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Mycologia, 94(5), 2002, pp. 823-833.
© 2002 by The Mycological Society of America

Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis


Soon Gyu Hong
Wonjin Jeong

     Korean Collection for Type Cultures, Korean Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, Korea

Hack Sung Jung 1

     School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Korea

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

There has been a systematic need to seek adequate phylogenetic markers that can be applied in phylogenetic analyses of fungal taxa at various levels. The mitochondrial small subunit ribosomal DNA (mt SSU rDNA) is generally considered to be one of the molecules that are appropriate for phylogenetic analyses at a family level. In order to obtain universal primers for polypores of Hymenomycetes, mt SSU rRNA genes were cloned from Bjerkandera adusta, Ganoderma lucidum, Phlebiopsis gigantea, and Phellinus laevigatus and their sequences were determined. Based on the conserved sequences of cloned genes from polypores and Agrocybe aegerita, PCR primers were designed for amplification and sequencing of mt SSU rDNAs. New primers allowed effective amplification and sequencing of almost full-sized genes from representative species of polypores and related species. Phylogenetic relationships were resolved quite efficiently by mt SSU rDNA sequences, and they proved to be more useful in phylogenetic reconstruction of Ganoderma than nuclear internal transcribed spacer (ITS) rDNA sequences.

Key words: Ganoderma, Hymenomycetes, information content, universal primers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Currently the taxonomy of polypores is primarily based on morphological characteristics, such as the shapes of basidiocarps and hymenophores, hyphal systems, and forms and sizes of basidiospores, and secondarily on mycological features like host relationships and rot types (brown versus white) (Donk 1964Citation, Ryvarden 1991Citation). However, overlapping and variable morphological characteristics have made the classification of polypores unreliable and unstable, which has been always a nuisance to mycologists (Alexopoulos et al 1996Citation, Hibbett and Donoghue 1995Citation).

Molecular systematics has been shown to be a valuable tool in modern fungal taxonomy (Bruns et al 1991Citation, Mitchell et al 1995Citation). Among various molecular methods including DNA-DNA hybridization, RFLP, and sequence analyses, phylogenetic analyses of amino acid or DNA sequences are known to have the highest resolving power (Bruns et al 1991Citation). Due to ubiquitous occurrence and essential functions, DNA sequence data of 18S, 26S, ITS, and mitochondrial rDNAs are most frequently used in recent phylogenetic studies of eukaryotic cells. Sequences of 18S rDNAs are conserved and have been used in phylogenetic analyses of fungi of higher taxonomic ranks such as classes or orders (Swann and Taylor 1993Citation, Wilmotte et al 1993Citation). On the other hand, ITS rDNAs are so variable that they often cannot be aligned accurately between genera and are now commonly used in the systematics of species within a genus (Moncalvo et al 1995aCitation, bCitation, Yan et al 1995Citation). However, mt SSU rDNAs were reported to evolve 16 times faster than 18S rDNAs (Bruns and Szaro 1992Citation), but are less variable than ITS rDNAs. Thus they are believed to have a potential to fill phylogenetic gaps at a family level between those available from 18S and ITS rDNAs.

The rDNA found in the nuclear genome of eukaryotes usually consists of tandem repeated units. It is generally considered that the rDNA arrays tend to be homogenized through concerted evolution (Hillis and Dixon 1991Citation). Some examples, however, showed that an organism could have multiple forms of a gene cluster of different sequences (Carranza et al 1996Citation, Tang et al 1996Citation, O'Donnell and Cigelnik 1997Citation, Fatehi and Bridge 1998Citation). Therefore, phylogenies based on 18S or ITS rDNA should be verified by other sources of data. Sequences of mt SSU rDNA serve this purpose.

Nearly full-length sequences of 18S and ITS rDNAs for fungi can be amplified by PCR, but only partial sequences of mt SSU rDNAs have been amplified (White et al 1989Citation, Hibbett and Donoghue 1995Citation) so far. For this reason, mt SSU rDNA sequences have not been popular among molecular systematists, and phylogenetic investigation of polypore fungi based on partial sequences of mt SSU rDNA has been found unsatisfactory (Hibbett and Donoghue 1995Citation).

In order to design universal primers for polypore fungi of Hymenomycetes, three polypore species, Bjerkandera adusta (Polyporaceae), Ganoderma lucidum (Ganodermataceae), and Phellinus laevigatus (Hymenochaetaceae), and one corticioid fungus, Phlebiopsis gigantea (Corticiaceae), were selected to clone mt SSU rDNAs. Based on conserved sequences of mt SSU rDNAs from B. adusta, G. lucidum, P. gigantea, P. laevigatus, and Agrocybe aegerita (Gonzalez et al 1997Citation), primers were designed for amplification and sequencing of mt SSU rDNAs. In order to assess the phylogenetic informativeness of mt SSU rDNA, similarity value, number of informative sites, skewness, and CI index were used.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Fungal strains – Strains used for PCR amplification and mt SSU rRNA gene cloning are listed in Table I. In order to compare information content of mt SSU rDNA with ITS rDNA, strains of Ganoderma for which ITS sequences are available (Moncalvo et al 1995aCitation) were selected for determining mt SSU rDNA sequences.


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  		TABLE I. Fungal strains used for PCR amplification and sequencing mt SSU rDNA

 
DNA isolation – For the isolation of mitochondrial DNA which was used in cloning the mt SSU rRNA gene, fungi were grown in 1 L of ME broth (1% malt extract, 0.5% yeast extract, 0.5% peptone) at 24 C for 2 wk with agitation. The mycelia were harvested by filtering, frozen with liquid nitrogen, and then homogenized with a mortar. Mitochondrial DNA was purified from total DNA by bisbenzimide-CsCl gradient centrifugation (Raeder and Broda 1985Citation). For the PCR amplification, nucleic acids were extracted from mycelia grown on ME agar covered with a cellophane disc according to the freeze-thawing method of Lecellier and Silar (1994)Citation.

Cloning of mt SSU rDNA – DNA fragments digested with EcoRI, HindIII, or HpaI were ligated with the plasmid pBluescript KS(-) (Stratagene, La Jolla, California, USA). The recombinant plasmids were transformed into competent Escherichia coli DH5{alpha}. Dot blotting or PCR screenings were performed to identify recombinant clones containing the mt SSU rRNA genes. For dot blotting, DNA fragments containing the mt SSU rDNA of Trimorphomyces papilionaceus (Hong et al 1993Citation, Jeong et al 1995Citation) were used as probes. For the PCR screening, three sets of primers were used: BAMS1 (5'-GTAAAAGCCTACCAAGCCGACG-3') and BAMS2 (5'-TTGACAGTGAGGGGTTCATGGG-3') for B. adusta, GLMS1 (5'-AACACAATAACCATTCCGCC-3') and GLMS2 (5'-TTCCTTCTTCATGCCTCCG-3') for G. lucidum, and PGMS1 (5'-TACTAGGGAGATTTCATTCC-3') and PGMS2 (5'-TTTAATTTTGGTTCHGATTGAACG-3') for P. gigantea (Fig. 1).



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  		 FIG.1. Organization and restriction map of the mt DNA region encoding SSU rRNA. Clones BAE16, GLE78, and PGH13 were selected by dot-blotting and BAHP1, GLHP1, and PGHP1 by PCR screening using primers indicated by arrows. Primers BAMS1, BAMS2, GLMS1, GLMS2, and PGMS1 were designed using flanking sequences between EcoRI and HpaI sites. PGMS2 was designed based on the conserved sequences between Bjerkandera adusta and Ganoderma lucidum. Sequences of primers were presented in the Materials and Methods. Restriction sites abbreviations: D, HindIII; E, EcoRI; H, HpaI.

 
SSU rDNA amplification and sequence determination – Mitochondrial SSU rDNA was amplified using BMS05 and BMS173 or BMS173B primers (Table II). Amplified mt rDNAs were purified using Wizard PCR prep kit (Promega, Madison, Wisconsin, USA) and cycle-sequenced using internal primers (Table II) and Top DNA sequencing kit (Bioneer, Chungju, Korea). Sequences of cloned DNAs were determined by the dideoxy termination method (Sanger et al 1977Citation) using Sequenase kit (Amersham, Buckinghamshire, England). Sequencing reactions were conducted following the supplier's guide.


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  		TABLE II. Primers used in PCR amplification and sequencing

 
Evaluation of information contents and phylogenetic potential – Nucleotide sequences of mt SSU rDNA were aligned using the molecular sequence editor PHYDIT 3.0 (formerly called AL16S, Chun 1995Citation) that allows editing, manual, and semi-computer alignment, and comparative analysis of rDNA nucleotide sequence data according to secondary structure model information (available at http://plaza.snu.ac.kr/~jchun/phydit/). Ambiguously aligned sites caused by multiple insertions and deletions found in nine variable domains and stem 15 of secondary structure (Fig. 2) (Neefs et al 1993Citation) were excluded from further analyses. The phylogenetic relationship of mt SSU rDNA sequences of Hymenomycetes were estimated by heuristic search option of PAUP*4.0 beta version (Swofford 1998Citation) with random sequence addition. Skewness values were calculated from 10 000 000 random trees and decay indices by procedures presented by Bruns et al (1992)Citation. Sequences of ITS rDNA were aligned using CLUSTALX program (Thompson et al 1997Citation) with a gap open penalty of 5 and a gap extension penalty of 1 and then manually adjusted. Phylogenetic relationship, consistency index, and skewness of tree distribution were estimated by exhaustive search of PAUP* 4.0 beta version (Swofford 1998Citation) for ITS and mt SSU rDNAs of the genus Ganoderma.



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  		 FIG. 2. Map of mt SSU rDNA from fourteen Hymenomycetes species and 16S rRNA of E. coli. Nine variable domains and stem 15 region were indicated as open boxes

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Cloning and sequencing of SSU rDNA – Screening of a B. adusta mitochondrial DNA library constructed in EcoRI site by dot blotting led to the selection of a plasmid carrying a 2.0 kb EcoRI fragment (BAE16, Fig. 1A). It was found that the clone lacked about 300 bp in the 5' region of the gene by the sequence alignment with the A. aegerita gene (Gonzalez et al 1997Citation). By PCR screening of HpaI library using two primers, BAMS1 and BAMS2, a recombinant plasmid carrying 2.5 kb HpaI fragment was selected (BAHP1, Fig. 1A).

Cloning of the mt SSU rRNA gene from G. lucidum was performed with the same procedure as the one for B. adusta. Two plasmids carrying mt SSU rRNA gene were selected; a plasmid carrying 7.0 kb EcoRI fragment (GLE78) and a plasmid carrying 3.7 kb HpaI fragment (GLHP1, Fig. 1B). The mt SSU rRNA gene of G. lucidum was found to have a 1506 nt group II intron between the positions 715 and 716 corresponding to the positions 788 and 789 of 16S rDNA of E. coli, which was deduced by comparing with the sequence alignment of E. coli and constructing a secondary structure model of the intron (data not shown).

Two clones encoding the SSU rRNA gene of P. gigantea were obtained from HindIII and HpaI libraries. The clones contained a 1.8 kb HindIII fragment (PGH13) and a 2.4 kb HpaI fragment (PGHP1) each (Fig. 1C). The sizes of the mt SSU rRNA of B. adusta, G. lucidum and P. gigantea were deduced as 1831, 1553, and 1892 bp respectively from sequence alignment with the mt SSU rDNA sequence of A. aegerita (Gonzalez et al 1997Citation).

Design of primers – The sequences of A. aegerita, B. adusta, G. lucidum, and P. gigantea were aligned with 16S rDNA of E. coli based on the secondary structure model, and it was found that each of the sequences had multiple insertions or deletions in different regions of the gene (Fig. 2). The mt SSU rRNA gene of B. adusta had long insertions in the V2, V4, V8, and V9 regions (Neefs et al 1993Citation). The gene was shorter in the V1, V3, V5, and V7 regions compared to that of E. coli. The V6 region of the gene was slightly longer than that of E. coli. The gene from G. lucidum had a long insertion in the V9 region. It was slightly longer in the V1, V5, V6, and V8 regions and shorter in the V2, V3, V4, and V7 regions than that of E. coli. The gene of P. gigantea had long insertions in the V2, V6, V8, and V9 regions. It was slightly longer in the V1 region and shorter in the V3, V4, V5, and V7 regions than that of E. coli. Although variable domains of the gene had multiple insertions or deletions as described above, the conserved domains were highly conserved among A. aegerita, B. adusta, G. lucidum, and P. gigantea.

Primers were designed to amplify and determine the sequence of SSU rRNA genes based on conserved sequences. Requirements for primers were as follows: They were conserved in all compared sequences, no significant hairpin loops were formed in secondary structure prediction, primer-dimers were not formed by each pair of forward and reverse primers, so that any DNA regions targeted could be amplified, and DNA sequences could be read bidirectionally assuming that 300 bps were read by one sequencing reaction (BMS05, BMS35, BMS55, BMS65, BMS105, BMS115, BMS145, BMS33, BMS53, BMS63, BMS103, BMS113, BMS133, BMS153, BMS173, Table II).

PCR reactions with primers BMS05 and BMS173 produced DNAs from 1.5 to 1.9 kb long for species of Table I from various families of Aphyllophorales except for Cytidia salicina, Phellinus laevigatus, and Pulcherricium caeruleum of the Corticiaceae and Hymenochaetaceae. In order to know which primer was fit for these template DNAs, we tested the fidelity of primers to template DNA by PCR reactions using four other primer sets, BMS05 and BMS113, BMS05 and BMS133, BMS55 and BMS173, and BMS65 and BMS173. Of the four sets of primers tested, two PCR reactions using BMS173 primer did not produce any DNA fragments, suggesting that the BMS173 primer did not work for some DNA templates from Aphyllophorales. In order to find DNA regions conserved in these species and to improve fidelity of primers, mt SSU rDNA from Phellinus laevigatus was cloned. The selected clone contained an 8.4 kb HindIII fragment. Alignment of P. laevigatus sequence with four previously determined mt SSU rDNA sequences revealed that second and third base positions (A and T) from the 3' end of BMS173 primer were not conserved in P. laevigatus.

Thirteen internal primers were generally good for determining sixteen mt SSU rDNA sequences from the strains listed in Table I except for C. salicina, P. laevigatus and P. caeruleum. However, sequencing reactions using two primers, BMS63 and BMS145, were not satisfactory for J. nitida, L. spadicea, M. nigra, and P. atropurpurascens. In the cases of BMS105, BMS103, BMS133, and BMS153, aligned sequences had non-conserved sites in the primer regions even though sequencing with these primers was successful. To improve fidelity of primers to template DNAs, primers were modified based on additional 17 mt SSU rDNA sequences (BMS105B, BMS155, BMS63B, BMS103B, BMS133B, BMS153B, and BMS173B, Table II). These primers were good for PCR amplification and sequencing of mt SSU rDNA from C. salicina, J. nitida, L. spadicea, M. nigra, P. laevigatus, P. caeruleum, and P. atropurpurascens.

Two external primers, BMS05 and BMS173B, were designed based on conserved sequences of five species of distant evolutionary relationships (Hibbett and Donoghue 1995Citation, Hibbett et al 1997Citation). PCR amplification tests using these two primers on Amanita verna (Amanitaceae, Agaricales), Pleurotus ostreatus (Pleurotaceae, Agaricales), Coniophora puteana (Coniophoraceae, Aphyllophorales) and Hymenochaete tabacina (Hymenochaetaceae, Aphyllophorales) were satisfactory (data not shown), suggesting a possibility that they work well for a wide range of fungi from Hymenomycetes, including Agaricales and Aphyllophorales.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
By aligning mt SSU rDNA sequences from fourteen fungal species of Hymenomycetes and 16S rDNA of E. coli based on the secondary structure model, it was found that the sequences had multiple insertions and deletions in the variable domains (Fig. 2). Large insertions or deletions were found in the V2, V4, V6, V8, and V9 domains. The other variable domains also had multiple insertions or deletions, so that sequence alignment was not possible except among the closely related taxa. Stem 15 of the secondary structure model was previously known to be a very conserved domain (Neefs et al 1993Citation). However, J. nitida and M. nigra had multiple insertions in this region. The two species did not form a monophyletic group in the phylogenetic analysis (Fig. 3A) and the two insertion sequences could not be aligned. Therefore, it is speculated that such large insertions in stem 15 of those two species could have been obtained by independent insertion events of two different origins.



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  		 FIG. 3. Phylogenetic analysis of full-length (A) and partial (B) mt SSU rDNA sequences of Hymenomycetes and distribution of tree lengths for 10 000 000 random trees. The consensus tree of three equally most parsimonious trees was presented for MS1/MS2 region of mt SSU rDNA. The phylogenetic tree was obtained with heuristic search of PAUP* 4.0 beta. Bootstrap values from a sample of 1000 replicates and decay values are shown at internal branches (bootstrap value/decay value)

 
Similarity values among fourteen species of Hymenomycetes for the 1085 aligned sites are presented in Table III. Similarity values ranged from 83.93% between A. aegerita and P. laevigatus to 99.22% between B. adusta and L. spadicea. High similarity values from 96.8% to 98.55% were found in four closely related species, C. salicina, G. lucidum, P. cinnabarinus, and T. versicolor (also see Fig. 3). Among these species, 435 additional sites were alignable in variable domains. Informative sites were increased three times, from seven to twenty-one.


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  		TABLE III. Similarity values (lower left) and different/total nucleotides (upper right) of conserved domains among 14 mt SSU rDNA sequences

 

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  		TABLE III. Extended

 
Phylogenetic analysis of 1085 aligned sites of fourteen mt SSU rDNAs yielded one most parsimonious tree of 764 steps, a skewness value of g1 = -0.8512 (p < 0.01) and a consistency index of 0.664. The number of informative sites was 221 (Fig. 3A). The deeply left-skewed data structure of the mt SSU rDNA indicates that the data have non-random structures that likely reflect phylogenetic signal (Hillis and Huelsenbeck 1992Citation). The high similarity values also indicated that mt SSU rDNA sequences of Hymenomycetes are not saturated with substitutions.

Two inferred monophyletic groups, which were supported by high bootstrap values and decay indices, were recognized by the phylogenetic analysis. One group includes C. salicina, G. lucidum, P. cinnabarinus, and T. versicolor. The species, M. nigra, was found to be related to this group by relatively high bootstrap value and decay index data. Two genera, Pycnoporus and Trametes, previously were recognized as closely related taxa based on the morphological characteristics such as a di-trimitic hyphal system, generative hyphae with clamps, hyaline, thin-walled, cylindric, smooth, and non-amyloid basidiospores, absence of cystidia, formation of white rot, tetrapolarity, and growth almost exclusively on angiosperms (Ryvarden 1991Citation). The genus Ganoderma also has the same morphological characteristics except that it forms double-wall basidiospores (Gilbertson and Ryvarden 1987Citation). The close phylogenetic relationship of C. salicina with three poroid fungi, G. lucidum, P. cinnabarinus, and T. versicolor, was not supported by morphological characteristics because it is a resupinate fungus with smooth hymenophores, monomitic hyphal system, numerous dendrohyphidia, and allantoid basidiospores. Concrete conclusions await analysis of other strains and related species. The genus Melanoporia has similar morphological characteristics except that it causes brown rot (Gilbertson and Ryvarden 1987Citation). However, it is premature to include M. nigra in this group because the similarity values among M. nigra and other species of this group were not high (Table III).

The other monophyletic group includes B. adusta, L. spadicea, P. caeruleum and P. gigantea. The genus Bjerkandera has been classified in the Polyporaceae by Donk (Donk 1964Citation) based on the poroid hymenophores. However, a close relationship of B. adusta to corticioid fungi has been presented in subsequent phylogenetic studies (Hibbett and Donoghue 1995Citation, Hibbett et al 1997Citation). It is evident that B. adusta, a poroid fungus, is closely related to three corticioid fungi, and C. salicina, a corticioid fungus, is closely related to three poroid fungi based on phylogenetic relationships (Fig. 3A) and similarity values (Table III).

Although it was not possible to reach concrete taxonomic conclusions with such a small number of taxa, it is obvious that mt SSU rDNA sequences contain considerable information, enough to resolve phylogenetic relationships among fungal species of Hymenomycetes. In terms of sequence lengths, partial mt SSU rDNA sequences amplified by MS1 and MS2 primers (White et al 1989Citation) for the set of same taxa had 68 informative sites out of total 319 unambiguously aligned sites. Heuristic search of the sequences produced three equally parsimonious trees of 257 steps (Fig. 3B). The consensus tree had polytomous unresolved branches. Bootstrap values and decay indices were much lower in a number of branches than those from the tree of full-length mt SSU rDNA. Distribution of random trees was less left-skewed than the one of full-length mt SSU rDNA sequences. From these results, it is apparent that sequences of full-length mt SSU rDNA are increasing phylogenetic information and resolving power considerably more than partial sequences.

Analysis of 476 aligned sites of ITS sequences from 9 strains of Ganoderma yielded 4 most parsimonious trees of 47 steps, a skewness value of g1 = -2.1366 (p < 0.01), a consistency index of 1.000 and 18 informative sites. The strict consensus tree was presented in Fig. 4A. Analysis of 1469 aligned sites of mt SSU rDNA for the same suite of taxa yielded one parsimonious tree of 111 steps, a skewness value of g1 = -1.5850 (p < 0.01) and a consistency index of 0.865. The number of informative sites was 62 (Fig. 4B). The number of most parsimonious trees itself was regarded as a poor indicator of data quality in phylogenetic analyses because a single solution can be found from random data with high probability (Hillis and Huelsenbeck 1992Citation). However, ITS rDNA sequences of Ganoderma have only 19 informative sites and the poor resolution of the phylogenetic relationship is likely to have originated from the poverty of phylogenetic information. On the other hand, parsimony analysis of mt SSU rDNAs produced one most parsimonious tree, which had a relatively high value of CI, bootstrap values, and decay indices. The number of informative sites was 3.3 times as many as that of ITS rDNA sequences. Eighty-four percent of informative sites (52/62) were distributed in nine variable domains. Average similarities among nine sequences of conserved domains and variable domains of mt SSU rDNA and ITS rDNA were 99.43, 94.51, and 96.67% each. The rate of evolutionary change of variable domains of mt SSU rDNA was found to be faster than ITS rDNA.



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  		 FIG. 4. Phylogenetic analyses of ITS (A) and mt SSU rDNAs (B) from Ganoderma and distribution of tree lengths for all possible trees. The consensus tree of 4 equally most parsimonious trees was presented for ITS sequences. The phylogenetic trees were obtained with exhaustive search of PAUP* 4.0 beta. Bootstrap values from a sample of 1000 replicates and decay indices are shown at internal branches (bootstrap value/decay index)

 
Multiple insertions or deletions were found in the V4 domain of mt SSU rDNA. The sequence alignment of these domains was presented in Fig. 5. The sister taxa of the genus Ganoderma, namely C. salicina, P. cinnabarinus, and T. versicolor, had homologous sequences with G. lucidum ATCC 46755 and 5 other strains with long sequences. Therefore, it is concluded that multiple deletion events caused the short V4 domain of G. oerstedii ATCC 52411, G. pfeifferi CBS 747.84 and G. resinaceum CBS 152.27. It is not certain whether additional deletion events occurred in G. oerstedii ATCC 52411 or multiple insertion events occurred in a common ancestor of G. pfeifferi CBS 747.84 and G. resinaceum CBS 152.27 after large deletions in a common ancestor of the three species. Multiple insertions or deletions of mt SSU rDNA were observed also within the genus Agrocybe (Gonzalez and Labarere 1998Citation) and Suillus (Bruns and Szaro 1992Citation). By comparative analysis of the secondary structures of variable domains from the genus Agrocybe, it was proposed that molecular studies of variable domains could be a good alternative method for determining relationships between species (Gonzalez and Labarere 1998Citation). In the preliminary study of the genus Ganoderma, we also conclude that the insertion or deletion events are congruent with the phylogenetic analysis of primary sequences, and that analysis of multiple insertion and deletion events could be a good phylogenetic marker.



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  		 FIG. 5. Sequence alignment of V4 domain (stem P23-1) from nine Ganoderma strains and three sister taxa. The boxed regions (1 and 2) base pair with those of opposite sites (1' and 2')

 
From phylogenetic analyses of several genera of Hymenomycetes and strains of the genus Ganoderma, it is clear that mt SSU rDNA sequences can be used as molecular markers to resolve phylogenetic relationships of both higher and lower rank of taxa, because the region is composed of conserved and variable domains. In addition, insertion and deletion events as well as the primary sequence can be used as important molecular markers to resolve phylogenetic relationships.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. K.S. Bae of KCTC (Korean Collection for Type Cultures), KRIBB (Korea Research Institute for Bioscience and Biotechnology), who kindly provided fungal strains for research collaboration. This work was supported by the Brain Korea 21 Project of the Ministry of Education and Human Resources Development and partly by the Young Scientist Research Grant of the Korea Research Foundation.


   FOOTNOTES
 
1 Corresponding author, Email: minervas{at}snu.ac.kr Back

Accepted for publication May 15, 2002.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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