Fungal Genetics and Biology 43 (2006) 376–387
www.elsevier.com/locate/yfgbi
Genetic linkage map and expression analysis of genes expressed in the
lamellae of the edible basidiomycete Pleurotus ostreatus 夽
Sang-Kyu Park, María M. Peñas, Lucía Ramírez, Antonio G. Pisabarro ¤
Department of Agrarian Production, Public University of Navarre, E-31006 Pamplona, Spain
Received 17 October 2005; accepted 16 January 2006
Available online 13 March 2006
Abstract
Pleurotus ostreatus is an industrially cultivated basidiomycete with nutritional and environmental applications. Its genome contains 35
Mbp organized in 11 chromosomes. There is currently available a genetic linkage map based predominantly on anonymous molecular
markers complemented with the mapping of QTLs controlling growth rate and industrial productivity. To increase the saturation of the
existing linkage maps, we have identiWed and mapped 82 genes expressed in the lamellae. Their manual annotation revealed that 34.1% of
the lamellae-expressed and 71.5% of the lamellae-speciWc genes correspond to previously unknown sequences or to hypothetical proteins
without a clearly established function. Furthermore, the expression pattern of some genes provides an experimental basis for studying
gene regulation during the change from vegetative to reproductive growth. Finally, the identiWcation of various diVerentially regulated
genes involved in protein metabolism suggests the relevance of these processes in fruit body formation and maturation.
2006 Elsevier Inc. All rights reserved.
Keywords: Edible Mushroom; Fruit body development; EST; Linkage map; Gene regulation; 26S proteasome; Arginase; Trehalose; Ubiquitin
1. Introduction
Pleurotus ostreatus (Jacq.: Fr.) Kumm. (Pleurotaceae,
higher Basidiomycetes) (Moncalvo et al., 2002) (oyster
mushroom) is a white rot, edible fungus industrially cultivated for food production because of its nutritional (Mattila et al., 2001) and health-stimulating (Hossain et al.,
2003) properties. Other applications such as its use in paper
pulp bleaching (Sigoillot et al., 2005), recycling of agricultural wastes (Aggelis et al., 2003), and bioremediation
(D’Annibale et al., 2005), among others (Cohen et al.,
2002), have fuelled the study of P. ostreatus biochemistry
and molecular biology.
夽
LR and AGP conceived and lead the project; MP constructed the
cDNA library; SKP analyzed the sequence and expression of the clones
and performed the linkage analysis; SKP and AGP wrote the manuscript
and produced the Wgures.
*
Corresponding author. Fax: +34 948 169 732.
E-mail address: gpisabarro@unavarra.es (A.G. Pisabarro).
1087-1845/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2006.01.008
Pulsed-Weld gel electrophoresis studies have determined
that the P. ostreatus genome contains 11 pairs of polymorphic chromosomes ranging in size from 1.4 to 4.7 Mbp, and
a total haploid genome size of 35 Mbp. The genetic cartography of P. ostreatus consists of a linkage map based on
anonymous molecular markers (RAPD and RFLP) and
some functional genes that identiWed 135 map positions in a
total size of 1000 cM (Larraya et al., 2000); and two maps
locating quantitative loci (QTLs) controlling growth rate
(Larraya et al., 2002) and industrial productivity (Larraya
et al., 2003). Genetic linkage maps based on RFLP, AFLP,
and RAPD molecular markers are available for a reduced
number of higher basidiomycetes (Callac et al., 1997; Kerrigan et al., 1993; Lind et al., 2005; Muraguchi et al., 2003;
Sonnenberg et al., 1996).
The identiWcation of the genes being expressed in a tissue
provides a picture of the corresponding developmental
stage, or of the response to a given environmental stress. In
P. ostreatus, a comparative study of sequences expressed in
vegetative growth and fruit bodies (Lee et al., 2002) emphasized the small number of genes simultaneously expressed
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
in both developmental stages. EST-based developmental
studies in fungi have been also published for Agaricus bisporus (De Groot et al., 1997; Ospina-Giraldo et al., 2000)
and Agocybe aegerita (Salvado and Labarere, 1991), and a
study of representational diVerence of developmentally regulated genes in Lentinula edodes has been recently published (Miyazaki et al., 2005). ESTs have also been used to
study the interaction between plants and mycorrhizal
(Morel et al., 2005) and phytopathogenic fungi (PosadaBuitrago and Frederick, 2005; Soanes et al., 2002), and in
the study of the phytopathogenic basidiomycetes Ustilago
maydis (Nugent et al., 2004; Sacadura and Saville, 2003)
and Phakopsora pachyrhizi (Posada-Buitrago and Frederick, 2005)
In this paper, we describe the identiWcation and
genetic linkage mapping of 82 P. ostreatus genes
expressed in the lamellae, and study individually their
expression in three developmental stages. The results
indicate that (i) most lamellae-speciWc genes code for
proteins for which a function cannot be assigned yet;
(ii) protein metabolism in fruit bodies presents some
peculiarities as deduced from the expression of some
genes, and (iii) most of the new genes mapped to map
positions where other previous genetic markers had
already been placed suggesting that the distribution of
genetic linkage markers on the chromosomes does not
depend solely of physical distances.
377
2.3. Total RNA isolation and Northern hybridization
2. Materials and methods
For total RNA isolation, samples were collected by
Wltration (vegetative mycelia) or dissection (lamellae and
fruit body), washed with sterilized distilled water, rapidly
frozen, pulverized on liquid nitrogen, and stored at ¡80 °C.
Total RNA was isolated from lamellae, mature fruit bodies
without lamellae (the fruit body stipes), dikaryotic vegetative mycelium N001, and monokaryotic vegetative mycelium (protoclones PC9 and PC15) by the hot phenol
procedure, followed by precipitation with lithium chloride
and denaturation with formamide and formaldehyde (Wessels et al., 1987). For Northern blot analysis, RNA concentrations were determined with a spectrophotometer, RNA
integrity was checked on 1% agarose/formaldehyde gel and
equal amounts of RNA were separated by electrophoresis
onagarose gels [1% agarose in 2.2 M formaldehyde, 20 mM
MOPS (morpholinepropanesulfonic acid), 5 mM sodium
acetate (pH 7.0), 1 mM EDTA], transferred onto Hybond
N+ membranes (Amersham, Little Chalfont, Buckinghamshire, UK) and Wxed to the membrane using an UV crosslinker with exposure of 120,000 J/cm2. Hybridization
reactions were performed at 65 °C as described by Church
and Gilbert (Church and Gilbert, 1984). The membranes
were hybridized with each cDNA probe labeled with [32
P]dCTP by using a Random Prime Labelling System Kit
(Amersham Bioscience, UK). Hybridized membranes were
exposed for autoradiography at ¡70 °C for 24–72 h on
X-ray Wlms.
2.1. Fungal strains and culture conditions
2.4. Plasmid DNA isolation and sequencing analysis
Pleurotus ostreatus strain N001 was used in this work.
Cultures were performed according to the standard protocols previously described (Larraya et al., 2000). The two
nuclei present in this strain had been previously separated
by de-dikaryotization, and the two corresponding protoclones (monokaryons carrying only one of the nuclei present in dikaryon N001) are deposited in the Spanish Type
Culture Collection (PC9: CECT20311, and PC15:
CECT20312) (Larraya et al., 1999).
A total of 133 clones were randomly picked from the
cDNA library and their inserts were partially sequenced.
All sequence reactions were performed with T7 sequencing
primer and some samples were also sequenced with T3
sequencing. The sequences were used as query to Wnd
homologs in the nucleotide and protein sequence databases
at the National Center for Biotechnology Information
(NCBI) using the BLASTX and BLASTN algorithms
(Altschul et al., 1997).
2.2. Construction of cDNA library
2.5. RFLP analysis and linkage mapping
Ten micrograms of the total RNA were extracted from
lamellae of mature fruit bodies using the protocol
described elsewhere (Sambrook et al., 1989) and were used
to construct a cDNA library using cDNA synthesis kit
(Stratagene, La Jolla, CA). The cDNAs longer than 0.3 kb
were selected, precipitated by standard methods (Sambrook et al., 1989) and cloned into EcoRI-digested pBluescript II SK (+) vector. Individual white colonies were
picked randomly and transferred to 0.5 ml microcentrifuge tubes containing LB medium (Lennox, 1955) with
50 g/ml ampicillin and 20% (v/v) glycerol, grown for 18 h
at 37 °C in a rotary shaker at 250 rpm, and stored at
¡80 °C until used.
For the RFLP analyses, genomic DNA was puriWed
from dikaryon N001, protoclones PC9 and PC15, and 80
monokaryons (haploid progeny) as described elsewhere
(Larraya et al., 2000). DNA samples were digested with the
restriction enzymes according to the suppliers speciWcations
and the digestion products were separated on agarose gels,
Southern blotted and probed with the corresponding
[-32P]dCTP-randomly labelled cDNA probes. Probe labeling was made the Random Prime Labelling System Kit
(Amersham Bioscience, UK). When a given enzyme-probe
combination detected polymorphism on protoclones PC9
and PC15, the enzyme was used to analyze the polymorphism segregation in the population of 80 monokaryons.
378
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
The linkage analysis was performed according to the methods previously described (Larraya et al., 2000) as described
by Ritter et al. (1990) and by Ritter and Salamini (1996)
using the MAPRF program.
2.6. Gene accession numbers
The sequences described in this paper have been deposited in the EMBL database under the accession numbers
indicated in Table 1.
3. Results and discussion
The aims of this study were to identify new genes
expressed diVerentially during vegetative and reproductive
growth in P. ostreatus, to study their expression in other
developmental stages, and to increase the density of the
existing genetic linkage map of this fungus. The work
focused on the genes expressed in the lamellae; but due to
the reduced number of P. ostreatus genes previously identiWed, we did not enrich the cDNA library in lamellae-speciWc sequences by subtraction. The relevance of these goals
stands on the peculiarities of the dikaryosis that occurs
throughout P. ostreatus life cycle, and on the usefulness of
mapping expressed genes (as well as other RFLP markers)
to classify BAC genomic clones of this fungus. This last
goal aims at the sequencing of relevant genomic regions in
this basidiomycete.
3.1. EST analysis
A total of 133 plasmids were single-pass partially
sequenced using the T7 and T3 primers (average sequence
read 449 bp, SD 151 bp), 12 of them (9.0%) had no insert,
and the remaining 121 insert-containing plasmids were
sorted in two classes: Class A included 18 plasmids (14.9%)
that had either vector (14 plasmids, 77.8%) or indeterminate inserts not amenable for sequencing (four plasmids,
22.2%), and Class B included 103 plasmids (85.1%) corresponding to hypothetical genes. Class B cDNA clones were
further classiWed on the basis of their BLASTX (Altschul
et al., 1997) similarity to database entries into three groups:
high similarity (Evalue < 10¡15, 62 clones, 60.2% of the
cDNAs), low similarity (10¡15 < Evalue < 10¡3, 10 clones,
9.7%), and no similarity (Evalue > 10¡3, 31 clones, 30.1%). In
summary, nearly one third of the sequences identiWed in
our study coded for previously unknown proteins.
The 103 Class B plasmids were grouped on the basis of
their redundancy in the sample and this allowed identifying
82 diVerent putative genes listed Table 1 along with their
manual annotation on the basis of the BLASTX results
(August 2005), the linkage group they map to, and their
expression proWle. A BLASTN search of the ESTs against
the non-human, non-mouse EST database revealed that 36
of them had signiWcantly similar counterparts
(Evalue < 10¡4), and that 22 of them had similarity to entries
of a P. ostreatus EST collection previously reported
(Lee et al., 2002) (accessions numbers that begin with “AT”
in Table 1).
The BLASTX similarity search indicated that 56.1% of
the genes identiWed in this study were highly similar to databases entries whereas 34.1% of them corresponded to
entirely new sequences. This value Wts with those previously
reported for other developmental studies carried in basidiomycetes where the fraction of genes similar to other present
in the databases ranged from 44.5 to 58% (Guettler et al.,
2003; Lacourt et al., 2002; Lee et al., 2002; Ospina-Giraldo
et al., 2000; Posada-Buitrago and Frederick, 2005);
although it is lower than the value found in the symbiotic
basidiomycete Hebeloma cylindrosporum (80%) (Wipf et al.,
2003).
Considering solely those genes for which BLASTX
counterparts were found, in most cases (86.8%) the most
similar sequences belong to the Kingdom Fungi wherein
the more frequent hits corresponded to Cryptococcus neoformans (21.7%, 10 genes) and Neurospora crassa (15.2%, 7
genes). The number of hits was higher to Basidiomycetes
than to Ascomycetes (58.7% vs. 41.3%), animals contributed with more hits than bacteria (11.3% and 1.9%, respectively), and no plants appeared among the highest scores.
These results Wt with the expectations due to the evolutive
neighborhood of fungi and animals.
The mean GC content of the EST collection was 53.35%
(SD 4.72). This value is slightly higher than the estimated
for the general genome (50.53%, our unpublished results)
suggesting a higher GC content in the coding regions in
comparison with the non-coding ones. In the white-rot fungus Phanerochaete chrysosporium strain RP78, the GC content of the coding regions (59 %) was also higher than the
overall value (57 %) (Martinez et al., 2004). GC values were,
however, higher in P. chrysosporium than in P. ostreatus.
3.2. Expression analysis
The expression of each one of the 82 genes was studied
in mature fruit bodies excluding lamellae (pileus), lamellae,
and vegetative monokaryotic (PC9 and PC15) and dikaryotic (N001) mycelia by Northern blot analysis. The expression proWle could not be determined for four of the ESTs
(4.9% of the total). The 78 genes whose expression pattern
was determined could be classiWed as described in Fig. 1.
Three major (A, general expression; B, fruit body-speciWc;
and C, lamellae-speciWc) and four minor expression patterns were found. For most of the lamellae-speciWc genes,
some expression could be also detected in the fruit body
lanes because of incomplete removal of the lamellae.
Previous reports on developmentally regulated genes
have been published in other higher basidiomycetes (De
Groot et al., 1997; Leung et al., 2000; Miyazaki et al., 2005;
Morel et al., 2005; Posada-Buitrago and Frederick, 2005;
Salvado and Labarere, 1991). In P. ostreatus, four genes
diVerentially expressed in the fruit body have been identiWed by diVerential display of RAPD (Sunagawa and
Magae, 2005), but none of them corresponded to those
Table 1
List of P. ostreatus ESTs
Clonea
Accession
General metabolism
MV082 MV087 MV088 AJ416500
Organism
BLASTXb EST
accessionc
BLAST Nd
Linkage
groupe
ProWlef
Arginase
Agaricus
bisporus
Cryptococcus
neoformans
Schizosaccharo
myces pombe
Schizosaccharo
myces pombe
Aspergillus
fumigatus
Neurospora
crassa
Neurospora
crassa
Cryptococcus
neoformans
Ustilago maydis
Amanita
muscaria
Pseudomonas
Xuorescens
5e-68
AT004496
0.0
I
AB
5e-56
AJ407642
3e-06
II
B
2e-49
BU638117
1e-05
XI
A
9e-32
AT002963
0.0
II
AB
3e-30
—
—
—
C
7e-30
AT004669
4e-15
I
B
6e-21
—
—
IV
A
1e-20
AT004381
e-124
I
C
3e-19
3e-19
—
AT002742
—
0.0
—
V
—
AB
3e-15
—
—
IV
C
Phanerochaete.
chrysosporium
Cryptococcus
neoformans
Coprinopsis
cinerea
Ciona savignyi
Schizosaccharo
myces pombe
Coprinopsis
cinerea
Leptosphaeria.
maculans
Aspergillus
niger
Cryptococcus
neoformans
e-136
AT002953
0.0
XI, VIII
A
2e-77
CB905291
3e-25
V, III
B
6e-57
CB010385
4e-67
IV, VII
A
8e-47
2e-45
AT003961
CD274093
0.0
1e-38
VI, VIII, V
III
A
A
2e-33
—
—
VI
A
2e-30
CK828255
1e-04
IV
A
1e-23
AT002886
2e-77
III, III
A
1e-19
AT005261
e-35
VI
C
Cryptococcus
neofornans
Nectria
haematococca
8e-68
AW444336
2e-23
—
—
7e-51
—
—
IV
C
MV080
AJ843921
Putative Zinc-Wnger
dehydrogenase
Putative C-4 methyl sterol oxidase
MV084
AJ843924
MV096 MV150
MV047
AJ416502
AJ843929
AJ843916
MV097
AJ843930
MV143
AJ843955
MV138
AJ843951
Trehalose-6-phosphate
phosphatase
Putative pyruvate dehydrogenase
kinase,
Hypothetical protein similar to a
peroxisomal dehydratase
Hypothetical protein similar to an
oxidoreductase
UDP-glucose dehydrogenase
MV124
MV152
AJ843949
AJ843959
C-3 sterol dehydrogenase
Phenylalanine ammonium lyase
MV028
AJ843909
Transthyretin like protein
Protein metabolism (synthesis and degradation)
MV100
AJ416499 Polyubiquitin
MV057
AJ843919
MV090
AJ843925
26S proteasome regulatory
subunit
Ribosomal protein L41
MV094
MV132
AJ843928
AJ417656
Ubiquitin
Ribosomal protein S19A
MV046
AJ843915
Serine protease
MV136
AJ417035
Aspartyl proteinase
MV133
AJ417655
Ribosomal protein L15
MV156 MV160
AJ843961
AJ843962
Putative endopeptidase
Cellular signaling
MV139
AJ843952
Splicing factor SF1
MV091
AJ416501
Ran 1-like protein kinase
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
Putative identiWcation
(continued on next page)
379
380
Table 1 (continued)
Accession
Putative identiWcation
Organism
BLASTXb EST
accessionc
BLAST Nd
Linkage
groupe
ProWlef
MV111
AJ843940
Homo sapiens
4e-38
—
—
X
E
MV045 MV175 MV181 AJ843914
Amyloid precursor protein
binding protein 1
GTP binding protein ypt1
AT002976
0.0
I
A
MV044 MV177 MV183 AJ843913
MV126
AJ417844
MAP kinase ubc3
MAPKK kinase ubc4
—
—
—
—
II
I
A
A
Cell structure and growth
MV058
AJ843920
Cellobiohydrolase 3
e-106
—
—
I
C
MV102
AJ843933
Calmodulin
3e-73
AT005110
0.0
X
A
MV105
AJ416503
7e-57
DR753368.1
1e-17
IV
A
MV128
AJ417659
Putative bud site selection-related
protein
Cyclophilin A
7e-42
AT004786
0.0
VII
A
2e-33
AT004009
5e-17
I
A
1e-15
AT004471
0.0
IV
C
1e-57
CD274935
2e-17
VI, I
B
4e-54
BU964180
8e-04
VI
C
3e-22
—
—
XI
B
Caenorhabditis
elegans
2e-13
AT004376
0.0
VI
A
Cryptococcus
neofornans
Neurospora
crassa
Cryptococcus
neoformans
Neurospora
crassa
Candida
albicans
9e-54
CK993156
3e-18
—
—
2e-52
BG278058
e-08
—
—
2e-45
—
—
I
C
2e-43
—
—
VIII
C
5e-29
CK992447
0.0
II
A
MV005 MV162
AJ843899
AJ843900
MV086 MV140 MV146 AJ843957
Glucan 1,3--glucosidase 1
precursor
Conidiation-speciWc protein
Transport
MV052 MV182
AJ843971
Mitochondrial carrier protein
MV054
AJ843918
Permease
MV110
AJ843939
MV106
AJ843936
N-acetylglucosaminylphosphatydyl inositol biosynthetic
protein
Transitional ER ATPase
Hypothetical proteins (Unclear function)
MV083
AJ843923 Hp CNBD3280
MV130
AJ417657
Hypothetical protein
MV021 MV022 MV032 AJ318523
Putative CAP64
MV093
AJ843927
Predicted protein
MV051
AJ843917
Hp Ca019.6835
Neurospora
2e-36
crassa
Ustilago maydis 2e-35
Ustilago maydis 1e-18
Agaricus
bisporus
Pleurotus
ostreatus
Cryptococcus
neoformans
Filobasidiella
neoformans
Agaricus
bisporus
Neurospora
crassa
Paxillus
involutus
Hebeloma
cylindrosporum
Neurospora
crassa
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
Clonea
MV019 MV169
Hp UM05775.1
Ustilago maydis 4e-23
Hypothetical protein CNBD4610
MV081
AJ843922
MV040 MV179 MV187 AJ843969
Hp UM03541.1
Hypothetical protein
MV109
AJ843938
Hp MG05250.4
Cryptococcus
neoformans
Ustilago maydis
Agaricus
bisporus
Magnaporthe
grisea
MV180
AJ843970
Hp Ca019.6835
MV015
MV112
AJ843905
AJ843941
Hp FG07570.1
Hp AN7069.2
MV148
AJ843958
Allergen
MV023
AJ843908
Putative galectin-3
MV098
AJ843931
Predicted protein
MV092
AJ843926
Putative mucin 1
Candida
albicans
Gibberella zeae
Aspergillus
nidulans
Malassezia
sympodialis
Oryctolagus
cuniculus
Tetraodon
nigroviridis
Macaca
mulatta
Genes without BLASTX similarity
MV119
AJ843947
MV118
AJ843946
MV116
AJ843945
MV114
AJ843943
MV115
AJ843944
MV141 MV163
AJ843953
No similarity
No similarity
No similarity
No similarity
No similarity
No similarity
AT005041
E-153
V
B
1e-22
—
—
III
B
1e-21
1e-17
—
—
—
—
XI
V
C
B
8e-15
—
—
V
C
3e-14
CCFBM9G11 e-118
—
A
5e-14
8e-09
—
—
—
—
III
I
C
B
6e-06
—
—
VIII
C
3e-04
—
—
III
A
5e-04
—
—
IX
C
e-03
—
—
VIII
C
AT004873
AT005258
AT004606
AT004736
AT004873
AT005258
0.0
E-177
E-176
E-108
6E-95
3E-25
III
V
VIII
IX
III
V, XI, XI, VII
C
C
A
A
C
C
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Genes without BLASTX or BLASTN similarity
Clone
Accession Linkage group
ProWle
Clone
Accession
Linkage group ProWle
Clone
Accession Linkage group ProWle
MV004
MV011
MV014
MV033
MV034
MV042
MV079 MV108
MV089
C
C
B
B
C
B
A
B
MV103
MV104
MV113
MV123
MV129
MV142
MV145
MV155
AJ843934
AJ843935
AJ843942
AJ843948
AJ417658
AJ843954
AJ843956
AJ843960
II
VI
IV
VIII
VII
III
IV
IV
MV157
MV161
MV165
MV168
MV171
MV172
MV174
AJ843963
AJ843964
AJ844894
AJ843965
AJ843966
AJ843967
AJ843968
a
b
c
d
e
f
AJ843898
AJ843901
AJ843904
AJ843910
AJ843911
AJ843912
AJ843937
AJ844893
VI, VI
VI, II
V
VIII
X
III
III
V
E
B
C
D
C
C
B
AB
—
X, I
V
X
—
III
VI
C
B
B
B
C
A
B
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
AJ843906
AJ843907
MV131 MV176 MV186 AJ843950
Clone name in the cDNA library. More that one clone number can correspond to the same EMBL accession number when the same sequence has been found redundantly.
Evalue of the best BLASTX match (August 2005). This identiWcation is used for the tentative annotation of the P. ostreatus gene.
Accession of the best BLASTN match to the non-human non-mouse EST database.
Evalue of the best BLASTN match to the non-human non-mouse EST database.
Linkage group the corresponding P. ostreatus EST maps to.
EST expression proWle. A, general expression; B, fruit-body-speciWc; C, lamellae-speciWc; A/B, fruit-body-speciWc but basal expression in vegetative mycelia; D and E, see text and Fig. 1 for details.
381
382
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
La
N001 PC9 PC15
Expression Number of
pattern
genes
F.b
% of
genes
.
MV094
A
24
30.8
B
19
24.4
C
28
35.9
AB
4
5.1
D
1
1.3
E
1
1.3
F
1
1.5
rRNA
MV019
rRNA
MV091
rRNA
MV087
rRNA
MV123
rRNA
MV103
rRNA
MV111
rRNA
Total
78
100
Fig. 1. Expression patterns P. ostreatus of genes. Total cellular RNA was
isolated and 20 g of total RNA was applied for Northern blot analysis.
5.8S ribosomal RNA probe of P. ostreatus var. Florida was used as internal control. La, lamellae; N001, dikaryotic vegetative mycelium; PC9, vegetative mycelium of protoclon PC9; PC15, vegetative mycelium of
protoclon PC15; Fb, mature fruit body without lamellae.
reported here. Other P. ostreatus genes diVerentially
expressed in vegetative and reproductive mycelia have been
previously reported (Asgeirsdóttir et al., 1998; Joh et al.,
2004; Peñas et al., 1998, 2005). The largest study of diVerentially expressed genes on this species has been reported by
Lee et al. (2002), which compared ESTs isolated from liquid
cultures and fruit bodies.
We studied the expression of the genes in diVerent
developmental stages using Northern analysis. The study
was made gene by gene in order to detect those whose
expression level was too low to be detected by more
insensitive approaches. Notwithstanding, the expression
of four genes (4.9% of the total) could not be determined,
probably because it was lower than the sensitivity threshold. They coded for proteins similar to a putative splicing
factor (MV139, BLASTX Evalue D 8 £ 10¡68), two genes
coding for products similar to C. neoformans and
N. crassa hypothetical proteins (MV083, Evalue D 9 £
10 ¡54; and MV130, Evalue D 2 £ 10 ¡52, respectively), and a
clone (MV124) similar to a C-3 sterol dehydrogenase
(Evalue D 3 £ 10¡19). Moreover, no RFLP polymorphism
was detected for clones MV083 and MV130 and, consequently, they could not be mapped to any linkage group.
In P. ostreatus, 30.8% of the genes identiWed in this
study as expressed in the lamellae were also expressed in
vegetative mycelia (55.8% in the case of the subpopulation
of genes expressed in fruit bodies), whereas Lee et al.
(2002) found that only 5.3% of the genes were expressed
simultaneously in both developmental stages, and Ospina-Giraldo et al. found that 12% of the genes were simultaneously detected in primordia and basidiome
A. bisporus samples (Ospina-Giraldo et al., 2000). These
authors conclude that gene expression must be quite
diVerent quantitatively and qualitatively during fruit
body formation. We consider that the statistical sampling
process involved in cDNA cloning can be responsible for
their results, and that Northern analysis is more accurate
in detecting gene expression. Consequently, our results
suggest that the number of genes diVerentially expressed
in the two developmental stages is smaller than that previously reported. These expression results partially support
the conclusion put forward by Zantinge et al. (1979) indicating that RNA sequences isolated from S Schizophyllum
commune fruiting and non-fruiting mycelia were identical
for at least 90%.
We have sorted the genes identiWed into Wve functional
classes, genes similar to hypothetical proteins and genes
coding for entirely new gene products (Table 2). These last
two groups represent 56.1% of the genes. Combining the
results of annotation and expression, it appears that the
genes coding for hypothetical proteins or new products represent 29.2% of the housekeeping genes (pattern A), 73.7%
of the fruit body (pattern B), and 71.5% of the lamellae
(pattern C) speciWc fractions. These results indicate that
most of the genes involved in fruit body and lamellae formation are yet completely unknown.
Table 2
Distribution of ESTs by functional classes
Expression
pattern
General
metabolism
Protein
metabolism
Cellular
signaling
Cell structure
and growth
Transport
Hypothetical
proteins
New genes
Total
A
B
C
AB
D
E+F
N.D.
Total
2 (8.3)
2 (10.5)
3 (10.7)
3
—
—
1
11 (13.4)
7 (29.2)
1 (5.3)
1 (3.6)
—
—
—
—
9 (11.0)
3 (12.5)
—
1 (3.6)
—
—
1
1
6 (7.3)
4 (16.7)
—
2 (7.1)
—
—
—
—
6 (7.3)
1 (4.2)
2 (10.5)
1 (3.6)
—
—
—
—
4 (4.9)
3 (12.5)
4 (21.1)
8 (28.6)
—
—
—
2
17 (20.7)
4 (16.7)
10 (52.6)
12 (42.9)
1
1
1
—
29 (35.4)
24 (100)
19 (100)
28 (100)
4
1
2
4
82 (100)
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
3.3. Overview of genes expressed in the lamellae
3.3.1. General metabolism
ESTs MV082, MV087, and MV088 code for a putative
arginase and were expressed mainly in fruit body and
lamellae although a basal expression was also observed
during vegetative growth (pattern AB). Wagemaker et al.
have cloned and studied an A. bisporus gene coding for an
arginase (Wagemaker et al., 2005) highly similar in
sequence (Evalue D 5 £ 10¡68) and expression pattern to that
described here. Moreover, P. ostreatus arginase is also
highly similar to a Coprinopsis cinerea arginase gene
expressed during senescence (DR907928, Evalue D 2 £ 10¡46)
but diVerent from that expressed in early meiotic prophase
(DR775205). The isolation of three independent ESTs corresponding to this gene suggests that it is highly expressed
in lamellae. It has been found that members of the Agaricaceae accumulate substantial amounts of urea in their fruit
bodies. Our results support a role for P. ostreatus arginase
in this process similar to that described for the A. bisporus
gene.
Clones MV096 and MV150 code for a putative trehalose–phosphatase similar (Evalue D 9 £ 10¡32) to that of
Schizosaccharomyces pombe TPS2. This enzyme is one of
the components of the trehalose-synthesizing complex. It
has been shown in A. bisporus cultures that trehalose is synthesized in the vegetative mycelium and later transported to
the emerging fruiting structures where it is eventually
metabolized (Wannet et al., 1999). Moreover, no trehalase
or trehalose synthase activities could be detected in this
fungus; instead, trehalose phosphorylase catalyzes the
reversible degradation and synthesis of trehalose (Wannet
et al., 1998). Our results indicate that the trehalose-6Pphosphatase gene has an AB expression pattern suggesting
the occurrence of trehalose synthesis at fruit bodies and
lamellae.
3.3.2. Protein metabolism
Most of the protein metabolism genes identiWed present
an A-type expression proWle (Table 1 and Fig. 1). There
were, however, some exceptions: clone MV057 codes for a
protein homologous to the RPT3 regulatory subunit of the
26S proteasome. This is one of the six proteasome regulatory subunits (Fu et al., 1999b), although its speciWc functions have not been fully clariWed. The use of clone MV057
as probe in Southern analyses revealed a gene duplication
(Table 1, Fig. 2). Duplication of genes coding for proteasome RPT particles has been reported in rice (Shibahara
et al., 2002) and Arabidopsis (Fu et al., 1999a); however, in
both cases, the duplication aVected all the genes except that
coding for RPT3. Moreover, the data available indicate
that most of the RPT genes in Arabidopsis are actively
expressed in diVerent tissues (Fu et al., 1999b); in contrast,
MV057 expression is only detected in fruit bodies and
lamellae (pattern B). In mammals, tissues and cells must
contain heterogeneous populations of 26S proteasome
complexes (Dahlmann et al., 2000). Our data suggest that in
383
the case of Wlamentous fungi, diVerent proteasome populations can be associated to diVerent developmental stages.
A similar result has been reported for the spatial distribution of the 26S proteasome in rice meristems and primordia
(Yanagawa et al., 2002).
Three genes coding for ubiquitin (MV094-1, -2, and -3)
and two coding for polyubiquitin (MV100-1 and -2) were
found. Both gene types displayed an A expression pattern
(Fig. 1). The core sequences of the P. ostreatus ubiquitin
and polyubiquitin genes were 83% identical and, consequently, cross-hybridization between them can occur and
they must be considered simultaneously. Clone MV094
coded for a conserved ubiquitin core fused to 58 aminoacids homologous to the S27 ribosomal protein. This protein
diVers from that described by Wang and Ng in P. ostreatus
(Wang and Ng, 2000), but its structure has been also found
in other fungi such as Ustilago maydis and N. crassa, animals and plants.
Clone MV100 corresponds to a polyubiquitin gene that
contains, at least, three absolutely conserved tandem repetitions of the ubiquitin core sequence. In C. neoformans, the
polyubiquitin gene UBI4 contains Wve copies of the ubiquitin core sequence (Spitzer and Spitzer, 1995). We cannot
discard a similar structure in P. ostreatus. The polyubiquitin gene described here displayed an A gene expression pattern (Fig. 1); however, Lee et al. (2002) isolated a
polyubiquitin gene highly expressed in the vegetative mycelium but not in fruit bodies, and Ospina-Giraldo et al.
obtained a similar result in A. bisporus Ospina-Giraldo
et al. (2000). Our results indicate that this gene is indeed
expressed in lamellae (since the cDNA library derives from
this tissue) and suggest that it is also expressed in fruit
bodies.
Clone MV111 codes for a protein with high similarity
(Evalue D 4 £ 10¡38) to the human amyloid precursor protein binding protein 1 (AppBp1). This protein is one of the
two monomers forming the Nedd8-speciWc activating heterodimer (Nedd8-speciWc E1 factor) (Walden et al., 2003).
Nedd8 is a ubiquitin-like protein involved in the posttranslational modiWcation of the Cullin subunits of SCF-type E3
ubiquitin-ligase complexes (Schwechheimer, 2004). Cullin
neddylation has been related to various cellular and developmental processes in organisms ranging from yeast to
mammals. In fact, putative proteins similar to that coded
for by MV111 are also found in C. neoformans and in Xenopus laevis. Osaka et al. (2000) report that the Nedd8-modifying system is essential for cell cycle progression in
Schizosaccharomyces pombe; our results indicate that
MV111 mediated neddylation is presumably diVerent in
reproductive than in vegetative mycelium as deduced from
the diVerential expression of this gene (see below).
3.4. Gene regulation during the change of growth phase
The expression analysis of each one of the cDNAs studied revealed four major patterns (Fig. 1, patterns A, B, AB,
and C) that accounted for 96.2% of the genes. The strategy
384
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
II
I
L8575
R72400
MV051
MV103
P121350
R121475
S172375
P9875*
vmh1
MV126
MV087
MV045
L15112 5
L11145 0 MV032-2
MV058
MV162
P71525
MV152*
P131550
L21850* MV019-2*
P31550*
MV138
MV054-2
R3362 5
R7222 5*
MV105*
MV143*
R202700
P11850
MV057-2
MV119
MV044
L182900
R151750
P11800
MV015-1
MV115
X
S17825
MV133-2*
P171425* ctr1*
MV023*
vmh3 MV102
MV161-1
SSR9
MV172*
MV142*
MV133-1*
R201400
L51475
matA * MV132-1*
MV131-1*
GP015
MV131-2
P41450
P51225
R42425
pox C
VII
MV046
S191475
MV028-2
MV004-1
MV104
L16875
MV163-4
R191300
L15152 5
S191250
R3925
P42550
P131250
R141750
R11475
P91400
S12800
MV148
R4425
MV052-3
P122150
P192000
MV129
P11825
MV163-3*
IX
vao
L141525 MV098-1
R102100
MV090-2
MV098-2
P62300*
R20650*
P12950
fbh1
P102325
P31375
L22475 / R161200
MV084
tel 3
L181200
MV131-3
XI
MV100-1
R167 75
pox 1 MV004-2
L191750
MV128
R13525
20 cM
MV111
P82775
P123525*
R32050
L11075 MV094-1
MV052-1
mnp2 MV156
MV034
R4117 5
VIII
R132175
R111 875
MV168
P61100
MV042*
R32475
P6950
L12600
P16400
R8575
MV150-1
MV054-1
MV109
R21675
S71200
P19550
L141800 MV150-2
MV174
MV165
R101375
MV113*
R171 500
P131625
P17425
P14650
MV106
R6950 MV011-1
MV014
R192 100
MV132-2
MV161-2*
MV163-1
R153100
MV028-1*
P15950
L7900 MV032-1
P151050
S192600
MV057-1
MV118
MV098-3
L14237 5
Rib MV011-2
P1750
L19252 5* MV145*
L10217 5
MV089
MV179
S71725
MV097
S122075
mnp3
L52525*
MV091*
P9950
L16110 0
AE-5
MV136*
R9130 0*
R3975
MV112
R151 025
hon2
R151200
MV155*
MV108
R1775 MV052-2
VI
MV090-1*
MV146*
MV080
R61525
R117 75
MV094-3*
L15625 MV019-1*
mnp1
P191125
R124 00
V
IV
III
R161250
L18217 5
L5850 Hind III*
MV033
MV092
P11600*
R201950
MV028-3
S112325
MV116
MV093
R193300 / vmh2
MV110
MV081
MV163-2
P22650*
R8300 MV094-2
R15575
R21600*
R14700
matB *
MV123
P22100
L61800
MV100-2
matB
MV114
L151275
Fig. 2. Genetic linkage map of P. ostreatus composed of 11 linkage groups. All of mapped EST clones were marked in colored boxes (Yellow, general
metabolism; red, protein metabolism; violet, cellular signalling; green, cell structure and growth; blue, transport; grey, unknown function; brown, PCRgenerated markers). Markers that deviated from the 1:1 segregation (P< 0.05) indicate with an asterisk.
followed for the isolation of the EST forced the expression
of the corresponding gene in the lamellae. Other genes isolated in our laboratory, however, showed expression patterns complementary to those described here: copper
transporter ctr1 (Peñas et al., 2005) was expressed in all
developmental stages except in the lamellae, fruit-body-speciWc hydrophobin fbh1 expression was detected by Northern analysis in fruit bodies, but the Fbh1 protein was not
detected in the lamellae using in situ immunodetection
(Peñas et al., 1998), and vegetative mycelium hydrpophobins vmh1 and vmh2 were expressed exclusively in vegetative mycelium whereas hydrophobin vmh3 was expressed in
both vegetative and fruit body mycelia (Peñas et al., 2002).
In summary, a number of developmentally regulated gene
promoters are now available in P. ostreatus to undertake
the study of developmental processes in this fungus.
In addition to the major gene expression patterns, some
minor patterns were found that must be explained. Gene
MV123 coded for a new gene expressed in all the developmental stages studied except in the vegetative mycelium of
monokaryotic protoclone PC15. The simplest explanation
for this behavior is the occurrence of a cis mutation preventing its expression in this protoclone. The expression
pattern of the genes MV103 and MV111 suggests a more
complex regulation as both are expressed in only one of the
monokaryotic protoclones and in the reproductive tissues
whereas their expression is not detected in the other protoclon or in the dikaryotic vegetative mycelium (Fig. 1). This
expression pattern suggests the occurrence of diVusible
transcription factors (repressors, in this case) produced by
one of the nucleus that regulates the gene expression in the
other nucleus within the dikaryon. This cross-talk between
nuclei within the dikaryon is currently being studied using
these genes as model in our laboratory.
3.5. Genetic linkage map of the cDNA clones
The 82 genes were mapped to the P. ostreatus linkage
map (Larraya et al., 2000) using the MAPRF software
described by Ritter et al. (1990); Ritter and Salamini (1996).
Seventy-four (90.2%) of the genes exhibited RFLP polymorphism and could be mapped, whereas eight clones
(9.8%) appeared as monomorphic and could not be
mapped. The 74 mapped genes determined 87 loci (Fig. 2)
since ten of them revealed various members of gene families
(Table 1). Twenty-Wve of the mapped genes exhibited
385
S.-K. Park et al. / Fungal Genetics and Biology 43 (2006) 376–387
Table 3
Pleurotus ostreatus genetic linkage map statistics
Chrom. Size % Total
Length % Total genome Kbp/cM
No.
(Mb) genome size (cM)
length
No. of No. of EST No. of EST No. of
No. of EST at No. of EST at No. of
EST
mapped per mapped per map
unique map
multiple map new map
mapped Mb
cM
positions positions
positions
positions
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
Total
13
8
13
11
12
12
7
8
3
6
6
99
4.7 13.2
4.4 12.4
4.6 13.0
3.6 10.1
3.5
9.9
3.1
8.7
3.2
9.0
3.0
8.5
2.1
5.9
1.8
5.1
1.5
4.2
35.5 100
107
164
183
63
92
58
72
107
76
41
98
1061
10.1
15.5
17.2
5.9
8.7
5.5
6.8
10.1
7.2
3.9
9.2
100
43.9
26.8
25.1
57.1
38.0
53.4
44.4
28.0
27.6
43.9
15.3
36.3 (13.0)
distorted segregation ratios; they were mostly assigned to
linkage groups III, IV, and IX where distorted segregations
had been previously reported (Larraya et al., 2000). The
cDNA clones mapped to all linkage groups, although they
appeared not evenly distributed across the diVerent chromosomes. Table 3 shows the distribution of EST loci
mapped per chromosome and the global statistics for this
linkage map.
To facilitate the mapping procedure, we evaluated the
use of PCR polymorphisms to map some of the cloned
cDNAs. For this purpose, speciWc primers deduced from
the cDNA sequence were used to amplify gene regions in
the search for size or restriction polymorphisms. The primers were selected, whenever it was possible, to anneal to
intron Xanking regions predicted on the basis of the
BLASTX results. Later, the identity of the PCR ampliWed
products was checked by Southern hybridization using
each corresponding cDNA as probe. In most cases, the
PCR products that could be mapped (i.e., those for which a
polymorphism could be detected) did not correspond to the
original gene. These products were presumably generated
by a RAPD-like reactions of one of the primers used and
were classiWed as anonymous PCR markers which mapped
to sites unlinked to those of the corresponding cDNA
mapped by RFLP (Fig. 2). The 12 loci identiWed by this
PCR-based approach increased the number of loci mapped
in this study to 99 (Table 3).
This map complements the Wrst P. ostreatus linkage map
with the addition of 99 new loci (Table 3). The addition of
the new genes did not change the general structure of the
linkage map previously reported. The total map length was
increased in 61 cM (6.1%) due to the mapping of the two
putative polyubiquitin loci that added 42 cM, and of locus
MV163-3 that added 18 cM (Fig. 2). The cDNA corresponding to clone MV163 codes for a lamellae-speciWc protein without signiWcant homologs in the gene databases and
corresponds to the largest gene family identiWed in this
study since it revealed four diVerent loci (Table 1).
The 99 new loci mapped in this work identiWed 46 new
genome positions (Table 3). Sixty-two percentage of the
2.8
1.8
2.8
3.1
3.4
3.9
2.2
2.7
1.4
3.3
4.0
2.9 (0.8)
0.12
0.05
0.07
0.17
0.13
0.21
0.10
0.07
0.04
0.15
0.06
0.11 (0.05)
18
19
24
14
18
19
16
15
13
12
13
181
1
4
4
5
5
4
2
5
1
2
5
38
12
4
9
6
7
8
5
3
2
4
1
61
2
4
7
6
6
4
4
5
1
2
5
46
genes mapped to positions already deWned by other markers. This accumulation of genetic markers at discrete
genome positions was already uneven in the Wrst map
released (Larraya et al., 2000). It is usually accepted that
wide genetic linkage distances reXect wide physical distances in the chromosome. However, as the genetic linkage
analysis is functional, long linkage distances can correspond to recombination hotspots that break the correlation
between genetic and physical distances. Some examples of
inXuence of the sequence structure on the recombination
frequency have been recently described in the literature: in
an analysis of the genetic structure of the matB locus in S.
commune, Fowler et al. (2004) found that the 8 kb region
separating the B3 and B2 loci contains 19 diVerent short
sequences with imperfect repeats as well as a 1 kb segment
where the GC content was highly biased, and that the 5 kb
region separating the loci B3 and B3 contains 17 short
imperfect direct repeats similar in length and number to
those of the B3–B2 complex. The ratio of physical to
genetic distance (up to 1 kb/cM) suggests that this region
forms a recombination hotspot. On the contrary, Espeso
et al. (2005) have found that recombination frequencies are
greatly reduced near the centromeres in two Aspergillus
nidulans chromosomes altering the physical to genetic distance ratio at these locations. The non-random distribution
of ESTs and anonymous markers on the genetic linkage
map suggest the presence of other recombination-prone
sequences scattered across the P. ostreatus genome.
Acknowledgments
This work was supported by research project AGL200204222-C03-01 of the Comisión Nacional de Ciencia y Tecnología. SKP was supported by a PhD fellowship from the
Spanish Ministerio de Asuntos Exteriores.
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