jamur karyo
TRANSCRIPT
-
8/18/2019 jamur karyo
1/13
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
Contents lists available at ScienceDirect
Fungal Genetics and Biology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y f g b i
http://dx.doi.org/10.1016/j.fgb.2015.07.013mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.fgb.2015.07.013http://www.sciencedirect.com/science/journal/10871845http://www.elsevier.com/locate/yfgbihttp://www.elsevier.com/locate/yfgbihttp://www.sciencedirect.com/science/journal/10871845http://dx.doi.org/10.1016/j.fgb.2015.07.013mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.fgb.2015.07.013http://crossmark.crossref.org/dialog/?doi=10.1016/j.fgb.2015.07.013&domain=pdf
-
8/18/2019 jamur karyo
2/13
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
M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250 239
http://-/?-http://-/?-http://-/?-
-
8/18/2019 jamur karyo
3/13
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
-
8/18/2019 jamur karyo
4/13
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.)
M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250 241
http://-/?-http://-/?-
-
8/18/2019 jamur karyo
5/13
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.
242 M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-
-
8/18/2019 jamur karyo
6/13
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.
3.4.1. Chromosome number
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.)
M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250 243
http://-/?-http://-/?-http://-/?-
-
8/18/2019 jamur karyo
7/13
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.
244 M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250
-
8/18/2019 jamur karyo
8/13
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.)
M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250 245
http://-/?-http://-/?-
-
8/18/2019 jamur karyo
9/13
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.
3.5.1. Chromosome number
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. hig- ginsianum 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:
246 M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250
http://-/?-http://-/?-http://-/?-http://-/?-
-
8/18/2019 jamur karyo
10/13
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.
M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250 247
http://www.broadinstitute.org/annotation/genome/neurospora/markers.html#correlationhttp://www.broadinstitute.org/annotation/genome/neurospora/markers.html#correlationhttp://-/?-http://-/?-http://www.broadinstitute.org/annotation/genome/neurospora/markers.html#correlationhttp://www.broadinstitute.org/annotation/genome/neurospora/markers.html#correlation
-
8/18/2019 jamur karyo
11/13
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
248 M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250
http://-/?-http://-/?-
-
8/18/2019 jamur karyo
12/13
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.
References
Akamatsu, H., Taga, M., Kodama, M., Johnson, R., Otani, H., Kohmoto, K., 1999.
Molecular karyotypes for Alternaria plant pathogens known to produce host-specific toxins. Curr. Genet. 35, 647–656.
Andras, S.C., Hartman, T.P.V., Alexander, J., McBride, R., Marshall, J.A., Power, J.B.,
Cocking, E.C., Davey, M.R., 2000. Combined PI–DAPI staining (CPD) reveals NOR
asymmetry and facilitates karyotyping of plant chromosomes. Chromosome
Res. 8, 387–391.Cannon, P.F., Damm, U., Johnston, P.R., Weir, B.S., 2012. Colletotrichum – current
status and future directions. Stud. Mycol. 73, 181–213.
Coleman, J.J., Rounsley, S.D., Rodriguez-Carres, M., Kuo, A., Wasmann, C.C.,
Grimwood, J., Schmutz, J., Taga, M., White, G.J., Zhou, S., Schwartz, D.C.,
Freitag, M., Ma, L., Danchin, E.G.J., Henrissat, B., Coutinho, P.M., Nelson, D.R.,
Straney, D., Napoli, C.A., Barker, B.M., Gribskov, M., Rep, M., Kroken, S., Molnár,
I., Rensing, C., Kennell, J.C., Zamora, J., Farman, M.L., Selker, E.U., Salamov, A.,
Shapiro, H., Pangilinan, J., Lindquist, E., Lamers, C., Grigoriev, I.V., Geiser, D.M.,
Covert, S.F., Temporini, E., VanEtten, H.D., 2009. The genome of Nectriahaematococca: contribution of supernumerary chromosomes to geneexpansion. PLoS Genet. 5, e1000618.
Cuomo, C.A., Güldener, U., Xu, J.R., Trail, F., Turgeon, B.G., Di Pietro, A., Walton, J.D.,
Ma, L.J., Baker, S.E., Rep, M., Adam, G., Antoniw, J., Baldwin, T., Calvo, S., Chang,
Y.L., Decaprio, D., Gale, L.R., Gnerre, S., Goswami, R.S., Hammond-Kosack, K.,
Harris, L.J., Hilburn, K., Kennell, J.C., Kroken, S., Magnuson, J.K., Mannhaupt, G.,
Mauceli, E., Mewes, H.W., Mitterbauer, R., Muehlbauer, G., Münsterkötter, M.,
Nelson, D., O’donnell, K., Ouellet, T., Qi, W., Quesneville, H., Roncero, M.I., Seong,
K.Y., Tetko, I.V., Urban, M., Waalwijk, C., Ward, T.J., Yao, J., Birren, B.W., Kistler,
H.C., 2007. The Fusarium graminearum genome reveals a link between localizedpolymorphism and pathogen specialization. Science 317, 1400–1402.
Crouch, J., O’Connell, R., Gan, P., Buiate, E., Torres, M.F., Beirn, L., Shirasu, K.,
Vaillancourt, L., 2014. The genomics of Colletotrichum. In: Dean, R.A., Lichens-Park, A., Kole, C. (Eds.), Genomics of Plant-Associated Fungi and Oomycetes:
Monocot Pathogens. Springer-Verlag, Berlin, pp. 69–102.
Eusebio-Cope, A., Suzuki, S., Garmaroodi, H.S., Taga, M., 2009. Cytological and
electrophoretic karyotyping of the chestnut blight fungus Cryphonectria parasitica. Fungal Genet. Biol. 46, 342–351.
Fincham, J.R.S., 1949. Chromosome numbers in species of Neurospora. Ann. Bot. 13,23–28.
Forgey, W.M., Blanco, M.H., Loegering, W.Q., 1978. Differences in pathological
capabilities and host specificity of Colletotrichum graminicola on Zea mays[maize]. Plant Dis. Rep. 62, 573–576.
Funabiki, H., Hagan, I., Uzawa, S., Yanagida, M., 1993. Cell cycle-dependent specific
positioning and clustering of centromeres and telomeres in fission yeast. J. Cell
Biol. 121, 961–976.
Gale, L.R., Bryant, J.D., Calvo, S., Giese, H., Katan, T., O’Donnell, K., Suga, H., Taga, M.,
Usgaard, T.R., Ward, T.J., Kistler, H.C., 2005. Chromosome complement of thefungal plant pathogen Fusarium graminearum based on genetic and physicalmapping and cytological observations. Genetics 171, 985–1001.
Gan, P., Ikeda, K., Irieda, H., Narusaka, M., O’Connell, R.J., Narusaka, Y., Takano, Y.,
Kubo, Y., Shirasu, K., 2013. Comparative genomic and transcriptomic analyses
reveal the hemibiotrophic stage shift of Colletotrichum fungi. New Phytol. 197,1236–1249.
Garrido, C., Carbú, M., Fernández-Acero, F.J., Vallejo, I., Cantoral, J.M., 2009.
Phylogenetic relationships and genome organization of Colletotrichumacutatum causing anthracnose in strawberry. Eur. J. Plant Pathol. 125, 397–411.
Grewal, S.I.S., Jia, S., 2007. Heterochromatin revisited. Nat. Rev. Genet. 8, 35–46 .
Hiruma, K., Onozawa-Komori, M., Takahashi, F., Asakura, M., Bednarek, P., Okuno, T.,
Schulze-Lefert, P., Takano, Y., 2010. Entry mode-dependent functionof an indole
glucosinolate pathway in Arabidopsis for nonhost resistance againstanthracnose pathogens. Plant Cell 22, 2429–2443.
Hliscs, R., Mühlig, P., Claussen, U., 1997. The spreading of metaphases is a slow
process which leads to a stretching of chromosomes. Cytogenet. Genome Res.
76, 167–171.
Hyde, K.D., Cai, L., Cannon, P.F., Crouch, J.A., Crous, P.W., Damm, U., Goodwin, P.H.,
Chen, H., Johnston, P.R., Jones, E.B.G., Liu, Z.Y., McKenzie, E.H.C., Moriwaki, J.,Noireung, P., Pennycook, S.R., Pfenning, L.H., Prihastuti, H., Sato, T., Shivas, R.G.,
M. Taga et al. / Fungal Genetics and Biology 82 (2015) 238–250 249
http://dx.doi.org/10.1016/j.fgb.2015.07.013http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0015http://refhub.elsevier.com/S1087-1845(15)30012-8/h0015http://refhub.elsevier.com/S1087-1845(15)30012-8/h0015http://refhub.elsevier.com/S1087-1845(15)30012-8/h0015http://refhub.elsevier.com/S1087-1845(15)30012-8/h0015http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0040http://refhub.elsevier.com/S1087-1845(15)30012-8/h0040http://refhub.elsevier.com/S1087-1845(15)30012-8/h0040http://refhub.elsevier.com/S1087-1845(15)30012-8/h0040http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0050http://refhub.elsevier.com/S1087-1845(15)30012-8/h0050http://refhub.elsevier.com/S1087-1845(15)30012-8/h0050http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0070http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0080http://refhub.elsevier.com/S1087-1845(15)30012-8/h0080http://refhub.elsevier.com/S1087-1845(15)30012-8/h0080http://refhub.elsevier.com/S1087-1845(15)30012-8/h0085http://refhub.elsevier.com/S1087-1845(15)30012-8/h0085http://refhub.elsevier.com/S1087-1845(15)30012-8/h0085http://refhub.elsevier.com/S1087-1845(15)30012-8/h0085http://refhub.elsevier.com/S1087-1845(15)30012-8/h0085http://refhub.elsevier.com/S1087-1845(15)30012-8/h0085http://refhub.elsevier.com/S1087-1845(15)30012-8/h0080http://refhub.elsevier.com/S1087-1845(15)30012-8/h0080http://refhub.elsevier.com/S1087-1845(15)30012-8/h0080http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0075http://refhub.elsevier.com/S1087-1845(15)30012-8/h0070http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0065http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0060http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0055http://refhub.elsevier.com/S1087-1845(15)30012-8/h0050http://refhub.elsevier.com/S1087-1845(15)30012-8/h0050http://refhub.elsevier.com/S1087-1845(15)30012-8/h0050http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0045http://refhub.elsevier.com/S1087-1845(15)30012-8/h0040http://refhub.elsevier.com/S1087-1845(15)30012-8/h0040http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0035http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0030http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0025http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0020http://refhub.elsevier.com/S1087-1845(15)30012-8/h0015http://refhub.elsevier.com/S1087-1845(15)30012-8/h0015http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0010http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://refhub.elsevier.com/S1087-1845(15)30012-8/h0005http://dx.doi.org/10.1016/j.fgb.2015.07.013
-
8/18/2019 jamur karyo
13/13
Tan, Taylor, P.W.J., Weir, B.S., Yang, Y.L., Zhang, J.Z., . Colletotrichum – names incurrent use. Fungal Diversity 39, 147–182.
Jin, Q.-W., Trelles-Sticken, E., Loidl, J., 1998. Yeast nuclei display prominent
centromere clustering that is reduced in nondividing cells and in meiotic
prophase. J. Cell Biol. 141, 21–29.
Johnson, G.D., Araujo, G.M.D.C.N., 1981. A simple method of reducing the fading of
immunofluorescence during microscopy. J. Immunol. Methods 43, 349–350.
Kato, S., Ohmido, N., Fukui, K., 2003. Development of a quantitative pachytene
chromosome map in Oryza sativa by imaging methods. Genes Genet. Syst. 78,155–161.
Kubo, Y., 2012. Appressorium function in Colletotrichum orbiculare and prospect forgenome based analysis. In: Pérez-Martín, J., Di Pietro, A. (Eds.), Morphogenesis
and Pathogenicity in Fungi, Topics in Current Genetics, vol. 22. Springer, New
York, pp. 115–131.
Kubo, Y., Takano, Y., 2013. Dynamics of infection-related morphogenesis and
pathogenesis in Colletotrichum orbiculare. J. Gen. Plant Pathol. 79, 233–242.Lucas, G.B., 1946. Genetics of Glomerella. IV. Nuclear phenomena in the ascus. Am. J.
Bot. 33, 802–806.
Ma, L.J., VanDer Does,H.C.,Borkovich,K.A.,Coleman, J.J., Daboussi, M.J., DiPietro, A.,
Dufresne, M., Freitag, M., Grabherr, M., Henrissat, B., Houterman, P.M., Kang, S.,
Shim, W.B., Woloshuk, C., Xie, X., Xu, J.R., Antoniw, J., Baker, S.E., Bluhm, B.H.,
Breakspear, A., Brown, D.W., Butchko, R.A., Chapman, S., Coulson, R., Coutinho,
P.M., Danchin, E.G., Diener, A., Gale, L.R., Gardiner, D.M., Goff, S., Hammond-
Kosack, K.E., Hilburn, K., Hua-Van, A., Jonkers, W., Kazan, K., Kodira, C.D.,
Koehrsen, M., Kumar, L., Lee, Y.H., Li, L., Manners, J.M., Miranda-Saavedra, D.,
Mukherjee, M., Park, G., Park, J., Park, S.Y., Proctor, R.H., Regev, A., Ruiz-Roldan,
M.C., Sain, D., Sakthikumar, S., Sykes, S., Schwartz, D.C., Turgeon, B.G., Wapinski,
I., Yoder, O., Young, S., Zeng, Q., Zhou, S., Galagan, J., Cuomo, C.A., Kistler, H.C.,
Rep, M., 2010. Comparative genomics reveals mobile pathogenicity
chromosomes in Fusarium. Nature 464, 367–373.Masel, A., Braithwaite, K., Irwin, J., Manners, J., 1990. Highly variable molecular
karyotypes in the plant pathogen Colletotrichum gloeosporioides. Curr. Genet. 18,81–86.
Mahmoud, A.M., Taga, M., 2012. Cytological karyotyping and characterization of a
410kb minichromosomein Nectria haematococca MPI.Mycologia 104, 845–856.McClintock, B., 1945. Neurospora I. Preliminary observations of the chromosomes of
Neurospora crassa. Am. J. Bot. 32, 671–678.Mehrabi, R., Taga, M., Kema, G.H., 2007. Electrophoretic and cytological karyotyping
of the foliar wheat pathogen Mycosphaerella graminicola reveals manychromosomes with a large size range. Mycologia 99, 868–876.
Narusaka, M., Shirasu, K., Noutoshi, Y., Kubo, Y., Shiraishi, T., Iwabuchi, M.,
Narusaka, Y., 2009. RRS1 and RPS4 provide a dual resistance-gene systemagainst fungal and bacterial pathogens. Plant J. 59, 672–683.
Neely, R.K., Deen, J., Hofkens, J., 2011. Optical mapping of DNA: single-molecule-
based methods for mapping genomes. Biopolymers 95, 298–311.
O’Connell, R., Herbert, C., Sreenivasaprasad, S., Khatib, M., Esquerré-Tugayé, M.T.,
Dumas, B., 2004. A novel Arabidopsis-Colletotrichum pathosystem for the
molecular dissection of plant–fungal interactions. Mol. Plant–MicrobeInteract. 17, 272–282.
O’Connell, R.J., Thon, M.R., Hacquard, S., Amyotte, S.G., Kleemann, J., Torres, M.F.,
Damm, U., Buiate, E.A., Epstein, L., Alkan, N., Altmüller, J., Alvarado-Balderrama,
L., Bauser, C.A., Becker, C., Birren, B.W., Chen, Z., Choi, J., Crouch, J.A., Duvick, J.P.,
Farman, M.A., Gan, P., Heiman, D., Henrissat, B., Howard, R.J., Kabbage, M., Koch,
C., Kracher, B., Kubo, Y., Law, A.D., Lebrun, M.H., Lee, Y.H., Miyara, I., Moore, N.,
Neumann, U., Nordström, K., Panaccione, D.G., Panstruga, R., Place, M., Proctor,
R.H., Prusky, D., Rech, G., Reinhardt, R., Rollins, J.A., Rounsley, S., Schardl, C.L.,
Schwartz, D.C., Shenoy, N., Shirasu, K., Sikhakolli, U.R., Stüber, K., Sukno, S.A.,
Sweigard, J.A., Takano, Y., Takahara, H., Trail, F., van der Does, H.C., Voll, L.M.,
Will, I., Young, S., Zeng, Q., Zhang, J., Zhou, S., Dickman, M.B., Schulze-Lefert, P.,
Loren, Ver., van Themaat, E., Ma, L.J., Vaillancourt, L.J., 2012. Lifestyle transitions
in plant pathogenic Colletotrichum fungi deciphered by genome andtranscriptome analyses. Nat. Genet. 44, 1060–1065.
O’Donnell, K., Rooney, A.P., Proctor, R.H., Brown, D.W., McCormick, S.P., Ward, T.J.,
Frandsen, R.J.N., Lysøe, E., Rehner, S.A., Aoki, T., Robert, V.A.R.G., Crous, P.W.,
Groenewald, J.Z., Kang, S., Geiser, D.M., 2013. Phylogenetic analyses of RPB1 and
RPB2 support a middle Cretaceous origin for a clade comprising all
agriculturally and medically important fusaria. Fungal Genet. Biol. 52, 20–31 .O’Sullivan, D., Tosi, P., Creusot, F., Cooke, M., Phan, T., Dron, M., Langin, T., 1998.
Variation in genome organization of the plant pathogenic fungus Colletotrichumlindemuthianum. Curr. Genet. 33, 291–298.
Perfect, S.E., Hughes, H.B., O’Connell, R.J., Green, J.R., 1999. Colletotrichum: a modelgenus for studies on pathology and fungal–plant Interactions. Fungal Genet.
Biol. 27, 186–198.
Perkins, D.D., 1992. Neurospora chromosomes. In: Federoff, N., Botstein, D. (Eds.),The Dynamic Genome: Barbara McClintock’s Ideas in the Century of Genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 33–44.
Peterson, D.G., Lapitan, N.L., Stack, S.M., 1999. Localization of single-and low-copy
sequences on tomato synaptonemal complex spreads using fluorescence in situhybridization (FISH). Genetics 152, 427–439.
Prusky, D., Freeman, S., Dickman, M.P., 2000. Colletotrichum: Host Specificity,Pathology, and Host-Pathogen Interaction. APS Press, St. Paul, Minnesota.
Raju, N.B., 1980. Meiosis and ascospore genesis in Neurospora. Eur. J. Cell Biol. 23,208–223.
Roca, M.G., Mendes-Costa, M.C., Davide, L.C., 2003. Cytogenetics of Colletotrichumlindemuthianum (Glomerella cingulata f.sp. phaseoli). Fitopat. Bras. 28, 367–373.
Rollins, J.A., 1996. The Characterization and Inheritance of Chromosomal Variation
in Glomerella Graminicola. Ph.D. Dissertation, Purdue University, West Lafayette,Indiana.
Rouxel, T., Grandaubert, J., Hane, J.K., Hoede, C., van de Wouw, A.P., Couloux, A.,
Dominguez, V., Anthouard, V., Bally, P., Bourras, S., Cozijnsen, A.J., Ciuffetti, L.M.,
Degrave, A., Dilmaghani, A., Duret, L., Fudal, I., Goodwin, S.B., Gout, L., Glaser, N.,
Linglin, J., Kema, G.H., Lapalu, N., Lawrence, C.B., May, K., Meyer, M., Ollivier, B.,Poulain, J., Schoch, C.L., Simon, A., Spatafora, J.W., Stachowiak, A., Turgeon, B.G.,
Tyler, B.M., Vincent, D., Weissenbach, J., Amselem, J., Quesneville, H., Oliver, R.P.,
Wincker, P., Balesdent, M.H., Howlett, B.J., 2011. Effector diversification within
compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations. Nat. Commun. 2, 202.
Schwartz, D.C., Samad, A., 1997. Optical mapping approaches to molecular genetics.
Curr. Opin. Biotechnol. 8, 70–74.
Shiraishi, Y., Yoshida, T., 1972. Banding pattern analysis of human chromosomes by
use of a urea treatment technique. Chromosoma 37, 75–83.
Shirane, N., Masuko, M., Hayashi, Y., 1988. Nuclear behavior and division in
germinating conidia of Botrytis cinerea. Phytopathology 78, 1627–1630.Shirane, N., Masuko, M., Hayashi, Y., 1989. Light microscopic observation of nuclei
and mitotic chromosomes of Botrytis species. Phytopathology 79, 728–730.Singleton, J.R., 1953. Chromosome morphology and the chromosome cycle in the
ascus of Neurospora crassa. Am. J. Bot. 40, 124–144.Smith, K.M., Galazka, J.M., Phatale, P.A., Connolly, L.R., Freitag, M., 2012.
Centromeres of filamentous fungi. Chromosome Res. 20, 635–656.
Spanu, P.D., Abbott, J.C., Amselem, J., Burgis, T.A., Soanes, D.M., Stüber, K., Loren,
Ver., van Themaat, E., Brown, J.K., Butcher, S.A., Gurr, S.J., Lebrun, M.H., Ridout,
C.J., Schulze-Lefert, P., Talbot, N.J., Ahmadinejad, N., Ametz, C., Barton, G.R.,
Benjdia, M., Bidzinski, P., Bindschedler, L.V., Both, M., Brewer, M.T., Cadle-
Davidson, L., Cadle-Davidson, M.M., Collemare, J., Cramer, R., Frenkel, O.,
Godfrey, D., Harriman, J., Hoede, C., King, B.C., Klages, S., Kleemann, J., Knoll, D.,
Koti, P.S., Kreplak, J., López-Ruiz, F.J., Lu, X., Maekawa, T., Mahanil, S., Micali, C.,
Milgroom, M.G., Montana, G., Noir, S., O’Connell, R.J., Oberhaensli, S., Parlange,
F., Pedersen, C., Quesneville, H., Reinhardt, R., Rott, M., Sacristán, S., Schmidt,
S.M., Schön, M., Skamnioti, P., Sommer, H., Stephens, A., Takahara, H., Thordal-
Christensen, H., Vigouroux, M., Wessling, R., Wicker, T., Reinhardt, R., 2010.
Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in
extreme parasitism. Science 330, 1543–1546.
Taga, M., Kaminakaya, H., Tanaka, K., Ogawa, M., Kubo, Y., 2014. Cytological analysis
of karyotype evoluation in the genus colletotrichum (abstract). In: XVIInternational Congress of Plant–Microbe Interactions.
Taga, M., Murata, M., 1994. Visualization of mitotic chromosomes in filamentous
fungi by fluorescence staining and fluorescence in situ hybridization.Chromosoma 103, 408–413.
Taga, M., Murata, M., Saito, H., 1998. Comparison of different karyotyping methodsin filamentous ascomycetes – a case study of Nectria haematococca. Mycol. Res.102, 1355–1364.
Taga, M., Tanaka, K., Kubo, Y., 2011. Mitotic chromosomes and karyotype of
Colletotrichum orbiculare (Abstract). In: The 26th Fungal Genetics Conference.Taga, M., Tsuchiya, D., Murata, M., 2003. Dynamic changes of rDNA condensation
state during mitosis in filamentous fungi revealed by fluorescence in situhybridisation. Mycol. Res. 107, 1012–102