Fungal Genetics and Biology 40 (2003) 93–102
www.elsevier.com/locate/yfgbi
A linkage map of the basidiomycete Coprinus cinereus basedon random amplified polymorphic DNAs and restriction
fragment length polymorphisms
Hajime Muraguchi,a,* Yasuhiro Ito,b Takashi Kamada,c and Sonoe O. Yanagia
a Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japanb Biological Function Division, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
c Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan
Received 25 March 2003; accepted 22 June 2003
Abstract
A genetic linkage map of the basidiomycete Coprinus cinereus was constructed on the basis of the segregation of 219 RAPD
markers, 28 RFLP markers and the A and B mating-type loci among 40 random basidiospore progeny from a single cross between a
wild-type homokaryon, KF3#2, and an AmutBmut strain, #326. Thirteen linkage groups covering a total of 1346 cM were identified
and correlated to the 13 chromosomes of this fungus by hybridization of RFLP and RAPD marker probes to CHEF blots. These
probes also revealed chromosome length polymorphisms (CLP), which could be associated with haplotype plots of the progeny. The
average kb/cM ratio in this cross was approximately 27.9 kb/cM. The AmutBmut strain undergoes sexual development without
mating, because of mutations in both A and B mating-type loci, and has been used to identify mutations affecting developmental
processes such as dikaryosis, fruit body morphogenesis, and meiosis. The markers in the map, especially the RAPD ones, would
facilitate mapping of genes responsible for such mutations induced in the AmutBmut strain.
� 2003 Elsevier Inc. All rights reserved.
Keywords: Coprinus cinereus; Genetic linkage map; Random amplified polymorphic DNAs; Restriction fragment length polymorphism;
Chromosome length polymorphism
1. Introduction
Coprinus cinereus (Schaeff. ex Fr.) S.F. Gray has a
typical homobasidiomycete life cycle and provides amodel system for studying fungal development. This
fungus belongs to the order Agaricales, culminating in
the development of a highly differentiated structure, the
fruit body (Kamada, 2002; K€uues, 2000; Moore, 1998).
Although the Agaricales include many species of edible
and medicinal mushrooms (Stamets, 2000), these
mushrooms are often poorly characterized or intractable
to genetic analysis. The life cycle of C. cinereus can becompleted in only two weeks and is easily manipulated
in the laboratory. It is possible to screen for mutations
affecting a wide spectrum of metabolic and develop-
* Corresponding author. Fax: +81-18-872-1676.
E-mail address: [email protected] (H. Muraguchi).
1087-1845/$ - see front matter � 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S1087-1845(03)00087-2
mental functions and to clone genes of interest by using
molecular genetic techniques (Pukkila and Casselton,
1991). Since many of the metabolic and developmental
pathways in C. cinereus appear to be identical or similarto those of other species of mushrooms, studies on this
fungus are likely to be helpful in the understanding of
mushrooms of commercial value.
A genetic map comprising closely-spaced DNA
markers covering entire chromosomes is a basic com-
ponent of the scientific infrastructure in model organ-
isms and serves as the underpinning for genome analysis
and gene cloning. The conventional linkage maps of C.cinereus have been constructed by a combination of
random spore and tetrad analysis (Day and Anderson,
1961; Moore, 1967; North, 1987; Takemaru, 1982). A
combined linkage map composed of 10 linkage groups
was presented afterward (Casselton, 1995; North,
1990). In C. cinereus, restriction fragment length
94 H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102
polymorphisms (RFLPs) (Botstein et al., 1980) havebeen commonly used as DNA markers to identify
chromosomes that carry genes of interest (Muraguchi
and Kamada, 1998, 2000; Zolan et al., 1992). In a va-
riety of filamentous fungi, random amplified polymor-
phic DNA (RAPD) markers, which share most of the
advantages of RFLP markers without many of their
disadvantages (Williams et al., 1990), have been used to
construct genetic maps and have been easily integratedinto the maps constructed with RFLP markers (Forche
et al., 2000; Kerrigan, 1993; Williams et al., 1991; Xu
and Leslie, 1996). In C. cinereus, however, RAPD
markers have been used only for discrimination of spe-
cies and strains (Ito and Yanagi, 1999; Ito et al., 1998).
C. cinereus has a total genome size of �37.5Mb (Dutta,
1974), which is organized into 13 chromosomes (Pukkila
and Lu, 1985), ranging in size from 5 to 1 megabasepairs (Mb) as separated by contour-clamped homoge-
neous electric field (CHEF) gel electrophoresis (Pukkila
and Casselton, 1991). The chromosomal DNAs were
only partially correlated with conventional linkage
groups (Pukkila, 1992).
In this study, a linkage map of C. cinereus was con-
structed on the basis of the segregation of 219 RAPD
markers, 28 RFLP markers and the mating type loci (Aand B) among 40 progeny from a single cross between a
wild-type strain, KF3#2, and an AmutBmut strain,
#326. The map is composed of 13 linkage groups, which
correspond to the 13 chromosomes. Although C. cine-
reus is a heterothallic species, homokaryotic strains that
develop fruit bodies without mating have been reported
(Muraguchi et al., 1999), and the AmutBmut strain is
one of such homokaryotic fruiting strains (Swamy et al.,1984). This strain was constructed through a cross be-
tween a strain carrying a mutation in the A mating type
locus and a strain carrying a mutation in the B mating
type locus, and has been used to screen for mutations
affecting dikaryotic phenotypes, such as clamp connec-
tion formation (Inada et al., 2001), fruit body develop-
ment (Chiu and Moore, 1990; Granado et al., 1997;
Kanda and Ishikawa, 1986; Kanda et al., 1989a,b) andmeiosis (Cummings et al., 1999). Thus, a large number
of mutations have been accumulated on the genetic
background of the AmutBmut strain. Therefore, a
comprehensive genetic map of the AmutBmut strain
would allow the rapid mapping of mutations and would
be of widespread interest and utility.
2. Materials and methods
2.1. Strains and growth conditions
KF3#2 and #326 were crossed to obtain progeny that
were used as a mapping population in this paper. KF3#2
(A91B91) is a wild-type homokaryon derived from a
fruit-body collected in Kyoto Prefecture, Japan. #326(AmutBmut pab1-1) is a homokaryotic fruiting strain
(Swamy et al., 1984). We refer to KF3#2 as parent A
and to #326 as parent B. The mapping population was
divided into two subpopulations: Set 1 (A91B91: #2,
#14, #15, #17, #20; A91Bmut: #3, #4, #8, #16, #22;
AmutB91: #7, #10, #13, #23, #29; AmutBmut: #1, #5,
#6, #9, #11) and Set 2 (A91B91: #25, #30, #36, #37,
#38; A91Bmut: #28, #45, #67, #74, #77; AmutB91: #31,#35, #46, #49, #52; AmutBmut: #12, #18, #19, #21,
#24). In this way, all members for each subpopulation
can be analyzed in one gel using a conventional appa-
ratus for electrophoresis, in which 22 samples can be
loaded at the maximum. The 10 A91B91 progeny were
subjected to CHEF electrophoresis for examination of
their karyotypes. A standard homokaryotic strain,
#5302 (A2B2), was used as a control for CHEF elec-trophoresis. MYG (1% malt extract, Difco, USA, 0.4%
yeast extract, Difco, USA, 0.4% glucose, 1.5% agar, Rao
and Niederpruem, 1969) was used for all experiments.
All other culture conditions were as described previously
(Kamada et al., 1984).
2.2. Mating-type determination
To test the mating-types of progeny from a cross
between KF3#2 (A91B91) and #326 (AmutBmut), all
progeny were first scored for the presence or absence of
clamp connections under a light microscope. Amut col-
onies were diagnosed by the presence of clamp connec-
tions. The Amut strains were then crossed with the
parental A91B91 strain, and Bmut colonies were diag-
nosed by dikaryotization in these crosses. Lastly, all A91strains not exhibiting clamp connections were crossed
with an AmutB91 tester strain (#7) to identify their B
mating-type allele.
2.3. Genomic DNA extraction
Four to six pieces of mycelia in 2mm agar cubes were
inoculated in 20ml of MYG liquid medium in a 9 cmpetri dish and incubated for 10 days. Resulting mycelial
lawns were harvested, washed with distilled water, and
then pressed between filter papers to be dried. The
pressed mycelia were lyophilized and powdered with a
glass rod. Genomic DNAs were extracted from the
powder as described (Zolan and Pukkila, 1986).
2.4. Random amplified polymorphic DNA analysis
Amplification reactions were performed in 15 ll vol-umes containing 10mM Tris–HCl, pH 8.3; 50mM KCl;
2.0mM MgCl2; 100 lM each dNTP (Takara); 0.2 lMprimer; 1 ng/ll genomic DNA; and 0.025 units/llTaq polymerase (Takara), overlaid with 1 drop of
mineral oil. A single 10-mer primer was used for the
Table 1
Sequences of RAPD primers designed
BA1 BG1
: 50-AGTGGAAGGT-30 : 50-GACTAGCCTC-30
BA2 BG2
: 50-ATGCCTACAG-30 : 50-GTCTACGAAC-30
BA3 BG3
: 50-AGGGCGTAAG-30 : 50-GTAGACCCGT-30
BA4 BG4
: 50-AGCGCCATTG-30 : 50-GTATCGCGGT-30
BA5 BG5
: 50-ACCGCGAAGG-30 : 50-GCACGGTACT-30
BT1 BC1
: 50-TGGGCACTGA-30 : 50-CACCGTATCC-30
BT2 BC2
: 50-TGGTCACCGA-30 : 50-CACCTAGTCC-30
BT3 BC3
: 50-TGCGTGCTTG-30 : 50-CTGTACATCT-30
BT4 BC4
: 50-TTCGAGCCAG-30 : 50-CCGCATCTAC-30
BT5 BC5
: 50-TTACCGGACG-30 : 50-CTCACCGTCC-30
H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102 95
amplification. Of 130 primers used, 100 are the Operon�sprimer sets (A, E, G, Q, and S sets, Operon Technolo-
gies) and 10 designated as R1-R10 are a Genosys kit,
GEN1-60 (Genosys Biotechnologies). The remaining 20
primers were designed (Table 1) and designated as BA1-
5, BT1-5, BG1-5, and BC1-5 (Ito and Yanagi, 1999; Ito
et al., 1998). Amplification was performed in a Perkin–
Elmer model 9700, programmed for preheating of 2min
at 94 �C, for 45 cycles of 1min at 94 �C, 1min at 36 �C,and 2min at 72 �C, using the fastest transitions available
between each temperature. Amplification products were
analyzed by electrophoresis in 1.4% agarose gels, de-
tected by staining for 30min in an ethidium bromide
solution (0.5 lg/ml) followed by destaining in water for
20min, and photographed on a UV-transilluminator.
RAPD markers were designated by beginning with the
primer number, followed by a dash, approximate size ofthe band, and a capital letter A or B. The A and B
represent a marker band that appears specifically in
KF3#2 and #326, respectively.
2.5. Restriction fragment length polymorphism analysis
Five micrograms of genomic DNAs were digested
with restriction enzymes, fractionated by electrophoresisin 0.8% agarose gels, and stained with ethidium bro-
mide. The fractionated fragments were transferred onto
nylon membranes (Hybond-Nþ) by capillary blotting
with 0.4N NaOH, and subjected to Southern hybrid-
ization. Probe-labeling and hybridization was per-
formed according to the instructions for the Gene
Images (Amersham–Pharmacia). Chromosome-specific
DNA clones, which were constructed from strain #5302(Arima et al., 1996), were a gift from T. Morinaga.
2.6. Linkage analysis and mapping
Segregation patterns of the markers were analyzed
using MAPMAKER software (Lander et al., 1987). The
data type was coded as ‘‘haploid.’’ Putative linkage
groups were initially determined by the results of pair-
wise comparisons of markers using the ‘‘Group’’ com-
mand with a minimum LODlinkage score of 4.0 for
statistical acceptance of linkage, and a maximum re-combination fraction of 0.4. Next, a first order ap-
proximation of each linkage group was obtained by the
‘‘First Order’’ command. When multiple markers ex-
hibited the same segregation pattern, one of them was
chosen for ordering the loci to avoid redundant com-
putations. The omitted markers were added to the map
after the ordering. The Kosambi mapping function was
used to convert recombinant fractions to map distances(Kosambi, 1944).
2.7. CHEF electrophoresis
Protoplasts were prepared from oidia of 5302,
KF3#2, #326, and 10 progeny of mating type A91B91
by the procedure described previously (Zolan et al.,
1992) with the modification of using 50mg/ml of Lysingenzyme (Sigma L1412) instead of Novozyme 234. Plugs
for CHEF electrophoresis were prepared as described
previously (Muraguchi and Kamada, 1998) and loaded
onto a 0.8% Pulse Field Certified Agarose (Bio-Rad) in
0.5� TBE buffer. CHEF electrophoresis was performed
on a CHEF mapper apparatus (Bio-Rad) at 14 �C ex-
changing the buffer daily. The chromosomal DNAs were
separated under a series of three conditions: switchinginterval, 25min increased linearly to 50min at 1.5V/cm
for 48 h; switching interval, 15min increased linearly to
25min at 1.8V/cm for 30 h, and switching interval, 8min
increased linearly to 15min at 2.4V/cm for 57 h. The gel
was stained in ethidium bromide (0.5 lg/ml) for 30min,
destained in distilled water for 1 h, and photographed
using a Polaroid system. The gel was subjected to blot-
ting as described above.
3. Results
3.1. Mapping population
The mapping population consisted of 40 progeny
from a cross between a homokaryotic wild-type strain,KF3#2, and a homokaryotic fruiting strain, #326. The
parents exhibited chromosome-length polymorphisms in
CHEF gel electrophoresis, and were predicted to carry a
lot of polymorphisms in DNA sequences that would
result in RAPD bands and RFLPs. The basidiospores
derived from the cross exhibited a high germina-
tion percentage (>90%). One hundred and seventy
96 H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102
basidiospore derivatives were examined for mating type.The four expected mating types, i.e., A91B91, A91Bmu,
AmutB91, and AmutBmut, occurred in a ratio of
52:30:39:48. As a mapping population, 40 strains were
selected from the basidiospore derivatives such that they
consisted of equal numbers of strains for the respective
mating types.
3.2. Marker development
3.2.1. Mating type loci
The mapping population consists of 40 members
whose mating types were diagnosed. The 40 members
were divided into two data sets, and the 20 members in
each set were arrayed such that the members with the
same mating type were grouped. In this way, RAPD or
RFLP markers linked to the A or B mating type locuswere recognized easily on the gels or blotting mem-
branes (Fig. 1).
3.2.2. RAPD markers
One hundred and thirty 10-mer primers were screened
for generation of RAPD bands between KF3#2 and
#326. Of the primers examined, 91 (70%) generated 234
RAPD marker-bands (an average of 2.57 RAPD perprimer). These RAPD markers were named after the
primer name, approximate size (bp) of the amplified
fragments and the parental origin of the marker bands,
in which A and B represent the marker band that appears
specifically in KF3#2 and #326, respectively. In addition,
the intensity of these bands, strong, middle or weak, was
recorded as +, ¼ , and ), respectively.Segregation patterns for 234 RAPD bands were ex-
amined in the mapping population. Of the markers
scored, segregation distortions were observed at 15 loci
(v2 > 3:84; P < 0:05). Of the 15 loci, four showed
highly distorted segregation (v2 > 10:8; P < 0:001), andthese four were not used for map construction.
Fig. 1. A segregation pattern of the RAPD bands generated by primer
R9. The first two lanes on the left present DNAs amplified from the
parents (KF3#2 and #326) and the other lanes contain DNAs ampli-
fied from the Set1 progeny, which were set in array according to
mating types. This pattern indicates that the marker R9-400B+ is
closely linked to B mating factor.
3.2.3. RFLP markers
Fifteen chromosome-specific DNAs (Arima et al.,
1996) were used to detect RFLPs between the parents.
Twelve of the 15 probes identified RFLPs and segrega-
tion of each RFLP was examined in the mapping pop-
ulation. In addition, 14 DNA fragments, which carry
genes cloned in previous studies, were also used as
RFLP markers to map their loci on the linkage groups.
Some of these RFLP probes were hybridized to CHEFblots in order to confirm the correlations of linkage
groups to chromosomes (Table 2).
3.2.4. rDNA locus
Since ribosomal DNA (rDNA) is organized in clus-
ters of repeated units on chromosome VI (Pukkila and
Skrzynia, 1993) and mitochondrial DNA (mtDNA) ex-
ists in high copy number in cells (Weber et al., 1986),complete digestion of whole-cell DNA with a restriction
enzyme usually generates an electrophoretic pattern
with intense DNA bands against the smear background
of the digestion products (Sagawa et al., 1998). EcoRI
digestion of whole-cell DNAs of KF3#2 and #326 ex-
hibited polymorphism in the electrophoretic pattern.
The RFLP in the intense bands segregated in a ratio of
22:18 in the mapping population. This segregation sug-gests that the intense bands result from rDNAs, because
the mitochondria of C. cinereus show typical mater-
nal inheritance (Casselton and Condit, 1972). This
RFLP was used to correlate a linkage group to
chromosome VI.
3.3. Map construction and linkage group analysis
Segregation patterns for 256 markers were scored on
the 40 members of the mapping population and ana-
lyzed by the MAPMAKER program (Lander et al.,
1987). Two hundred and twenty two markers were ini-
tially grouped into large 13 linkage groups by selecting a
minimum LOD of 4.0 and a maximum recombination
fraction of 0.4. Some of the markers that were not in-
corporated into the initial linkage groups were added tothe linkage groups by relaxation of the minimum LOD
to 3.0. When robust RAPD markers were not incorpo-
rated even in a minimum LOD of 3.0, the RAPD bands
were labeled and hybridized to CHEF blots to determine
the chromosomes carrying the RAPD marker loci. Of
the probes derived from RAPD bands, probes of E17-
2200B+ and E9-1200B+ hybridized only to chromo-
somal DNAs originated from #326 (data not shown),indicating that the regions amplified in these markers are
deleted in KF3#2. The map is depicted for the linkage
groups containing these markers added (Fig. 2).
The published numbers of chromosomes were re-
spected as much as possible in the nomenclature for the
chromosomes in this study. The markers and genes used
to correlate the linkage groups to the chromosomes are
Table 2
Markers for identification of chromosomes
Chromosome Marker (gene) Type Clone name (probe) Confirmation References
I A Phenotype — — Mutasa et al. (1990)
I his5 RFLP Cosmid: I-8H3 inserta — Muraguchi, unpublished
I H1#54 RFLP ca. 1 kbp EcoRI fragment — Arima et al. (1996)
II Q20-1500B+ RAPD — CHEF blots This study
III H3#7 RFLP ca. 1 kbp EcoRI fragment CHEF blots Arima et al. (1996)
IV trp1 RFLP LLC5200 CHEF blots Pukkila (1992)
IV H1-3#72 RFLP ca. 1 kbp EcoRI fragment — Arima et al. (1996)
IV H5#46 RFLP ca. 1 kbp EcoRI fragment CHEF blots Arima et al. (1996)
V trp2 RFLP Entire plasmid CHEF blots Pukkila (1992)
V H1-2#84 RFLP ca. 1 kbp EcoRI fragment — Arima et al. (1996)
V spo11 RFLP Cosmid: 207A7 inserta — Celerin et al. (2000)
V pcc1 RFLP Cosmid: V-6E4 inserta — Murata et al. (1998)
VI H2#11 RFLP ca. 1 kbp EcoRI fragment CHEF blots Arima et al. (1996)
VI clp1b RFLP Cosmid: IV-3A3 inserta — Inada et al. (2001)
VI rDNA RFLPc — — Pukkila (1992)
VII A8-800A+ RAPD — CHEF blots This study
VII E9-1200B+ RAPD — CHEF blots This study
VII R7-500A+ RAPD — CHEF blots This study
VIII rad12 (rad50) RFLP Cosmid: VIII-23G1 insert — Acharya and Zolan, unpublished
VIII H6#9 RFLP ca. 1 kbp EcoRI fragment — Arima et al. (1996)
VIII H6#25 RFLP ca. 1 kbp EcoRI fragment CHEF blots Arima et al. (1996)
IX 2D5 RFLP Cosmid: IX-2D5 inserta CHEF blots Kamada, unpublished
IX msh6 RFLP Cosmid: 1D8 inserta — Celerin and Zolan, unpublished
X H7-2#10 RFLP ca. 1 kbp EcoRI fragment CHEF blots Arima et al. (1996)
X S6-700B+ RAPD — CHEF blots This study
X B Phenotype — — O�Shea et al. (1998)
XI msh2 RFLP Cosmid: 202C5 inserta — Celerin and Zolan, unpublished
XI H7-3#19 RFLP ca. 1 kbp EcoRI fragment CHEF blots Arima et al. (1996)
XI G3-1100A+ RAPD — CHEF blots This study
XII ich1 RFLP 5.4 kbp PstI fragment — Muraguchi and Kamada (1998)
XII H8#6 RFLP ca. 1 kbp EcoRI fragment CHEF blots Arima et al. (1996)
XIII benA RFLP Cosmid: 219K6 inserta CHEF blots Matsuo et al. (1999)
a LLC5200 cosmid vector was used in these cosmid clones and the inserts were excised with NotI digestion.b clp1 has been mapped on chromosome IV of strain 5302 (Inada et al., 2001).cRFLP in the rDNA region was detected by EcoRI digestion of genomic DNA.
H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102 97
listed in Table 2. The mating-type A locus has been
mapped on linkage group I, which is the largest chro-
mosome (O�Shea et al., 1998; Pukkila, 1992), and was
also used to identify chromosome I in this study. Two
linkage groups were assigned to chromosomes II and
III, based on the Southern hybridization of RAPD
marker Q20-1500B+ and RFLP marker H3#7,respec-
tively, to the CHEF blots (Fig. 2, Table 2). The trp1,trp2, and rDNA genes were used to identify chromo-
somes IV, V, and VI, respectively. These genes had
previously been assigned to chromosomes IV, V, and VI
in strain 218 (Pukkila, 1992). The chromosomes VII to
XIII were numbered in order of decreasing size in our
standard strain #5302 (Fig. 3). The genes spo11 and
rad12 (rad50) have been mapped to chromosomes V
and VIII, respectively, of strain Okayama 7 (Celerin and
Zolan, personal communication; Zolan et al., 1993).
These genes were similarly mapped to the same chro-
mosomes in this study (Fig. 2; Table 2), suggesting that
the chromosome numbers in this study are consistent
with those in strain Okayama 7. In this study, the
mating-type B locus was mapped to chromosome X,
consistent with the observation that the cloned B-linked
sequence hybridized to one of the smallest chromosomesof approximately 2.2Mb (O�Shea et al., 1998).
3.4. Inheritance of chromosome length polymorphisms
The parental strains and the 10 progeny with mating
type A91B91 in the mapping population were examined
for their karyotypes by using CHEF electrophoresis
(Fig. 3). The parental strains exhibited numerous
b
Fig. 3. Electrophoretic karyotypes of the parents and their ten progeny in the mapping population. Center panel shows chromosomal DNAs sep-
arated by CHEF electrophoresis. Strains used are shown on the upper side. The Set 1 progeny of A91B91 are strains #2, #14, #15, #17, and #20, and
the Set 2 progeny of A91B91 are strains #25, #30, #36, #37, and #38. Approximate sizes are shown on the right side. Left and right panels represent
hybridization signals of chromosomes with odd, except chromosome I, and even numbers, respectively. To distinguish homologous chromosomes,
the hybridization signals on the CHEF blots are colored and layered with Photoshop (Adobe) based on probes used, which are shown on both the
sides of the panels with chromosome numbers.
H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102 99
chromosome lengthpolymorphisms (CLPs). Theprogeny
examined also exhibited unique karyotypeswith extensiveCLPs. For example, the homologs of chromosome III
differed in size by about 1 mega base between the parents.
The relationship between size of homologous chro-
mosomes and haplotype plots was examined for the
same progeny. Fig. 4 represents the relationship for
chromosomes III and XIII. Chromosome III is larger in
parent B (#326) than in parent A (KF3#2). The segre-
gation patterns show that two progeny have one cross-over breakpoint in the region covered by the markers on
chromosome III. The size of the chromosome in these
two progeny was similar to that of parent B, implying
that the region below BA2-900A) might give rise to the
CLP. The progeny isolate shown on the far right also
had a parent B type of chromosome, despite the fact that
the chromosome was from parent A at least in the re-
gion covered by the markers. These results suggest thatthe region responsible for the CLP is the distal region of
the chromosome that is outside the most distal marker,
E9-3600A). Chromosome XIII is larger in parent A
than in parent B. Four of 10 progeny examined for
karyotype had one cross-over breakpoint at different
regions along the chromosome (Fig. 4, XIII). The rela-
tionships between the cross-over events and the resulting
chromosome sizes suggest that the region causing theCLP exists between A3-1500A¼ and E14-900A¼ , not
a distal end of the chromosome.
Fig. 2. A linkage map of Coprinus cinereus, which was constructed mainly
MAKER version 2.0 for the Macintosh using the Kosambi mapping function.
followed by a dash and approximate size of the band. The intensity of the
respectively. RFLP markers were designated by beginning with the clone or
restriction enzymes are represented by Bm, BamHI; Ec, EcoRI; Ps, PstI; S
unpublished. Skewed segregations are shown on the right of the markers. M
4. Discussion
In this report, we present a genetic map of C. cine-
reus, which was constructed based on the segregation of
219 RAPD markers, 28 RFLP markers and two mating
type loci in a mapping population comprising 40 prog-
eny. The conventional linkage maps of this fungus have
been constructed using biochemical and morphological
markers (Day and Anderson, 1961; Moore, 1967;
North, 1987; Takemaru, 1982) and a combined linkagemap composed of 10 linkage groups was presented af-
terward (Casselton, 1995; North, 1990). These maps
represented only part of the 13 chromosomes in this
fungus. Although the mapping population used in this
study was relatively small, it was enough to identify 13
linkage groups corresponding to the 13 chromosomes,
providing a framework to map genes of interest to
particular chromosomes.
4.1. Map utilization
This map is the first comprehensive genetic map of
the genome of C. cinereus. The map will be useful for
rapid mapping of genes involved in complicated sexual
processes, because the mapping population was ob-
tained from a cross involving the AmutBmut strain as aparent, and the map provides information about the
genome of the AmutBmut strain. The AmutBmut strain
based on RAPD markers. Data analyses were performed with MAP-
RAPD markers were designated by beginning with the primer number,
RAPD bands, strong, middle, and weak, is shown as +, ¼ , and ),gene name, followed by a slash and the restriction enzyme used. The
a, SalI, and Ev, EcoRV. The num1, his5, SdhB, and SdhC genes are
arkers used for Southern hybridization of CHEF blots are boxed.
Fig. 4. Relationship between chromosome length polymorphism and haplotype plots for chromosomes III and XIII. The upper panels are Southern
hybridizations of CHEF blots using a probe of the underlined marker in the lower panels. The CHEF gel is the same as that shown in Fig. 3. The
lower panels show haplotype plots on the respective chromosomes. (A) and (B) represent genomic regions that come from KF3#2 and #326, re-
spectively. The presumed structure of the parental chromosomes are shown on the right.
100 H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102
exhibits dikaryotic phenotypes in spite of being a ho-
mokaryon (Swamy et al., 1984). Any mutation induced
on the genetic background of the AmutBmut strain can
now be easily mapped to a particular chromosome by
screening RAPD markers from the map for those linked
to the phenotype.
This linkage map should facilitate the construction of
a physical map of this fungus. The clones carrying RAPDmarkers could be identified by PCRwith RAPD primers.
Clones with large inserts, such as bacterial artificial
chromosomes (BAC), may carry more than two markers.
Therefore, contigs of BACs could be assigned to chro-
mosomal regions based on markers in the linkage map.
4.2. Reproducibility of RAPD markers
The RAPD markers in the map were scored with the
intensity of the amplified bands, providing information
of reproducibility. Although RAPD markers are known
to suffer from irreproducibility, intense RAPD bands
scored in this study should also be observed in other
laboratories. Therefore, intense RAPD markers would
be available as landmarks on the chromosomes, when
mutations are mapped onto the chromosomes bysearching for markers linked to the phenotypes.
The RAPD markers of weaker bands as well as intense
bands were observed three times, when primers were
selected and when segregation of RAPD bands was ex-
amined in two sets of progeny (as shown in Fig. 1). Most
of the RAPD bands scored exhibited 1:1 segregation,
suggesting that the RAPD bands were derived from a
single loci. The differences in band intensity might result
from the degree of matching between primers and ge-nomic sequences (Williams et al., 1990). The weaker
RAPD bands may be misscored in the progeny and re-
sult in expansion of the map distance.
4.3. Size of the map
The map consists of 13 linkage groups covering a
total of 1346 cM. This distance is in good agreementwith an estimated minimum genetic length of 1300 cM
(Holm et al., 1981). The haploid genome size of
C. cinereus has been estimated to be 37.5Mbp (Dutta,
1974). Averaged over the entire genome, the ratio of
these two values is �27.9 kbp/cM. This ratio is lower
than those reported in other agaricales: 48.5 kbp/cM in
Agaricus bisporus (Kerrigan, 1993); and 35.1 kbp/cM in
Pleurotus ostreatus (Larraya et al., 2000). This suggeststhat the number of the markers is high enough to cover
H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102 101
a large part of the chromosomes in this fungus, althoughit is possible that there exist chromosomal regions not
detected in the present study and that those regions may
be found by mapping of more markers.
The recombinational sizes of the chromosomes do
not necessarily correspond to the physical sizes of the
chromosomes. That is, because at least one cross-over is
necessary per pair of homologs for proper disjuction
during meiosis, the ratio of physical distance to mapdistance will always be smaller for smaller chromosomes
(Zickler and Kleckner, 1999). Consistent with this idea,
we found that the kb/cM ratio ranged from 59.1 kb/cM
for chromosome I to 16.0 kb/cM for chromosome XI. In
addition, the recombinational sizes of the chromosomes
would be influenced by the presence of regions that
suppress recombination, such as centromeres and het-
erochromatin regions (Grewal and Klar, 1997).
4.4. Nomenclature for chromosomes
The linkage groups presented here were correlated
with chromosomal DNAs resolved via CHEF gel elec-
trophoresis. In C. cinereus, the linkage groups carrying
the mating-type A and B loci have been numbered as I
and II, respectively (Casselton, 1995; North, 1990;Takemaru, 1982). On the other hand, the chromosomes
have been numbered on the basis of physical size in a
standard strain 218 (Pukkila, 1992), with the largest
chromosome being I and the smallest XIII. Linkage I is
fortunately the largest chromosome (O�Shea et al., 1998;
Pukkila, 1992), while the mating-type B locus was
mapped on chromosome X when numbered in order of
size in this study. Thus there are discrepancies betweengenetic linkage and chromosome numbers.
Since C. cinereus strains exhibit extensive CLP, the
order of chromosomes varies with the strain, especially
for larger chromosomes (Fig. 3). Therefore, a single
strain could not be selected to define the numbers for
chromosomes. Although a nomenclature based on the
markers is proposed (Table 2), the chromosome num-
bers in this study correspond to approximately de-creasing sizes of chromosomes (Fig. 3).
4.5. Chromosome length polymorphism
CLPs have been reported in a diversity of fungal
species including C. cinereus (Zolan, 1995). Mechanisms
causing CLPs are largely unknown, although it has
been shown in Candida albicans that CLPs result fromexpansion and contraction of the repeated genomic
DNA sequence (RPS) (Chibana et al., 2000) and it has
been suggested in C. cinereus that translocation
(Pukkila and Casselton, 1991) might be a possible cause
of CLP (Zolan et al., 1994). In the present study, the
linkage map, together with its assignment to the chro-
mosomes, has been shown to be useful to analyze re-
gions in a chromosome that are responsible for CLPs.Fig. 4 shows examples of such analysis: possible chro-
mosomal regions causing CLPs are deduced to be distal
and intermediate in chromosome III and XIII, respec-
tively, from the chromosome size and the haplotype
plots. Since we do not map telomere markers, we can-
not rule out the possibility that additional chromosome
fragments are present, for example, above the Q18-
900B¼ marker on the linkage group XIII. If this is thecase, there remains a room for another interpretation of
the data shown in Fig. 4. Further study with this map
will provide a clue to clarifying the mechanisms for
CLPs in this fungus.
Acknowledgments
We thank T. Morinaga for chromosome-specific
DNAs,L.A.Casselton for the cloned trp2 gene,M.Zolan,
M. Celerin, and S. Acharya for sending cosmid DNAs
carrying the rad12 (rad50), msh2, msh6, and spo11 genes,
and M. Zolan for critial reading of the manuscript.
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