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: muraguchi@akita-pu.ac.jp (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

98 H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102

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.

References

Arima, T., Okida, T., Morinaga, T., 1996. Behavior of chromosomes

after meiosis in Coprinus cinereus. Mycoscience 37, 111–115.

Botstein, D., White, R.L., Skolnick, M.H., Davis, R.W., 1980.

Construction of a genetic map in man using restriction fragment

length polymorphisms. Am. J. Hum. Genet. 32, 314–331.

Casselton, L.A., 1995. Genetics of Coprinus. In: K€uuck, U. (Ed.), The

Mycota II. Springer, Berlin, pp. 35–48.

Casselton, L.A., Condit, A., 1972. A mitochondrial mutant of

Coprinus lagopus. J. Gen. Microbiol. 72, 521–527.

Celerin, M., Merino, S.T., Stone, J.E., Menzie, A.M., Zolan, M.E.,

2000. Multiple roles of Spo11 in meiotic chromosome behavior.

EMBO J. 19, 2739–2750.

Chibana, H., Beckerman, J.L., Magee, P.T., 2000. Fine-resolution

physical mapping of genomic diversity in Candida albicans.

Genome Res. 10, 1865–1877.

Chiu, S.W., Moore, D., 1990. A mechanism for gill pattern formation

in Coprinus cinereus. Mycol. Res. 94, 320–326.

Cummings, W.J., Celerin, M., Crodian, J., Brunick, L.K., Zolan,

M.E., 1999. Insertional mutagenesis in Coprinus cinereus: use of a

dominant selectable marker to generate tagged, sporulation-defec-

tive mutants. Curr. Genet. 36, 371–382.

Day, P.R., Anderson, G.E., 1961. Two linkage groups in Coprinus

lagopus. Genet. Res. 2, 414–423.

Dutta, S.K., 1974. Repeated DNA sequences in fungi. Nucleic Acids

Res. 11, 1411–1419.

Forche, A., Xu, J., Vilgalys, R., Mitchell, T.G., 2000. Development and

characterization of a genetic linkage map of Cryptococcus neofor-

mans var. neoformans using amplified fragment length polymor-

phisms and other markers. Fungal Genet. Biol. 31, 189–203.

Granado, J.D., Kertesz-Chaloupkova, K., Aebi, M., Kues, U., 1997.

Restriction enzyme-mediated DNA integration in Coprinus cine-

reus. Mol. Gen. Genet. 256, 28–36.

Grewal, S.I.S., Klar, A.J.S., 1997. A recombinationally repressed

region between mat2 and mat3 loci shares homology to centromeric

repeats and regulates directionality of mating-type switching in

fission yeast. Genetics 146, 1221–1238.

102 H. Muraguchi et al. / Fungal Genetics and Biology 40 (2003) 93–102

Holm, P.B., Rasmussen, S.W., Zickler, D., Lu, B.C., Sage, J., 1981.

Chromosome pairing, recombination nodules and chiasma forma-

tion in the basidiomycete Coprinus cinereus. Carlsberg Res.

Commun. 46, 305–346.

Inada, K., Morimoto, Y., Arima, T., Murata, Y., Kamada, T., 2001.

The clp1 gene of the mushroom Coprinus cinereus is essential for A-

regulated sexual development. Genetics 157, 133–140.

Ito, Y., Yanagi, S.O., 1999. Discrimination of basidiomycete species

and strains by random amplified polymorphic DNA (RAPD)

analysis. JARQ 33, 149–154.

Ito, Y., Fushimi, T., Yanagi, S.O., 1998. Discrimination of species and

strains of basidiomycete genus Coprinus by random amplified

polymorphic DNA (RAPD) analysis. Mycoscience 39, 361–365.

Kamada, T., 2002. Molecular genetics of sexual development in the

mushroom Coprinus cinereus. BioEssays 24, 449–459.

Kamada, T., Katsuda, H., Takemaru, T., 1984. Temperature-sensitive

mutants of Coprinus cinereus defective in hyphal growth and stipe

elongation. Curr. Microbiol. 11, 309–312.

Kanda, T., Ishikawa, T., 1986. Isolation of recessive developmental

mutants in Coprinus cinereus. J. Gen. Appl. Microbiol. 32, 541–

543.

Kanda, T., Ishihara, H., Takemaru, T., 1989a. Genetic analysis of

recessive primordiumless mutants in the basidiomycete Coprinus

cinereus. Bot. Mag. Tokyo 102, 561–564.

Kanda, T., Goto, A., Sawa, K., Arakawa, H., Yasuda, Y., Takemaru,

T., 1989b. Isolation and characterization of recessive sporeless

mutants in the basidiomycete Coprinus cinereus. Mol. Gen. Genet.

216, 526–529.

Kerrigan, R.W., 1993. Meiotic behavior and linkage relationships in

the secondarily homothallic fungus Agaricus bisporus. Genetics

133, 225–236.

Kosambi, D.D., 1944. The estimation of map distances from recom-

bination values. Ann. Eugen. 12, 172–175.

K€uues, U., 2000. Life history and developmental processes in the

basidiomycete Coprinus cinereus. Microbiol. Mol. Biol. Rev. 64,

316–353.

Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J.,

Lincoln, S.E., Newburg, L., 1987. MapMaker: an interactive

computer package for construction primary linkage maps of

experimental and natural populations. Genomics 1, 174–181.

Larraya, L.M., Perez, G., Ritter, E., Pisabarro, A.G., Ramirez, L.,

2000. Genetic linkage map of the edible basidiomycete Pleurotus

ostreatus. Appl. Env. Microbiol. 66, 5290–5300.

Matsuo, T.H.Y., Muraguchi, H., Kamada, T., 1999. Effects of amino-

acid substitutions in b tubulin on benomyl sensitivity and micro-

tubule functions in Coprinus cinereus. Mycoscience 40, 241–249.

Moore, D., 1998. Fungal Morphogenesis. Cambridge University Press,

New York.

Moore, D., 1967. Four new linkage groups in Coprinus lagopus. Genet.

Res. 9, 331–342.

Muraguchi, H., Kamada, T., 1998. The ich1 gene of the mushroom

Coprinus cinereus is essential for pileus formation in fruiting.

Development 125, 3133–3141.

Muraguchi, H., Kamada, T., 2000. A mutation in the eln2 gene

encoding a cytochrome P450 of Coprinus cinereus affects mush-

room morphogenesis. Fungal Genet. Biol. 29, 49–59.

Muraguchi, H., Takemaru, T., Kamada, T., 1999. Isolation and

characterization of developmental variants in fruiting using a

homokaryotic fruiting strain of Coprinus cinereus. Mycoscience 40,

227–235.

Murata, Y., Fujii, M., Zolan, M.E., Kamada, T., 1998. Molecular

analysis of pcc1, a gene that leads to A-regulated sexual morpho-

genesis in Coprinus cinereus. Genetics 149, 1753–1761.

Mutasa, E.S., Tymon, A.M., G€oottgens, B., Mellon, F.M., Little,

P.F.R., Casselton, L.A., 1990. Molecular organisation of an A

mating type factor of the basidiomycete fungus Coprinus cinereus.

Curr. Genet. 18, 223–229.

North, J., 1987. Linkage map of Coprinus cinereus (Schaeff. ex Fr.) S.F.

Gray. In: O�Brien, S.J. (Ed.), Genetic Maps 1987, vol. 4. Cold

Spring Harbor Laboratory Press, New York.

North, J., 1990. Linkage map of Coprinus cinereus (Schaeff. ex Fr.) S.F.

Gray. In: O�Brien, S.J. (Ed.), Genetic Maps, Book 3 Lower

Eukaryotes. Cold Spring Harbor Laboratory Press, New York.

O�Shea, S.F., Chaure, P.T., Halsall, J.H., Olesnicky, N.S., Leibbrandt,

A., Connerton, I.F., Casselton, L.A., 1998. A large pheromone and

receptor gene complex determines multiple B mating type specific-

ities in Coprinus cinereus. Genetics 148, 1081–1090.

Pukkila, P.J., 1992. Methods of genetic manipulation in Coprinus

cinereus. In: Chang, S.T. et al. (Eds.), Culture Collection and

Breeding of Edible Mushrooms. Gordon and Breach, Philadelphia,

pp. 249–264.

Pukkila, P.J., Lu, B.C., 1985. Silver staining of meiotic chromosomes

in the fungus, Coprinus cinereus. Chromosoma 91, 108–112.

Pukkila, P.J., Casselton, L.A., 1991. Molecular genetics of the agaric

Coprinus cinereus. In: Bennett, J.W., Lasure, L.L. (Eds.),MoreGene

Manipulations in Fungi. Academic Press, San Diego, pp. 126–150.

Pukkila, P.J., Skrzynia, C., 1993. Frequent changes in the number of

reiterated ribosomal RNA genes throughout the life cycle of the

basidiomycete Coprinus cinereus. Genetics 133, 203–211.

Rao, P.S., Niederpruem, D.J., 1969. Carbohydrate metabolism during

morphogenesis of Coprinus lagopus (sensu Buller). J. Bacteriol. 100,

1222–1228.

Sagawa, I., Moriyama, Y., Yanagi, S.O., Ando, A., Nagata, Y., 1998.

Rapid discrimination of mitochondrial DNA type and use of

results to study mitochondrial inheritance in Pleurotus spp.. Biosci.

Biotech. Biochem. 62, 769–772.

Stamets, P., 2000. The gilled mushroom. In: Growing Gourmet and

MedicinalMushrooms.Ten SpeedPress, Berkeley, CA, pp. 207–348.

Swamy, S., Uno, I., Ishikawa, T., 1984. Morphogenetic effects of

mutations at the A and B incompatibility factors in Coprinus

cinereus. J. Gen. Microbiol. 130, 3219–3224.

Takemaru, T., 1982. Genetic map of Coprinus macrorhizus. In:

Ishikawa, T. (Ed.), Experimental Methods in Microbial Genetics.

Kyoritsu, Tokyo, pp. 355–356.

Weber, C.A., Hudspeth, E.S., Moore, G.P., Grossman, L.I., 1986.

Analysis of the mitochondrial and nuclear genomes of two

basidiomycetes, Coprinus cinereus and Coprinus sterorarius. Curr.

Genet. 10, 515–525.

Williams, J.G.K., Kubelik, A.R., Rafalski, J.A., Tingey, S.V., 1991.

Genetic analysis with RAPD markers. In: Bennett, J.W., Lasure,

L.L. (Eds.), More Gene Manipulations in Fungi. Academic Press,

San Diego, pp. 431–439.

Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey,

S.V., 1990. DNA polymorphisms amplified by arbitrary primers

are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535.

Xu, J.-r., Leslie, J.F., 1996. A genetic map ofGibberella fujikuroimating

population A (Fusarium moniforme). Genetics 143, 175–189.

Zickler, D., Kleckner, N., 1999. Meiotic chromosomes: integrating

structure and function. Annu. Rev. Genet. 33, 603–754.

Zolan, M.E., 1995. Chromosome-length polymorphism in fungi.

Microbiol. Rev. 59, 686–698.

Zolan, M.E., Pukkila, P.J., 1986. Inheritance of DNA methylation in

Coprinus cinereus. Mol. Cell. Biol. 6, 195–200.

Zolan, M.E., Heyler, N.K., Ramesh, M.A., 1993. Gene mapping using

marker chromosomes in Coprinus cinereus. In: Baltz, R.H. et al.

(Eds.), Industrial Microorganisms: Basic and Applied Molecular

Genetics. American Society for Microbiology, Washington, pp. 31–35.

Zolan, M.E., Heyler, N.K., Stassen, N.Y., 1994. Inheritance of

chromosome-length polymorphisms in Coprinus cinereus. Genetics

137, 87–94.

Zolan, M.E., Crittenden, J., Heyler, N.K., Seitz, L.C., 1992. Efficient

isolation and mapping of rad genes of the fungus Coprinus cinereus

using chromosome-specific libraries. Nucleic Acids Res. 20, 3993–

3999.