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. 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