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Cytological analyses of the karyotypes and chromosomes of three

Colletotrichum species, C. orbiculare, C. graminicola and C. higginsianum

Masatoki Taga a,⇑, Kaoru Tanaka b,1, Seiji Kato c, Yasuyuki Kubo b

a Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japanb Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japanc Yamanashi Prefecture Agritechnology Center, 5644 Asao, Akeno-cho, Hokuto 407-0201, Japan

a r t i c l e i n f o

Article history:

Received 24 May 2015

Revised 28 July 2015

Accepted 31 July 2015

Available online 1 August 2015

Keywords:

Colletotrichum

Chromosome

Karyotype

Heterochromatin

Cytology

a b s t r a c t

In contrast to the recent accomplishments of genome projects, cytological information on chromosomes

and genomes of the genus Colletotrichum is very scarce. In this study, we performed mitotic cytological

karyotyping for the three species, C. orbiculare, C. graminicola, and C. higginsianum by fluorescence micro-

scopy and compared the results with those from genome projects. Chromosome number (CN) of C. orbic-

ulare was determined for the first time to be n = 10 with no minichromosomes (MCs) in the genome,

while CNs of C. graminicola and C. higginsianum were consistent with those from their genome project

including the number of MCs. Regarding chromosome features, C. orbiculare was peculiar in that each

chromosome was distinctly partitioned into a highly AT-rich pericentromeric region and the remaining

highly GC-rich regions, and the pericentromeric region was judged to be constitutive heterochromatin.

Integrating all the discernible morphological characteristics such as chromosome length, nucleolar orga-

nizing region, and DAPI-stained regions, idiograms were constructed for the three species. The overall

cytological features of the chromosomes and genomes fit well with the data from the genome projects

in terms of genome size, GC-content, and the occurrence of AT-rich regions. This study represents the

most comprehensive and detailed mitotic cytological karyotyping of fungi ever reported.

2015 Elsevier Inc. All rights reserved.

1. Introduction

Colletotrichum is a large genus comprising many imperfect spe-

cies classified as Glomerella (subdivision Ascomycotina) in their

perfect state (Hyde et al., 2009). Colletotrichum fungi occur world-

wide and cause anthracnose diseases in a wide range of dicotyle-

donous and monocotyledonous hosts, posing vast losses in food

production (Prusky et al., 2000). Thus, Colletotrichum is recognized

as one of the most important genera of plant-pathogenic fungi.

Besides their economic importance, members of this genus have

provided excellent experimental models in both fungal biologyand plant pathology. For instance, C. orbiculare and C. lindemuthi-

anum have been used for many years to study the molecular and

cellular bases of differentiation of infection structures and estab-

lishment of hemibiotrophic infection in fungi (Kubo, 2012; Kubo

and Takano, 2013; Perfect et al., 1999). Plant–pathogen

interactions such as host resistance and signal transduction have

also been studied with Colletotrichum species (Hiruma et al.,

2010; Narusaka et al., 2009; Tanaka et al., 2009).

Information of karyotype is fundamental for the analysis of

eukaryotic genomes. In Colletotrichum, karyotyping has been

attempted using cytology, pulsed-field gel electrophoresis (PFGE),

and optical mapping. Regarding cytology, as far as we know, only

two papers have been published on Colletotrichum: one for G. cin-

gulata (Colletotrichum stage not mentioned in the paper) (Lucas,

1946) and the other for C. lindemuthianum (Roca et al., 2003). In

both papers, four meiotic chromosomes were observed using con-ventional light microscopy. On the other hand, karyotyping by

PFGE was used to estimate chromosome number (CN), size of chro-

mosomal DNA (chDNA), and chromosome polymorphism of C.

gloeosporioides (Masel et al., 1990; Garrido et al., 2009), C. linde-

muthianum (O’Sullivan et al., 1998) and C. acuatum (Garrido

et al., 2009). Curiously, there was a great discrepancy between

the results from PFGE and cytology for C. lindemuthianum, that is,

n = 9–12 by PFGE (O’Sullivan et al., 1998) vs. n = 4 from meiotic

cytology (Roca et al., 2003). Recently, optical mapping, a physical

mapping method that is increasingly used in microbial genome

projects (for reviews, see Neely et al., 2011; Schwartz and Samad,

http://dx.doi.org/10.1016/j.fgb.2015.07.013

1087-1845/ 2015 Elsevier Inc. All rights reserved.

⇑ Corresponding author.

E-mail addresses: [email protected] (M. Taga), [email protected].

jp (K. Tanaka), [email protected] (S. Kato), [email protected] (Y. Kubo).1 Present address: Central Research Institute, Ishihara Sangyo Kaisha, Ltd., Shiga,

Japan.

Fungal Genetics and Biology 82 (2015) 238–250

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1997), was used in the genome projects for C. graminicola and C.

higginsianum, which we believe provided conclusive data of CN,

size of chDNAs and total genome size of the two species

(O’Connell et al., 2012). Overall, only a few species have been sub-

jected to karyotyping in Colletotrichum, and reliable information of

karyotype is limited.

In this study, we performed mitotic cytological karyotyping for

C. orbiculare, C. graminicola

, and C. higginsianum

. These three spe-

cies were chosen because their genome projects were completed

ahead of other Colletotrichum species (Gan et al., 2013; O’Connell

et al., 2012), and we expected that cytological data from our study

would complement sequence-based results from genome projects

to lead to more comprehensive understanding of the genomes of

Colletotrichum. An added merit for using these species was that

we might discover some aspect of karyotype evolution in this

genus by comparing their karyotypes in relation to the latest

molecular phylogenetic tree that placed the three species in differ-

ent major clades (Cannon et al., 2012). So far, no cytological kary-

otyping has been published for fungi from such a point of view.

For obtaining reliable cytological karyotypes in this study, we

used nonconventional cytological techniques instead of conven-

tional techniques. They were the germ tube burst method (GTBM)

to prepare slide specimens of spread chromosomes (Shirane et al.,

1988; Taga et al., 1998), fluorescent staining and fluorescence

microscopy to visualize chromosomes, and fluorescence in situ

hybridization (FISH) to detect any chromosomal region(s) with cer-

tain DNA sequences (for reviews in fungi, see Tsuchiya and Taga,

2010). The superiority of these techniques for karyotyping filamen-

tous fungi hasbeenproven, for instance, in Nectria (Tagaet al., 1998;

Mahmoud and Taga, 2012), Alternaria (Akamatsu et al., 1999),

Cochliobolus (Tsuchiya and Taga, 2001), and Cryphonectria

(Eusebio-Cope et al., 2009). Consequently, cytological karyotypes

of the three species that incorporated detailed information of chro-

mosome morphology as well as definitive CN were determined. To

our knowledge, this is the most detailed cytological analysis of fun-

gal karyotype with mitotic chromosomes ever reported.

2. Materials and methods

2.1. Fungal strains

Two strains of C. orbiculare and one strain each of C. graminicola

and C. higginsianum were used. C. orbiculare strain 104-T

(MAFF240422) was a gift from Y. Takano. It was isolated in Japan

from cucumber (Yasumori, 1962) and has served as a representa-

tive lab strain of this species for more than 50 years. Another strain

SGN04-20 of C. orbiculare was isolated from cucumber in 2004 in

Japan and obtained from the culture collection of Kyoto

Prefectural University. C. graminicola strain M1.001 (also known

as M2) was collected in Missouri from maize (Forgey et al., 1978)

and sent to us by L. Vaillancourt. C. higginsianum strain IMI349063 was obtained from R.J. O’Connell. It was isolated from

Brassica rapa (pak-choi) in Trinidad and has been used for molecu-

lar biological study (O’Connell et al., 2004). The entire genomes of

these strains, except SGN04-20, have already been sequenced; the

results for M1.001 and IMI 349063 were published in 2012

(O’Connell et al., 2012) and in 2013 for 104-T (Gan et al., 2013).

The overall features of these strains and species including their

habitats and genomic data were described in a review by Crouch

et al. (2014). All strains were maintained on potato dextrose agar

(PDA) as slant cultures at 24 C.

2.2. Protoplast preparation and pulsed-field gel electrophoresis

Small mycelial agar plugs cut from a PDA plate culture of 104-Twere incubated at 24 C for 2 days in 200 ml of potato sucrose

broth supplemented with yeast extract (PSY; broth from 200 g of

potato, 20 g of sucrose, and 2 g of yeast extract per liter) (Takano

et al., 2001) on a rotary shaker at 120 rpm. Mycelia were harvested

by filtration on gauze, rinsed with distilled water, and treated with

enzyme solution [10 mg of Lysing Enzymes from Trichoderma har-

zianum (Sigma–Aldrich, St. Louis, MO), 5 mg Kitalase (Wako Pure

Chemicals, Osaka), and 10 mg of driselase (Kyowa Hakko, Tokyo)

per ml of 1.2 M MgSO4

amended with 10 mM Na2

HPO4

] at 30 C

for 5 h with gentle agitation to release the protoplasts.

Protoplasts were then filtered through three layers of Kimwipe

(Nippon Paper Crecia) into a plastic tube. Trapping buffer (0.6 M

sorbitol) was overlaid on the protoplast suspension and cen-

trifuged at 600 g for 7 min to collect the protoplasts at the interface.

The harvested protoplasts were washed in 1 M sorbitol twice by

centrifugation, first at 650 g for 7 min and second at 450 g for

7 min. The final pellet was suspended in SE (1 M sorbitol, 50 mM

EDTA, pH 8.0), and agarose plugs containing ca. 1.7 108 proto-

plasts/ml were made according to Taga et al. (1998).

ChDNAs were separated in 0.8% agarose gel (pulsed-field certi-

fied grade agar, Bio-Rad, Hercules, CA) using a contour-clumped

homogeneous electric field (CHEF) type of apparatus (CHEF DR II,

Bio-Rad) with the running conditions of Taga et al. (1998). DNA

size markers from Schizosaccharomyces pombe and Hansenula win-

gei were purchased from Bio-Rad.

2.3. Germ tube burst method (GTBM)

A conidial suspension for the GTBM was prepared as follows.

Strains 104-T and SGN04-20 were cultured on PDA plates and

strain IMI 349063 was grown on oatmeal agar plates (30 g of pow-

dered Quaker brand oats and 15 g of agar per liter) for 1 week at

22–24 C in the dark. The plate cultures were then flooded with

PSY (ca. 2 ml per culture in 9-cm diameter Petri dish), followed

by repeated pipetting with a Pasteur pipette to harvest conidia in

PSY. Conidial concentration was then adjusted to ca. 3 105/ml

with PSY. For strain M1.001, PSY in a flask was inoculated with

small mycelial agar plugs cut from PDA plates and shaken on arotary shaker at 50 rpm for 2 days at 24 C. Conidia produced in a

budding-like manner in the medium were filtered through two lay-

ers of Kimwipe, pelleted by centrifugation, and finally resuspended

in PSY at ca. 3 105/ml.

This conidial suspension was then subjected to the GTBM to

make slide preparations of mitotic chromosomes as described by

Tsuchiya and Taga (2010) with some modifications. Briefly,

150ll of conidial suspension was incubated for germination on a

poly-L-lysine-coated slide at 28 C in a humid chamber. After incu-

bation for 18–19 h for 104-T and SGN04-20, 9–10 h for M1.001,

and 10 h for IMI 349063, the PSY on each slide was replaced with

fresh PSY containing 100lg/ml of thiabendazole (TBZ), with 1

more hour of incubation. The slide was then dipped in water to

wash off the medium, immersed in fixative (9:1 ethanol to aceticacid) for 30 min at room temperature, flame-dried, and stored in

a desiccator at room temperature until use.

2.4. Fluorescence staining and fluorescence microscopy

Slide specimens were stained with a mixture of DAPI (1 lg/ml)

and propidium iodide (PI) (0.5lg/ml) (hereafter, called DAPI/PI)

dissolved in antifading mounting solution ( Johnson and Araujo,

1981), then observed with an epifluorescence microscope

(Olympus BH-2/BHS-RFC) and a 100 oil immersion objective lens

(N.A 1.3). For fluorescence observation, either UV or G excitation or

a triple band pass filter (D/F/R 612 BP405, Chroma Technology,

Bellows Falls, VT) was used. Images were captured with an

Olympus DP70 CCD camera attached to the microscope. When nec-essary, separately captured images using UV or G excitation were

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merged using the Olympus DP Manager software supplied with the

CCD camera. Other image processing including cutting and align-

ing individual chromosomes of a spread was done using Adobe

Photoshop CS5 (Adobe Systems).

2.5. Fluorescence in situ hybridization (FISH)

Slide specimens to be used for FISH were first treated with

100lg/ml RNase A in 2 SSC (1 SSC: 0.15 M NaCl, 0.015 M

sodium citrate) for 1 h, followed by a brief rinse with 2 SSC,

and dehydrated through an ethanol series (70–85–99%, each for

5 min). The air-dried specimens were stained with DAPI/PI and

photographed as mentioned. Then, cover glasses were removed

and the slides gently rinsed with 2 SSC for 1 h on a shaker to

wash off the mounting solution. Finally, the slide specimens were

dehydrated with ethanol as described, air-dried, and subjected to

hybridization. As the probe for FISH, a plasmid clone pABM1

(Tsuge et al., 1989), which contains almost half of 18S and 28S

rRNA genes as well as the entire of 5.8S rRNA gene in the repeating

unit of ribosomal DNA (rDNA) of Alternaria alternata, was used.

Probe labeling was done with BioNick Labeling System

(Invitrogen, Life Technologies, Carlsbad, CA) according to the man-

ufacturer’s protocol. Hybridization and hybridization detection

with avidin-FITC (Boehringer Mannheim Biochemicals,

Indianapolis, IN) were done as described previously (Taga et al.,

2003). For excitation of FITC, an Olympus DMIB cube was used.

2.6. Giemsa staining

For Giemsa staining of the GTBM-prepared specimens, the con-

ventional method called HCl-Giemsa and a novel method first

reported in fungi here (hereafter, called urea-Giemsa) were used.

The HCl-Giemsa procedure was that of Shirane et al. (1988) with

some modifications. Briefly, GTBM-prepared slides were sequen-

tially dipped in 95% for 10 min and 70% ethanol at room tempera-

ture for 3 h, and air-dried. The slides were then treated according

to the method of Shirane et al. (1988) (1 M HCl for 5 min at roomtemperature and then for 10 min at 60 C) or ours (0.2 M HCl for

10 min at 60 C). After rinsing with water, the slides were stained

with 3.5% Giemsa solution (Merck KGaA, Darmstadt) diluted with

1/15 M Sørensen’s phosphate buffer (pH 7.0) for 3.5 h, followed

by brief washing with water and air-drying. The slides were finally

mounted in Entellan (Merck).

Urea-Giemsa staining followed a modified version of the tech-

nique of Shiraishi and Yoshida (1972) that was originally devel-

oped for human chromosomes as a substitute for ordinary

G-banding technique. Slides were treated with 6 M urea dissolved

in distilled water for 10 min at 37 C and immediately transferred

to 5% Giemsa solution diluted with 1/15 M Sørensen’s phosphate

buffer (pH 7.0). After staining for 8.5 h at room temperature, the

slides were briefly washed with water, air-dried, and mounted inEntellan. Giemsa-stained specimens were observed with an

Olympus BH2 bright-field microscope with a 100 oil immersion

objective lens (N.A 1.35). Micrographs were taken using an

Olympus DP70 CCD camera attached to the microscope.

For serial staining with DAPI/PI and urea-Giemsa, slides were

first stained with DAPI/PI and observed with a fluorescence micro-

scope. Then, cover glasses were carefully removed from the slides,

and the slides soaked in 2 SSC for 5 min to wash off the antifade

mounting solution. Finally, the slides were stained and observed

using the urea-Giemsa technique.

2.7. Chromosome alignment and idiograms

Chromosomes were aligned by cutting each chromosome fromthe original image using Adobe Photoshop CS5. The cut

chromosomes were first arranged by longitudinal axial length

(hereafter, referred to as chromosome size). Then, the order of

chromosomes in the arrangement was modified so that chromo-

somes sharing similar features other than size were ranked the

same among alignments of different nuclei. Finally, the aligned

chromosomes were numbered in ascending order. The chromo-

some sizes were measured using ImageJ v.1.38x (National

Institutes of Health, Bethesda, MD, USA; http://rsbweb.nih.gov/

ij/download.html). For constructing idiograms, staining intensity,

margin of DAPI- or PI-stained regions and position of constriction

were decided by eye-inspection, and axial lengths of the regions

were measured with ImageJ. Integrating information of relative

chromosome size, constrictions of putative centromeres, DAPI-

and PI-stained regions and nucleolar organizing region (NOR), idio-

grams were drawn with Microsoft PowerPoint 2007.

3. Results

3.1. Pulsed-field gel electrophoresis (PFGE) of C. orbiculare

While CN and physical sizes of chromosome complements for C.

graminicola strain M1.001 and C. higginsianum strain IMI 349063had already been determined by optical mapping (O’Connell

et al., 2012), no information of chromosome complements was

available for C. orbiculare even if its genome project was completed

with the strain 104-T. Therefore, C. orbiculare strain 104-T was ana-

lyzed with PFGE before performing cytological karyotyping. Using

the running conditions to separate a wide size range of DNA from

ca. 6 Mb to


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partitioned into distinctive segments that were preferentially

stained with either DAPI or PI (Fig. 3A and B). Thus, superimposing

the separate images taken by UV and G excitation with the aid of a

computer software (Fig. 1C) or using a multiple band path filter

that can simultaneously excite both DAPI and PI (Fig. 1D) was

necessary to capture the whole image of each chromosome in C.

orbiculare. In the subsequent analyses of C. orbiculare, therefore, a

triple band pass filter was used for observation. Contrary to the

case of C. orbiculare, UV excitation gave a much clearer image of

the whole chromosome than using the triple band path filter for

observing DAPI/PI-stained specimens of C. graminicola and C. hig-

ginsianum. Hence, most observations were made by UV excitation

for these species.

3.3. Karyotyping of C. orbiculare

The two strains, 104-T and SGN04-20, were karyotyped with

metaphase spreads of good quality that were selected based on

that chromosomes were not overlapped and retained undistorted

morphology. The representative alignments for the two strains

and idiograms that diagrammatically illustrate the features of each

chromosome are shown in Fig. 4. The results are summarized

below.

3.3.1. Chromosome number

When chromosomes in more than 20 metaphase spreads for

each strain were counted, the count was consistently 10. Hence,

CN was unambiguously determined to be n = 10 for both strains.

3.3.2. AT-rich segment

In each strain, 8 of 10 chromosomes, i.e., chromosomes 1–8 of

104-T and chromosomes 1–7 and 9 of SGN04-20, had a conspicu-

ous, internal segment that preferentially stained with DAPI (here-

after, called DAPI-band) (Fig. 4). Because of the binding

specificity of DAPI to A-T, the DAPI-bands were thought to be

AT-rich, and in total occupied around 40% of the sum of the longi-

tudinal axial length of all chromosomes in each strain. Although

less conspicuous than the DAPI-bands just described, relatively

intense DAPI-stained regions were recognizable in the remaining

two chromosomes (also called DAPI-bands). Highly contrasting

with the DAPI-bands, the other parts of the chromosomes were

intensely stained in red, indicating preferential binding of PI tothese regions. Taking into account that PI is known to bind to

DNA in a non-base-specific manner, this result indicates that these

regions are GC-rich with low affinity to DAPI to lead to dominant

binding with PI instead of DAPI. As a result, it is reasonable to

say that chromosomes of C. orbiculare are largely partitioned into

segments of an AT-rich or GC-rich nature.

In addition to metaphase, nuclei in other stages were observed

with attention to the DAPI-bands. Interestingly, DAPI-bands in the

interphase nucleus were clustered to occupy a distinct area at the

periphery of the nucleus (Fig. 5A, also see Fig. 2A). In early pro-

phase, DAPI bands remained at the hemispherical periphery of

the nucleus, while the chromosome ends were at the other hemi-

sphere (Fig. 5B). In late prophase, the cluster of DAPI-bands had

dissolved in accordance with the dissociation of chromosomes(Fig. 5C). Given that centromeres of at least several chromosomes

reside in the DAPI-bands (see 3.3.3 below), the observed clustering

of DAPI-bands and the orientation of the chromosome ends likely

represents the Rabl orientation of chromosomes.

3.3.3. Chromosome morphology

The condensed metaphase chromosomes were rod-shaped.

Although they are thought to comprise two sister chromatids,

structures indicative of sister chromatids were not discernible.

The longitudinal axial lengths of the largest and smallest chromo-

some were ca. 3–4 lm and 1–2 lm, respectively, depending on the

specimens of the two strains. For 5 randomly chosen specimens of

104-T, size ratios of the largest to the smallest ranged from 2.2:1 to

2.7:1. Constrictions probably representing centromeres were rec-ognized within DAPI-bands on several chromosomes. Also, some

Fig. 1. Ethidium-bromide-stained CHEF–PFGE gel separating chromosomal DNA of

Colletotrichum orbiculare strain 104-T. An agarose plugs containing 1.7 108

protoplasts/ml was used. Running conditions were described in Section 2.

Schizosaccharomyces pombe (Sp) and Hansenula wingei (Hw) were used as size

standards, and selected chromosome sizes are shown to the left.

Fig. 2. Enlargement of the size of metaphase chromosomes by using the germ tube

burst method (GTBM). Specimens of Colletotrichum orbiculare strain 104-T were

prepared with the GTBM, double-stained with DAPI and PI and observed with a

triple band pass filter. (A) Interphase nuclei in a germ tube. (B) Metaphase nuclei in

a germ tube. (C) Metaphase nuclei discharged outside a germ tube. In (A–C),

whitish-blue to blue fluorescence and red fluorescence indicate preferential

staining with DAPI and PI, respectively. All figures are at the same magnification.

Scale bar: 2 lm. (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)


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chromosomes were bent within or at the margin of the DAPI-band

(for example, chromosomes 3 of the two strains in Fig. 4).

3.3.4. Nucleolar organizing region (NOR)

In each metaphase spread of both strains, a particular chromo-

some had a relatively large region that stained distinctively

dark-brownish. These regions were located interstitially in 104-T

and distally in SGN04-20 (see chromosome 2 in Fig. 4A and chro-

mosome 5 in Fig. 4B). Subsequent FISH analysis of 104-T showed

that this region specifically hybridized with the rDNA probe

(Fig. 6A and B), indicating that it is NOR. Thus, we concluded thatthe NOR is distinguishable by its distinctive color and that both

strains have only one NOR in their genomes. Interestingly, the

NOR of SGN04-20 was long and thread-like at late prophase

(Fig. 6C) and club-like at presumable prometaphase (Fig. 6D).

3.3.5. Chromosome identification

In each spread of the two species, chromosome sizes of individ-

ual chromosomes had some degree of sample variation (examples

shown in Supplementary Fig. 1). For instance, the size of chromo-

some 2 (NOR-chromosome) including the region of the NOR was

the largest in some samples of 104-T (Fig. 4A), whereas it ranked

third or fourth largest in the other samples (Supplementary

Fig. 1). Therefore, definite chromosome identification relying solely

on chromosome size was not possible. To reliably identify individ-ual chromosomes, integration of chromosome size, DAPI-band and

NOR was useful for identifying chromosomes 2 and 7–10 in 104-T

and chromosomes 1 and 6–10 of SGN04-20 (see idiograms in

Fig. 4).

3.3.6. Karyotype polymorphism

Comparing relative chromosome sizes, DAPI-bands and

NOR-chromosomes, intraspecific variation of karyotypes was evi-

dent between 104-T and SGN04-20. Especially for the

NOR-chromosomes, the difference in the position of NOR (intersti-

tial vs. distal) and chromosome size (large vs. middle size) was evi-

dence of chromosome rearrangements.

3.3.7. Giemsa staining

To assess the stainability of GTBM-prepared specimens with

conventional stains, specimens of 104-T were stained with

HCl-Giemsa or with the urea-Giemsa staining technique. For

HCl-Giemsa staining, the commonly used hydrolytic condition

with 1 M HCl and much weaker hydrolysis with 0.2 M HCl yielded

similar results. That is, chromosomes were stained along the entire

lengths except for the NOR region, that was faintly stained

(Fig. 7A and B). Segments such as DAPI-bands that were observed

using fluorescence staining were not clearly distinguishable with

HCl-Giemsa. Urea-Giemsa, on the other hand, somewhat differen-

tially stained chromosomes; regions that probably corresponded toDAPI-bands were densely stained with vague delineation (Fig. 7C).

Fig. 3. Compartmentalization of chromosomes into AT- and GC-rich regions in Colletotrichum orbiculare strain 104-T. A metaphase nucleus double-stained with DAPI and PI

was observed using different excitation methods. (A) UV excitation for DAPI staining. (B) G excitationfor PI staining. (C) Superimposition of the images of A and B. (D) Images

obtained with a triple band path filter. Scale bar: 2 lm.

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Regarding urea-Giemsa, serial staining coupled with DAPI/PIstaining was also attempted to correlate the results from the two

staining methods. In this serial staining, DAPI-bands were shown

to correspond to the regions densely stained by urea-Giemsa

(Fig. 7D–G). Since urea-Giemsa is known to visualize G-bands in

mammalian chromosomes, our result suggests that DAPI-bands

of C. orbiculare have a G-band-like nature.

Overall, the images with the two Giemsa staining techniques

were rather vague and ill defined compared to those of the fluores-

cence staining. Thus, detailed observation of chromosome mor-

phology was difficult with these techniques.

3.4. Karyotyping of C. graminicola

The karyotype of the standard strain, M1.001, was analyzedwith the DAPI/PI-stained specimens. Since UV-excitation enabled

visualization of the chromosomes in more detail than the use of a triple band path filter (compare Fig. 8A with Fig. 8B), we used

UV-excitation for the karyotyping. Chromosome alignments with

spreads at prometaphase (Fig. 8A) and metaphase (Fig. 8C) are

illustrated in Fig. 9 in combination with an idiogram for the prome-

taphase alignment. Interphase nuclei were also observed to assess

the configuration of chromosomes in this stage (Fig. 8D). The

results are summarized below.


For 14 selected spreads, the CN of M1.001 was analyzed. In

every spread, chromosomes were categorized into two types, i.e.,

ordinary, rod-shaped chromosomes and dot-like MCs. Counts for

the ordinary type of chromosomes were consistently 10, whereas

those for the MCs varied from one to three depending on thespreads (9 spreads with 2 MCs, 4 with 3 MCs, 1 with 1 MC) as

Fig. 4. Cytological karyotypes of Colletotrichum orbiculare strains 104-T and SGN04-20. Mitotic metaphasechromosomes double-stained withDAPI and PI wereobserved with

a triple band path filter. (A) Karyotype of 104-T. (B) Karyotype of SGN04-20. Left image in each panel is the original micrograph of a spread used for chromosome alignment.

Idiograms show relative chromosome length, putative centromeric constriction, position and size of DAPI-bands and nucleolar organizing region (NOR). DAPI-bands with

different fluorescence intensities are in different colors. The numbers 1–10 below the idiogram are chromosome numbers assigned to individual chromosomes. The numerals

below chromosome alignment indicate relative ratios of chromosome length. Arrowheads indicate NORs. Scale bars: 2 lm.

Fig. 5. Interphase and prophase nuclei of Colletotrichum orbiculare strain 104-T. (A) Interphase nuclei. A distinct bluish white DAPI-stained area, formed by the clustered

DAPI-bands, is seen at the periphery of each nucleus. (B) Early prophase nucleus. Note that DAPI-bands and chromosome ends are at opposite sides of the nucleus. (C) Late

prophase nucleus. Chromosomes are more condensedand partitioned intohighly AT-rich (DAPI-bands) and highly GC-rich regions. Scale bars:2 lm. (Forinterpretation of the

references to colour in this figure legend, the reader is referred to the web version of this article.)


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Fig. 6. Nucleolar organizing region (NOR) of Colletotrichum orbiculare. (A and B) Detection of NOR by fluorescence in situ hybridization (FISH) in strain 104-T. (A) DAPI/PI-

stained images of a nucleus at late prophase nucleus and a metaphase chromosome cut from the image of a different nucleus (inset) before FISH. (B) FISH signals on

specimens shown in A. (C and D) NOR in strain SGN04-20. C: Late prophase nucleus. (D) Presumable prometaphase nucleus and a metaphase chromosome cut from the other

nucleus (inset). Arrowheads in A, C and D mark the dark-brownish fluorescence that distinguishes the NOR. The arrow in C shows a small reddish knob on a distal end of a

chromosome, suggesting that the true chromosome end is outside the NOR. Scale bars: 2 lm (1 lm in inset). (For interpretation of the references to colour in this figure

legend, the reader is referred to the web version of this article.)

Fig. 7. Giemsa staining of mitotic metaphase chromosomes of Colletotrichum orbiculare strain104-T. (A andB) Metaphase nucleus stained by theHCl-Giemsa technique using

1 M HCl (A) or 0.2 M HCl (B) for hydrolysis. Arrowheads indicate NORs. (C) Metaphase spread stained by the urea-Giemsa technique. (D–G) Serial staining with DAPI/PIfollowed by urea-Giemsa of the same specimens. (D and E) Metaphase. (F and G) Interphase. Scale bars: 2 lm.



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Fig. 8. Spread specimens of Colletotrichum graminicola strain M1.001. Prometaphase (A and B), metaphase nuclei (C) and interphase nuclei (D) were stained with DAPI/PI and

observed under UV-excitation (A, C and D) or with a triple band pass filter (B). Arrows indicate NORs, arrowheads mark minichromosomes. Scale bars: 2 lm.

Fig. 9. Chromosome alignments of Colletotrichum graminicola strain M1.001. The two alignments were made using the prometaphase and metaphase spreads shown in

Fig. 8A and C. In the idiogram for the prometaphase spread, relative chromosome length, position and size of DAPI-bands and nucleolar organizing region (NOR) are

integrated. DAPI-bands with higher fluorescence intensity are shown in blue; those with lower fluorescence intensity are in pale blue. Numbers 1–13 are the chromosome

numbers assigned to the individual chromosomes. Numerals below the chromosome alignment in A and at the bottom in B indicate relative ratios of chromosome length,

where NORwas omittedfrom size measurement. Arrows indicate knobs, arrowheads markNORs. Scale bars:2 lm.(For interpretationof the references to colour in this figurelegend, the reader is referred to the web version of this article.)


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shown in Fig. 8A and C. Because the spreads with 2 MCs formed the

majority and artifacts such as loss of minute chromosomes like

MCs from the spread during slide preparation were likely, we

determined the cytological CN of this strain to be n = 13, the same

as the result of optical mapping (O’Connell et al., 2012).

3.4.2. Morphological features of chromosomes

In the prometaphase alignment, chromosomes aside from theMCs could be classified into two groups based on their sizes, i.e.,

large chromosomes (chromosomes 1–5) and mid-sized chromo-

somes (chromosomes 6–10) (Fig. 9A). In the metaphase alignment,

on the other hand, the sizes of chromosomes except MCs decreased

more or less continually from chromosome 1 to chromosome 10 in

the alignment (Fig. 9B). Hence, size-based grouping of chromo-

somes, as was possible in the prometaphase alignment, was diffi-

cult in the metaphase alignment.

In both the prometaphase and metaphase alignments, chromo-

some 10 was designated as the NOR-chromosome, based on the

presence of a thread-like protrusion from the chromosome apex

(arrowheads in Fig. 9A and B).

3.4.3. Fluorescent bands and knobsChromosomes other than MCs were characterized by fluores-

cent bands (hereafter, called DAPI-bands as in C. orbiculare) or

knobs (called DAPI-knobs) that were intensely stained with DAPI.

The DAPI-bands were more distinct in prometaphase chromo-

somes than in metaphase chromosomes (Fig. 9A and B), and distri-

bution of the bands was unique to each chromosome. As to the

DAPI-knobs, the ends of chromosomes 3 and 9 were accompanied

by the conspicuous DAPI-knobs, which could serve as a reliable

marker for identifying these chromosomes. Integrating this infor-

mation and chromosome size, we constructed an idiogram of the

prometaphase chromosome complements (upper panel, Fig. 9A).

In interphase nuclei, many DAPI-stained speckles of various

shapes and sizes that probably correspond to DAPI-bands or

DAPI-knobs were scattered across the nucleus (Fig. 8D), which con-

trasted to the interphase nuclei of C. orbiculare that showed clus-

tering of DAPI-bands in a region (Fig. 5A).

3.5. Karyotyping of C. higginsianum

The karyotype of the standard strain, IMI 349063, was analyzed

using DAPI-PI-stained specimens and UV-excitation. A selected

specimen and chromosome alignment are illustrated in Figs. 10

and 11.


In each of 22 good specimens examined, chromosomes were

either ordinary, rod-shaped chromosomes or dot-like MCs as in

C. graminicola (Figs. 10B and 11). The number of ordinary chromo-somes was consistently 10, while the number of MC varied from 0

to 3 depending on the spread. Of the 24 spreads, 18 had 2 MCs, 3

had 1 MC and 1 had 0 MC. Since the spreads with 2MCs formed

the overwhelming majority, the CN of IMI 349063 was determined

to be n = 12, consistent with the result from optical mapping

(O’Connell et al., 2012).

3.5.2. Features of chromosomes

In the selected specimen shown in Fig. 11, chromosome sizes,

except for two MCs, were 2.4–1.0lm. Of the 12 chromosomes,

chromosome 7 was designated as the NOR-chromosome because

of its thread-like protrusion. In contrast to the cases in C. orbiculare

and C. graminicola, neither conspicuous DAPI-bands nor

DAPI-knobs were seen in the condensed metaphase chromosomes(Fig. 11). Also, speckles in the interphase nucleus as observed in C.

graminicola were not present except for an intensively stained spot

(Fig. 10C).

4. Discussion

4.1. Chromosome number

In this study, the CN for C. orbiculare, C. graminicola, and C. higginsianum was determined to be 10, 13, and 12, respectively; the

last two CNs also include MCs. According to the genome projects

of C. graminicola and C. higginsianum, the MCs of these species

are enriched in repetitive DNA (O’Connell et al., 2012) and can be

regarded as supernumerary or B chromosomes (Bs) (Crouch

et al., 2014). Therefore, it is rational to describe the CN of the

two species in the form of 10 + 3B for C . graminicola and 10 + 2B

for C . higginsianum, where the first numeral denotes the number

of ordinary or core chromosomes (hereafter, the core-CN).

Confining the CN to the core chromosomes, therefore, we can con-

clude that the three species have the same core-CN. In addition to

these species, we recently analyzed C. gloeosporioides (gloeospori-

oides clade) and C. truncatum (truncatum clade) using GTBM and

found that their core-CN is also 10 (Taga et al., 2014). Takentogether, the five species examined so far had the same core-CN

in spite of their belonging to different major clades in a recent

molecular phylogeny (Cannon et al., 2012). This finding is highly

contrasting to the case of the genus Fusarium, which exhibits

extensive species diversification as in the case of Colletotrichum

(O’Donnell et al., 2013): the core-CN in Fusarium species varies

from 4 for F. graminearum (Cuomo et al., 2007) to 14 for F. solani

(Coleman et al., 2009). Considering that a major mechanism

responsible for the range in core-CNs in the Fusarium is chromo-

some fusion (Ma et al., 2010), conservation of the core-CN in the

Colletotrichum species may be in part due to the lack of mecha-

nisms involved in chromosome fusion events. As to core-CN, it is

intriguing to know whether homologous or syntenic relationships

of chromosomes are present among the three species analyzedhere. Presently, the answer to this issue is limited to C. graminicola

and C. higginsianum, for which only 35% of the two genomes were

shown to be syntenic (O’Connell et al., 2012). Because C. orbiculare

is far-distantly related to the other species in the molecular phy-

logeny of Colletotrichum (Cannon et al., 2012), synteny between

C. orbiculare and C. graminicola or C. higginsianum is thought to

be significantly low, making identification of homologous chromo-

somes among these species difficult. Obviously, an analysis of syn-

teny and chromosome homology is beyond the reach of the

cytological method used here, and large-scale comparative geno-

mics will be needed. Regarding Bs, their occurrence is believed to

be a common feature in Colletotrichum (Crouch et al., 2014).

Although the two strains of C. orbiculare studied here did not con-

tain Bs, more isolates need to be surveyed to conclude that C. orbi-

culare does not have Bs as a rule.

4.2. Morphology of Colletotrichum chromosomes

Of the morphological features of chromosomes, chromosome

size measured in longitudinal axial length served to identify and

align chromosomes in our karyotyping. Apart from karyotyping,

the measurements of chromosome sizes are notable in that they

are to some extent proportionally correlated to the physical sizes

of chDNAs determined by optical mapping (Supplementary

Table 1 of O’Connell et al., 2012). Namely, with the exception of

the MCs, relative chromosome sizes in our measurements and opti-

cal maps are 2.3: 2.1: 2.0: 2.0: 1.8: 1.2: 1.2: 1.1: 1: 1 (Fig. 9A) vs.

2.1: 2.1: 1.9: 1.9: 1.8: 1.4: 1.3: 1.2: 1.1: 1 in C. graminicola, and2.1: 2.0: 1.9: 1.6: 1.5: 1.5: 1.4: 1.4: 1.3: 1 vs. 2.0: 2.0: 2.0: 1.9:


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1.7: 1.7: 1.5: 1.4: 1.2: 1 in C. higginsianum (Fig. 11). A similar pro-

portional relationship is also recognizable between the total value

of all chromosome sizes in a nucleus and the genome size: The

total sizes for C. orbiculare (Fig. 4A), C. graminicola (Fig. 9B), andC. higginsianum (Fig. 11) are in the ratio of 1.7: 1.2: 1, while the

ratios of genome sizes are 1.7 (88.8 Mb): 1.1 (57.4 Mb): 1

(53.4 Mb) (O’Connell et al., 2012; Gan et al., 2013). These data seem

to suggest that cytological measurements may have additional

uses other than karyotyping for analyzing Colletotrichum genomes.

Actually, by assuming a linear correlation between cytological

chromosome length and chDNA size in C. orbiculare, we succeeded

in estimating the genome size of this species to be 80–100 Mb

based on our cytological measurements (Taga et al., 2011). In fungi,

a correlation between cytological chromosome size and the size of

chDNA is also known for the meiotic pachytene chromosomes of

Neurospora crassa (Perkins, 1992; http://www.broadinstitute.org/

annotation/genome/neurospora/markers.html#correlation).

However, the use of mitotic specimens is thought to be more prac-tical in terms of the ease of preparing specimens and applicability

to a wide range of species.

In this study, the NOR served as a reliable morphological marker

to identify a specific chromosome in the genome called the

NOR-chromosome. The NOR in C. graminicola and C. higginsianum

extended as a long protrusion from the chromosome apex in meta-

phase, while the NOR of C. orbiculare in the same stage was rather

condensed and detectable by its distinctive color with

DAPI/PI-staining. The long protrusion of NOR has already been

reported in various filamentous ascomycetes (Shirane et al.,

1988, 1989; Taga and Murata, 1994; Taga et al., 1998; Tsuchiya

and Taga, 2001; Gale et al., 2005; Mahmoud and Taga, 2012) as

well as in fission yeast (Umesono et al., 1983), suggesting that it

is a prevailing feature of the fungal NOR. From the view of chro-matin structure, such a protrusion is thought to reflect a

less-condensed state of chromatin and may be underlain by the

chromatin architecture unique to the NOR as discussed by Taga

et al. (2003). While such protrusion of the NOR has been rarelyfound in plants and animals, the distinctive staining of NOR with

DAPI/PI as observed in C. orbiculare has been reported in plants

(for instance, see Andras et al., 2000). The relatively GC-rich nature

of NOR and the interaction modes of DAPI and PI with DNA may be

responsible for this distinctive staining (Peterson et al., 1999). It

seems reasonable to suppose that the same staining mechanism

is present for the NOR of C. orbiculare.

Besides chromosome size and the NOR, constriction and bend-

ing constituted morphological features of the chromosomes. We

presumed from the morphological criterion that constrictions rep-

resent centromeres, but we still need evidence for their being bona

fide centromeres. The widely accepted molecular proof for cen-

tromere identity is the association of a centromere-specific histone

H3 variant (CenH3 in the case of N. crassa) to the cetromeric DNA(Smith et al., 2012). In future studies, therefore, the constriction of

chromosomes should be examined for a histone H3 variant. As to

chromosome bending, it is common in chromosome specimens of

plants and animals, in which centromeres appear to behave like

a hinge for bending. In contrast, chromosome bending has rarely

been noted in fungi. Exceptionally, positions of centromeres were

assigned in Neurospora to the bending positions on the mitotic

metaphase chromosomes of the third division in ascus

(McClintock, 1945; Fincham, 1949; Singleton, 1953). Since bending

in C. orbiculare was linked to DAPI-bands encompassing cen-

tromeres and seems to be usable as a marker for centromere posi-

tion, bending might be similarly useful in other species that do not

have discernible pericentromeric DAPI-bands.

4.3. AT-rich segments (DAPI-bands) of C. orbiculare

The distinguishing feature of chromosomes in C. orbiculare was

the partitioning of each chromosome into a large AT-rich segment

(DAPI-bands) and the remaining GC-rich regions, which respec-

tively occupied around 40% and 60% of the genome as assessed

by the longitudinal axial length. Compatible with this result is

the finding of the genome project of this species that its genome

is constituted of AT-rich regions named AT blocks and the remain-

ing GC-rich parts; AT blocks make up 43.4 Mb (49.2%) of the gen-

ome with an average GC% of 19.25% (Gan et al., 2013). Although

the chromosomal locations of the AT blocks were not elucidated

in the genome project, considering the data from this study and

the genome project leads to the conclusion that the AT blocksreside predominantly in the DAPI-bands.

Fig. 10. Spread specimens of Colletotrichum higginsianum strain IMI 349063. Metaphase (A and B) and interphase nuclei (C) were double-stained with DAPI and PI and

observed under UV-excitation. A is the original image of B, in which one chromosome marked by an asterisk is moved from the original location in A. In B, arrow indicates

nucleolar organizing region, and arrowheads mark minichromosomes. Scale bars: 2 lm.

Fig. 11. Chromosome alignments of Colletotrichum higginsianum strain IMI 349063

for the spread shown in Fig. 10B. Below the chromosome alignment, chromosome

numbers (1–12) and relative ratios of chromosome lengths are shown, where

nucleolar organizing region was omitted from size measurement. Arrowhead

indicates nucleolar organizing region. Scale bar: 2 lm.


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The next issue to address then is how the DAPI-bands are

assembled with AT-blocks, though not much information is avail-

able to resolve this issue other than the AT blocks are largely

gene-sparse regions of low complexity sequences (Gan et al.,

2013). Further efforts to molecularly characterize the AT blocks

in more detail and allocate each identified AT block to the specific

site of a certain DAPI-band is necessary. To perform such an anal-

ysis, the fiber-FISH technique of Tsuchiya et al. (2002) for other

fungi should be exploited as well as an in silico analysis. Before

the present study, compartmentalization of chromosomes into AT

blocks and GC blocks has been reported in Leptosphaeria maculans

(Rouxel et al., 2011). However, the AT blocks of L. maculans are

mainly composed of transposons, and their average size is much

larger than those of C. orbiculare (38.6 kb vs. 7.8 kb). In addition,

the AT and GC blocks of L. maculans appear alternately in repeti-

tious fashion on the chromosomes. Thus, the types of chromosome

compartmentalization apparently differ between the two species

and cannot be treated collectively.

We judged the DAPI-bands of C. orbiculare to be constitutive

heterochromatin because they remained condensed throughout

mitotic cell cycles. The DAPI-bands may also be referred to as peri-

centromeric (or pericentric) heterochromatin because they encom-

pass presumable centromeres. In fungi, cytological detection of

these types of heterochromatin has rarely been reported. As far

as we know, constitutive heterochromatin was cytologically shown

only in the mitotic chromosomes of Cryphonectria parasitica

(Eusebio-Cope et al., 2009) and pericentromeric heterochromatin

in the meiotic chromosomes of N. crassa (McClintock, 1945;

Singleton, 1953). Compared with those cases of heterochromatin,

the DAPI-bands of C. orbiculare stand out for its unprecedented

large size. Aside from a cytological viewpoint, constitutive hete-

rochromatin including pericentromeric heterochromatin can also

be characterized molecularly (for reviews, see Grewal and Jia,

2007). Typically, they are enriched for repetitive satellites DNAs

and transposable element remnants and have relatively low gene

density. At the protein level, they are marked with hypoacetylated

histones, methylated H3K9 and heterochromatin protein 1 (HP1).In future studies, therefore, DAPI-bands should be analyzed with

respect to these attributes. Provided that DAPI-bands are pericen-

tromeric, we suggested that C. orbiculare chromosomes take on the

so-called Rabl orientation at interphase. Rabl orientation has been

repeatedly demonstrated in budding and fission yeasts using

sophisticated FISH experiments (for instance, see Funabiki et al.,

1993; Jin et al., 1998), and in filamentous fungi, clustering of cen-

tromeres in interphase has been reported at interphase II and III in

asci of N. crassa without attention to Rabl orientation (Raju, 1980).

Thus, that the chromosomes of C. orbiculare assume the Rabl orien-

tation should be considered unusual.

Besides the importance for chromosome architecture,

DAPI-bands should be evaluated for their contribution to the gen-

ome size. That is, the genome project and this study indicated thatthe strikingly large genome size of C. orbiculare compared with that

of C. graminicola, C. higginsianum and C. gloeosporioides (88.3 Mb vs.

57.4 Mb, 53.4 Mb, and 55.6 Mb) is attributable to the concentrated

accumulation of AT blocks as DAPI-bands in the genome. Presently,

no evidence that the AT blocks are composed mainly of trans-

posons has been obtained, and so how an enormous amount of

AT blocks accumulates in the genome remains unclear. This uncer-

tainty of the involvement of transposons in the genome expansion

of C. orbiculare is contrasting to the case of the powdery mildew

Blumeria graminis f.sp. hordei, in which extraordinary

genome-size expansion (the genome of this fungus is ca. 120 Mb)

was shown to be caused by the massive proliferation of trans-

posons that were evenly distributed throughout the genome with-

out clustering (Spanu et al., 2010). Supposedly, proliferation of repetitive sequences such as transposons is a common mode of

genome-size expansion in eukaryotes including fungi. In this

regard, C. orbiculare may have a novel way of genome-size

expansion.

4.4. Chromosome architecture of C. graminicola and C. higginsianum

In C. graminicola, all chromosomes aside from the MCs had

DAPI-bands or DAPI-knobs. In accordance with this observation,

many speckles intensely stained with DAPI were observed in

the interphase nuclei. Presently, direct association of these obser-

vations with the data of genome project is difficult because we

have not established a one-to-one correspondence of cytologi-

cally identified chromosomes to the optically mapped chromo-

somes or scaffolds of contigs. In spite of that, some inference

concerning the content of DAPI-bands of C. graminicola can be

made in comparison with the distribution map of transposons

and GC content for optically mapped Chromosome 1 (the largest

chromosome in the optical map) produced in the genome project

(see Supplementary Fig. 3 in O’Connell et al., 2012). In this map,

several distinctive AT-rich regions containing transposon clusters

are scattered on the chromosome. Since Chromosome 1 should

correspond to one of the five large chromosomes in our align-

ment that contain 4–7 DAPI-bands (Fig. 9A), DAPI-bands are

likely to correspond to the AT-rich regions of Chromosome 1

and hence contains clusters of transposons. As to the cytological

nature of DAPI-bands and DAPI-knobs of C. graminicola, it is not

certain whether they are constitutive heterochromatin.

Considering that they seemed to remain condensed in the inter-

phase nuclei as speckles and that similar bands and knobs of

Cryphonectria parasitica were shown to be constitutive hete-

rochromatin (Eusebio-Cope et al., 2009), it is probable that they

are constitutive heterochromatin.

In C. higginsianum, the genome project showed that repetitive

DNA makes up 1.22% of the assembled sequences compared with

12.23% in C. graminicola, and GC-content (%) of the scaffolds is

55.1% compared with 49.12% in C. graminicola (O’Connell et al.,

2012). Although these values of C. higginsianum should be regardedas underestimates (O’Connell et al., 2012), our observations of the

absence of DAPI-bands and DAPI-knobs on the metaphase chromo-

somes and DAPI-stained speckles in the interphase nuclei seem to

be compatible with the data of the genome project.

4.5. Comparison of karyotyping techniques for Colletotrichum

In this study, we established protocols for cytological karyotyp-

ing using GTBM for the three Colletotrichum species. Considering

the similarity in the formation and germination of conidia within

this genus, the protocols should be applicable to various

Colletotrichum species without major modifications. Of the various

merits of the GTBM, the good separation of chromosomes and

enlargement of chromosome size were crucially important in thisstudy. Regarding the mechanisms of these two events, chromo-

some separation is explainable by the release of chromosomes

from a nucleus that has little space to allow full chromosome

spreading, whereas the reason for size enlargement is unknown.

In the case of human chromosomes, real-time tracking with a video

camera revealed that stretching of a chromosome that leads to

chromosome enlargement is a very slow process that occurs after

cell bursting, suggesting that cell bursting is not the direct cause

of chromosome enlargement (Hliscs et al., 1997). Similar analysis

may elucidate the mechanism of chromosome enlargement of

the GTBM-prepared fungal specimens.

In addition to the GTBM, fluorescence staining was also vital to

our karyotyping. With the DAPI or DAPI/PI staining, clearer chro-

mosome images were acquired than with Giemsa staining, andthe DAPI-bands in C. orbiculare and C. graminicola could be


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visualized. Even so, the method still needs improvement for ana-

lyzing the captured images. Concretely, we relied on visual inspec-

tion to detect or evaluate DAPI-bands, constrictions and

fluorescence intensity of bands. By contrast, computer-aided image

analysis, which enables qualitative and quantitative analyses of the

color and intensity of fluorescence in objective terms, is commonly

used in the recent karyotyping of plants and animals (for an exam-

ple of DAPI/PI-stained specimens, see Kato et al., 2003). For exclud-

ing subjectivity from idiograms, such image analysis techniques

will be useful in the field of fungal cytogenetics.

Before our study, the mitotic cytology reported for C. linde-

muthianum (Roca et al., 2003) and meiotic cytology for G. cingulata

(Lucas, 1946) and C. lindemuthianum (Roca et al., 2003) were done

by bright-field microscopy with conventional fixation and staining

techniques. Compared with our study, these previous studies

reported much smaller and simple-shaped chromosome. For

instance, the mitotic chromosomes of Roca et al. (2003) were

0.26–0.57 lm long and dot- or oval-shaped. Furthermore, the

CNs determined in those studies were inconsistent with those from

PFGE (Masel et al., 1990; O’Sullivan et al., 1998; Garrido et al.,

2009). Thus, a reliable karyotype is thought to be difficult to obtain

for Colletotrichum by conventional cytology.

PFGE is one choice for karyotyping fungi. In this study, we

used PFGE for strain 104-T of C. orbiculare, but determination of

EK was hampered by clumping of chromosomes and the upper

limit of resolution of PFGE. For the three species analyzed in

the present study, there has been only one instance of PFGE, for

analyzing strain M1.001 of C. graminicola, and its CN was con-

cluded to be n = 9, comprising 6 ordinary chromosomes and 3

MCs (Rollins, 1996). Considering our present study and optical

mapping (O’Connell et al., 2012), we conclude that n =9 is an

underestimate due to incomplete resolution of similar-sized large

chromosomes. This example, as well as ours for strain 104-T of C.

orbiculare, illustrates the limitation of PFGE to correctly deter-

mine CN. In Colletotrichum, CNs have so far been derived from

PFGE analyses for C. gloeosporioides (Masel et al., 1990), C. linde-

muthianum (O’Sullivan et al., 1998) and C. acutatum (Garridoet al., 2009). Cytological reexamination of these species with

our method may help validate previous conclusions from PFGE.

Despite its limitation, PFGE has an advantage in detecting minute

chromosomes. In Colletotrichum, for instance, MCs of 0.1 Mb and

0.27 Mb have been reported for C. acutatum (Garrido et al.,

2009) and C. gloeosporioides (Masel et al., 1990) with PFGE.

Such minute MCs are likely to be missed from cytological detec-

tion because the smallest chromosome we have detected by

cytology is ca. 0.35 Mb for Mycosphaerella graminicola (currently

called Zymoseptoria tritici) (Mehrabi et al., 2007). Thus, cytological

results on the occurrence/absence of MCs should be confirmed by

PFGE. Considering the merits and demerits of cytology and PFGE,

we recommend the combined use of both methods to karyotype

Colletotrichum.While optical mapping is a powerful tool to construct a phys-

ical genome map of various fungi, the cost and time for comple-

tion may restrict its application to the species with large

genomes rich in repetitive DNAs (Neely et al., 2011) and thus pre-

cludes its use for C. orbiculare. The present study demonstrated

the utility of cytology as an alternative to optical mapping for

analyzing the genomes of species such as C. orbiculare. In the

future, optical mapping will be improved to deal with large, com-

plex genomes and continue to play a central role in fungal kary-

otyping. Even so, this technique inherently cannot provide the

kind of morphological information on chromosomes, except for

chromosome size, that we obtained in this study. If optical maps

and cytological karyotypes could be integrated, we could gain

important insights into the architecture of the chromosomesand genomes of fungi.

Acknowledgments

We thank Richard C. O’Connell, Yoshitaka Takano and Lisa J.

Vaillancourt for fungal strains. We also thank Beth E. Hazen for

carefully reading the manuscript and giving valuable suggestions.

This work was supported in part by Grants-in-Aid for Scientific

Research from the Ministry of Education, Culture, Sports, Science,

and Technology (Grant Nos. 24248009 and 20140023).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.fgb.2015.07.013.

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