis

• Fluorescence histograms can be

analysed for calculating the relative
difference in nuclear DNA content
between the specimens – ploidy
levels
• To estimate the absolute value of
genome size it is necessary to
compare the study object with a
standard whose DNA content is
already known.
• By including an internal standard, Nuclear DNA content in a.u.
relative DNA content is converted to
absolute DNA content in Mbp or in
pg (1pg = 978 Mb). Fig. P. ostreatus (standard) and P. punctatus (unknown).
Spores of P. ostreatus (denoted as M1, CV 10%) and
Unknown = standard X M2unknown/M1standard spores of P. punctatus (denoted as M2, CV 11%).
 Calibration standards for C-values of fungal DNA

• An organism in which genome size has been measured by

some other means, can be used as a standard.
• Applicability of chicken erythrocytes (2C=2.33pg) is generally
accepted, but this value is too high for measuring fungal
genome size (1C = 0.01 - 0.06pg / 10 Mb - 60 Mb mostly).
• It is desirable to select a standard taxon with genome size
which is similar to that of the unknown taxon (the difference in
genome size can be two-to threefold.
 Calibration standards for
C-values of fungal DNA

Veselska et al., (2014) introduced a new

standard, Aspergillus fumigatus CEA10

The genome size of A. fumigatus CEA10

estimated by FC (29.7 Mb), using
S. cerevisiae as a standard, is in
agreement with its genome size acquired
by genome sequencing (29.2 Mb) (E,G) PI-stained S. cerevisiae BY4743aa,
(F, H) PI-stained A. fumigatus CEA10.
Veselska et al., 2014. Cytometry Part A 85A: 854861
 Which method to use depends on
the specific nature of the fungus.

Veselska et al. 2014, when optimizing the FC protocol for estimating

genome size in Geosmithia, evaluated the effects of different
RNAse A concentrations and incubation times,
different types and durations of fixation, and
various buffers and the localization of PI to nuclei.
They chose the genus Geosmithia as a model filamentous fungus.
 Calibration standards for C-values of fungal DNA

• Anderson et al. (2010) have proposed Puccinia graminis f.sp. tritici

and Sclerotinia sclerotiorum as standards for rust fungi, with a larger
genomes

• In an ideal case, only one specimen /strain of a standard species from

a single source should be used for calibration, which improves
comparability of the results obtained at different laboratories

• e.g strains of S. cerevisiae differ in ploidy and genome size

 Calibration standards for C-values of fungal DNA

I have tested different methods to find appropriate candidates for

standards.
Nuclear DNA content was measured in the same fungal specimens
at different laboratories using different cytometers -
Becton Dickinson FACSort, FACSDiva, Accuri 6;
MACSQuant®, Particle Analysing System (PAS).
 Methods of preparation of intact cells,
spores and conidia for FC
Nuclei can be stained in the cell or isolated chemically, or mechanically.
The material is either fixed or unfixed.
Different sets of reagents are used.
For dissolving cell membrane lipids, for eliminating the cell cytoskeleton and
nuclear proteins and for digesting cellular RNA, nonionic detergent Tween, Triton
X-100 and the enzymes zymolyase, trypsin, proteinase K and pepsin were used.
For stabilizing nuclear chromatin, spermine is used.
Stained samples should be analysed within some days of staining with PI.
After adding the fluorochrome DAPI SR101, optimum time for measurement is
35±10 min.
Kullman, 2000
Method used for ploidy analysis of Saccharomyces carlsbergensis with FC

Cells were grown overnight (o/n) at room temperature to an end-

exponential growth state.
For the staining of cells with PI, cultures were first washed and resuspended
in 1× SSC buffer before fixation in 70% ethanol at −20° o/n.
Samples were then treated with RNAse o/n at 37° followed by proteinase K
treatment at 50° for 1 hr.
A final concentration of 3 µg/mL PI was added to each sample and incubated
for 18 hr in darkness before FS analysis

Walter et al. 2014 Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager
Yeast. G3, 4 (5): 783-793. http://g3journal.org/content/4/5/783.full
 Ploidy analysis of Saccharomyces carlsbergensis with FC
DNA content of the
S. carlsbergensis
S. carlsbergensis 1C=19.5 Mb
and S. pastorianus S. cerevisiae 1n S. pastorianus WS34/70
Weihenstephan 1C=23 Mb
compared to 1n and 2n
S. cerevisiae 2n
laboratory S. cerevisiae
1C=12 Mb strains.

The ploidy of S. carlsbergensis

was determined on the basis of
sequence data using FC .

This strain is basically triploid with

a diploid S. eubayanus and haploid
S. cerevisiae genome content.
Walter et al. 2014 Genome Sequence of Saccharomyces carlsbergensis, the World’s
First Pure Culture Lager Yeast. G3, 4 (5): 783-793. http://g3journal.org/content/4/5/783.full
 FC is appropriate for single-cell uninucleate fungi

Yeast, conidia and spores

- If cells in the G1 cell cycle phase are dominating.
In the case of yeast, progression through the cell cycle makes identification of G1
sometimes quite difficult. It is important to always use G1 nuclei.
When using spores you have to find out in which cell cycle station the nuclei are
arrested.

- if cells are without autofluorescence (AF)

Autofluorescence is the term used to describe the fluorescent signals derived
from the cells in the absence of fluorescent probes.
 Calibration standards for C-values of fungal DNA

Examined candidates:
• Conidia from a pure culture of the ascomycete
Trechispora hemisphaerioides (TFC 97-71 from TAA 147708)
• Cells of the yeast Saccharomyces cerevisiae (YAC strain),
• Spore print of the oyster mushroom Pleurotus ostreatus (TAA 142824),
Dicarya - Ascomycota and Basidiomycota cells are dikaryotic with haploid
nuclei.
In these fungi the G1 phase of the cell cycle corresponds to unreplicated
haploid genome size (1C-value).
 Cell-cycle study by FC using fluorescence histogram

• About 100,000 stained cells are needed to

study their cell-cycle phase distributions.
• The method relies on a single-time
measurement of cell populations of one
sample.
• Kinetic information can be inferred from
DNA content (position of the cell cycle).
• Progression through the S-phase and
mitosis is expressed by changes in cellular
DNA content.
Nuclear DNA content of a conidium
• The position of a cell in the cycle can be
estimated on the basis of measurement of Fig. T. hemisphaerioides. Most of the conidia
DNA content. are in cell cycle phase G1 (denoted as RN1.
Ratio RN2/RN1=2.0.
G1= 1C, genome size
 FC data analysis

Fungal nuclei in hyphae and spores are haploid, i.e. mean G1=1C

The genome size in Mbp (1C) :

Mean G1 fluorescence of sample nuclei
Mean G1 fluorescence of reference standard

Also a median or peak (mode) value is used.

 Calibration standard T. hemisphaerioides for C-values of fungal DNA

• Conidia of T. hemisphaerioides
seem first as the most suitable
standard - vegetative reproduction
serves as a guarantee of uniform
DNA contet, the wall of conidae is
thin and easily permeable by
enzymes.
• However, cultivation of many years
may change genome size. DNA content-frequency histogram
Fig. T. hemisphaerioides conidia. Most of the
conidia are in cell cycle phase G1
(denoted as M1, CV 8%). Ratio M2/M1=2.0
 Calibration standard S. cerevisiae for C-values of fungal DNA

Dividing cells of S. cerevisiae

may cause problems related
to the obtaining of cells of
the G1 phase of the cell
cycle due to presence of an
active cell cycle.

Fig. 2. S. cerevisiae cells (Y1). Cells in cell cycle phase G1

are represented by the first peak and cells in cell cycle
phase G2M, by the second peak

Cells can be used as a Fig. 3. S. cerevisiae. Before fixing the cells were stored for
standard when cell cycle has some days at 4°C. Most of the cells are in cell cycle phase
stopped. G1 (denoted as RN1, CV 14%). Ratio RN2/RN1=2.1
Kullman 2000
Calibration standard Pleurotus ostreatus
for C-values of fungal DNA

The spore print of Pleurotus ostreatus (TAA 142824)

served as the standard, with a DNA content of 25 Mb
estimated by FC using S. cerevisiae (YAC 13,1 Mb) Kullman 2000

If you cut off a fruitbody, turn it gill

side down on a piece of paper and
leave for a couple of hours, the
falling spores will produce a
spore print

The intraspecific C-value variability in P. ostreatus is reported

to be 20.8 to 35.6 Mb (34.3 and 35.6 Mb with total sequencing)
http://www.zbi.ee/fungal-genomesize/
YES
Some examples of searches:
Spores of P. ostreatus treated with methanol and RNAse and stained with PI

P1 P2

P1 P2

FACSDiva Version 6.1.3

RNAse has digested cellular RNA

 Spores of P. ostreatus treated with methanol,
but not with RNAse, and stained with PI

P1 P2

P1 P2

FACSDiva Version 6.1.3

Methanol fixation is not sufficient in order to degrade cellular RNA

 Some examples of searches:
Spores of P. ostreatus treated with Zymolyase

Fig. 13. Clean nuclei gated on the dot

plot of FSC/SSC to exclude aggregates.
Nuclei in cell cycle phase G1 are
represented by a peak (NR1).

Fig. 14. damaged spores which have

been gated on the dot plot of
FSC/SSC. The fluorescence intensity of
clean nuclei is denoted by RN1 as in
Fig. 13.
This treatment has removed also the AF and of
spores. Some nuclei are released from spores.
Using this method three ploidy levels (2x, 3x, 6x) were found in
the genus Cystoderma :
2x C. amianthinum (29.5 Mb)
C. carcharias (25.3 Mb)
C. jasonis (25.9 Mb)

3x C. adnatifolium (35.9 Mb)

C. granulosum (37.3 Mb)

6x C. terreii (65.4 Mb)

Surprisingly, the correlation between DNA count and spore sizes (volume, length and width)
within the genus is negative;
it means that in these case species with larger spores have lower DNA content than species
with smaller spores.
Saar & Kullman 2000. Folia Cryptog. Estonica, Fasc. 36: 87.94
Calibration standard P. ostreatus for C-values of fungal DNA

Spores of P. ostreatus serve as a better standard -

division of nuclei stops in the G1 phase of the cell cycle.

In a powder form, they can be used through many decades.

But only the spore print of a tested specimen can be used as
a standard due to intraspecific variability of genome size.
 PV PU
Staining with DAPI SR101 improves resolution

Simultaneous two-parameter analysis was

carried out with DAPI and SR 101 (as a protein
fluorochrome) in combination.
Protein content-frequency histogram
Two specimens of P. ostreatus PV and PU

Fig.15. DNA content histogram of spore print PV

Fig. 16. Protein content histogram of spore print PV
Fig. 17. DNA content histogram of spore print PU
Fig. 18. Protein content histogram of spore print PU DNA content-frequency histogram

Spore print PV denoted as R1

Two sub-populations of spore print PU are
denoted as R2 and R3
 Staining with DAPI SR101 improves resolution
Bi-parametric analysis of two specimens
of P. ostreatus
Two sub-populations of spore print PU
are denoted as R2 and R3 differing in

Protein content
DNA content by 4.9 Mb (20%) which
were not detected when only PI
staining or DAPI staining was used.

Staining with DAPI SR101 is essential

in the study of aneuploidy and
especially in genetic characterising of
heteroploids derived by intraspecific
hybridisation.
DNA content
 Staining with DAPI SR101

Bi-parametric analysis the

P. ostreatus and P. pulmonarius complex
can indicate polyploidisation and ongoing
speciation in this complex.
One fruitbody of P. ostreatus may produce two
distinct spore populations one of which is
comparable to those of P. pulmonarius.
Species P. pulmonarius is polyploid..

The difference in genome size and chromosome number within P. ostreatus appears as heteroploidy
(see Fungal Genome Size Database http://www.zbi.ee/fungal-genomesize/).
We presume that the divergence that arises from a spore print reflects the fate of a hybrid genome in meiosis.
Our results seem to confirm that parental genomes of different sizes segregate in meiosis.
Zygotic meiosis can occur even in the case of low density of homology between chromosomes
(CLP and aneuploidy) and may ensure distribution of highly different strains.

Kullman & Greve, 2007

 FC of filamentous fungi
For rapid and accurate analysis of the nuclear DNA content of flowering
plants the following are sufficient:
• Propidium iodide - a stock solution (1 mg/mL) in deionized water
stored in aliquots at -20°C
• RNAse A (10 mg/mL in water) stored at 4°C
• Nylon filters notional porosity of 30 μm
• Razor blades
However, it is not so easy in the case of filamentous fungi that have
fruitbodies with autofluorescence (AF)

David W. Galbraith and Georgina M. Lambert

 Autofluorescence (AF) of ascomycetes
fruitbody of Scutellinia sp.

hairs hymenium
AF of ascospores
Sarcoscypha austriaca
Stained with DAPI
 Sarcoscypha austriaca
• Teleomorph sexually reproductive form in the life cycle • Anamorph The asexual reproductive form in the life cycle
Fruitbody nuclei Spores and conidia

Spore nuclei

Nuclei in conidium

Bacteria

A few intact nuclei for FC

In most cases it is not
possible to use the
fruitbodies for FC.
Fig. 1. Closed mitosis. DNA is
concentrated into brightly
stained poles. a- a photo of a
DAPI-stained nucleus; b and c -
analysis with Image PRO.
The y axis denotes fluorescence
intensity. Bar = 5 ųm.

Fig. 2. Nuclei of paraphyses:

a - endopolyploid nuclei of
paraphyses;
b - unequal haploidization of the Fruitbody nuclei of Neottiella vivida
endopolyploid nucleus (ratio of TAA 179520 stained with DAPI.
DNA content 1:7). Bar = 10 ųm.
 It is more promising to measure genome size in pure cultures

Peziza lobulata TAAM179671 Peziza micropus TAAM179653

Fig. 3. Relative nuclear DNA content in the hyphae of a pure culture of Peziza lobulata TAA179671. The ultimate nucleus has
higher growth potential compared with its sister nucleus. Replication in the nucleus of the top cell is faster than division,
while the posterior nuclei can be divided without replication and stop at the 1C value.
As a result, there arise endopolyploidization (in the apical cells) and haploidization (in the posterior cells).
C – the C-value which corresponds to genome size. Bar = 5 ųm.
 Heterokaryosis through anastomosis and parasexuality
leading to diploidization and subsequently to haploidization
can play a role in the lability of nuclear DNA content in hyphae

Nuclei of
conidia are
the most
reliable for 2C
measuring
genome size 1C+1C=?C
anastomosis
endopolyploid 1C
1C
 Plant pathogenic fungi
Puccinia on a leaf
Basidiomycetes
FC of Rust fungi

• The application of the nuclear isolation protocol, developed

for plant tissues Galbraith et al. (1983), to rust-infected
host tissues enabled the release of intact nuclei from both
the host plant and the fungal cells
• According to the clearly defined G1 peaks of both organisms
30 rust species were analyzed

Tavares et al., 2014. Genome size analyses of

Pucciniales reveal the largest fungal genomes
 Plant pathogenic fungus
Releasing of intact nuclei from both Puccinia on a leaf

the host plant and the fungal cells

FC histograms of fluorescence intensities of PI-stained
Spore nuclei
nuclei simultaneously isolated from plant and hyphae:

(A) Hemileia vastatrix (Hv) and its host plant,

Coffea arabica (Ca; 2C = 2.49 pg DNA)
(B) Coffea arabica (Ca) and the plant DNA reference
standard, Raphanus sativus (Rs, 2C = 1.11 pg DNA)

(C) Hemileia vastatrix (Hv) 1C=?

Fungal nuclei
Host plant with Host plant with
obtained upon
fungal pathogen referent plant
germination of
spores in water
(A1) represents the gating in the dot-plot of SSC vs.FL
to exclude as maney partial nuclei as possible and
other types of debris. Tavares et al., 2014. Genome size analyses of
Pucciniales reveal the largest fungal genomes
Rapid and simple method for nuclear extraction from a single plant root tip,
can it be used for fungal hyphae as well?
• Enzymatic digestion and mechanical isolation of
nuclei during homogenization with a
commercial hand mixer
• Sufficient nucleare amounts are extracted with
the special buffer Partec GmbH (CyStain UV
Ploidy, code number 05-5004
One-step staining protocol of cellular DNA
http://www.sysmex-partec.com/reagents-
accessories/agroscience-aquaculture.html
Partec GmbH is now Sysmex Partec GmbH,
On the slide, four diamond-cut slide pieces were attached
with company head Office in Görlitz
with commercial glass glue so that they could form two
fissures—the longitudinal one, where the root is allocated,
and the narrow transversal one, for the passage of the razor
blade (b) 2-mL microtube. (c) hand mixer. Bars = 1 cm.
Silva et al. 2010. Improved and ReproducibleFlowCytometry
Methodology for NucleiIsolation from SingleRoot Meristem. Use a brand new razor blade under a stereoscopic
Journal of Botany microscope.
Rapid and simple way to extract nuclei from a plant root tip,
can it be used for hyphae as well ?

Morphological analysis of DAPI-stained nuclei

after 2- (a), 4- (b) and 6- (c) pulse homogenizations.

After running of 6 pulses the nuclei were well preserved,

isolated and showing no residual cytoplasm (CV 3.2%).
Bar = 10 μm.

Silva et al. 2010. Improvedand ReproducibleFlowCytometry Methodology for Nuclei Isolation from SingleRoot Meristem.
Journal of Botany
 Conclusions

Flow cytometry (FC) is a useful and an adequate method for measurement of

fungal genome size and for characterisation of fungal species.
Precise methods have been developed for quantitative evaluation of the DNA
content of fungal spores, conidia and intact cells using propidium iodide
or/and DAPI dyes.
When used correctly, FC can also provide exact information of genome size of
filamentous fungi. Nevertheless, Image Analysis proves essential in some
cases.
Experimental series obtained with the use of the same staining technique are
the most suitable in estimation of genome size.
Analyses for finding out slight differences in fungal genome size should be
made using the same fluorochrome and the same reference organism.
 Conclusions

• In estimation of fungal genome size the spores of Pleurotus ostreatus as

well as the conidia of Aspergillus fumigatus CEA10 can be suggested as the
primary standards.
• FC is also a convenient tool in measurement of relative spore dimensions
and in the study of relationship between spore/nuclear size and DNA
content.
• FC of spore prints should prove powerful in determination of the genome
size of a large number of similar specimens, collected from different
ecotypes and geographic areas, with the aim to study intraspecific
variability and speciation.
• The method has been increasingly used in mycology as evidenced by the
rapidly growing number of relevant articles.
 Relevant references
D'Hondt L., Hofte M., Van Bockstaele E., Leus L. 2011. Applications of flow cytometry in plant pathology for
genome size determination, detection and physiological status. Mol. Plant Pathol. 12, 815–828

Kullman B. 2000. Application of flow cytometry for measurement of nuclear DNA content in fungi. Folia Cryptog.
Estonica 36 : 31-46. http://www.ut.ee/ial5/fce/folia.html

Tavares S., Ramos A.P., Pires A.S., et al. 2014. Genome size analyses of Pucciniales reveal the largest fungal
genomes. Frontiers in Plant Science 5: 422.

Veselska T., Svoboda J., Ruzickova Z., Kolarık M. 2014. Application of Flow Cytometry for Genome Size
Determination in Geosmithia Fungi:
A Comparison of Methods. Cytometry Part A 85A: 854861.
http://www.slideshare.net/ediersen1/CSI-Labs-Training-Seminar-2007?related=1