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Mycologia, 96(2), 2004, pp. 240-248.
© 2004 by The Mycological Society of America

Molecular biology

Variations in sequence and occurrence of SSU rDNA group I introns in Monilinia fructicola isolates


Marie-José Côté 1

     Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield), Centre for Plant Quarantine Pests, 3851 Fallowfield Road, Ottawa, Ontario, K2H 8P9 Canada

Mireille Prud’homme

     Health Canada, Food Directorate, Building No. 7, Tunney’s Pasture, P.L. 0700E1, Ottawa, Ontario, K1A 0L2 Canada

Allison J. Meldrum

     Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield), Centre for Plant Quarantine Pests, 3851 Fallowfield Road, Ottawa, Ontario, K2H 8P9 Canada

Marie-Claude Tardif

     Health Canada, Food Directorate, Building No. 7, Tunney’s Pasture, P.L. 0700E1, Ottawa, Ontario, K1A 0L2 Canada

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

A group I intron of 418 base pairs in the Monilinia fructicola ribosomal small-subunit sequence was characterized. The absence of such an intron in M. laxa and M. fructigena led to a PCR test for M. fructicola identification based on the presence of this intron. The failure to amplify a PCR fragment for some isolates of M. fructicola recently lead to speculation that the intron might not be present always in M. fructicola. In this study, we analyzed 13 isolates of M. fructicola and found that the intron was absent in four isolates and we determined from sequence analysis that there are several nucleotide variations that allow the M. fructicola ribosomal SSU intron to be grouped into 6 polymorphic types.

Key words: 18S rRNA, brown rot, group I intron sequences


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Monilinia fructicola is one of the three Monilinia species that cause brown rot of fruit. M. fructicola and M. laxa attack mainly stone fruit, whereas M. fructigena attacks stone fruit but favors pome fruit (Batra 1991Go). In North America, brown rot of fruit is caused mainly by M. fructicola and to a lesser extent by M. laxa. In Europe, the main causal agents of the disease are M. fructigena and M. laxa (Byrde and Willetts 1977Go). To aid in preventing the entry of M. fructigena into North America and entry of M. fructicola into Europe, efforts were made toward establishing a molecular method for Monilinia species differentiation, which resulted in Fulton and Brown (1997)Go locating a group I intron in the small-subunit (SSU) ribosomal DNA gene of M. fructicola.

Group I introns are recognized by a particular secondary structure and splicing pathway (Cech and Herschlag 1996Go) and are common in rDNA sequences of fungi and algae ( Johansen et al 1996Go). It also was demonstrated that a common characteristic of group I introns was their insertion in the same position of the SSU independent of the phylogeny of the host organism (Bhattacharya et al 1996Go, Gargas et al 1995Go, Hibbett 1996Go). The group I intron present in the SSU rDNA of M. fructicola, which is located at position 943 relative to Escherichia coli ribosomal SSU (GenBank accession: ECORRD) or position 1165 relative to Saccharomyces cerevisiae ribosomal SSU (GenBank accession: YSCRGEA) is not found in M. fructigena or M. laxa. However, several more-distant fungi, green algae and amoebae have a group I intron in the same position of the SSU rDNA (Gargas et al 1995Go).

PCR primers for the M. fructicola SSU rDNA intron and some of the SSU sequence were developed for species identification (Fulton and Brown 1997Go). Recently there have been reports that the intron-containing PCR product does not always occur in isolates thought to be M. fructicola, suggesting that some isolates lack the intron (Förster and Adaskaveg 2000Go, Hughes et al 2000Go). The objective of this study was to look for the SSU rDNA intron in 13 isolates of M. fructicola by using PCR with different primer pairs and, when the intron was present, to analyze the sequence. Four M. fructicola isolates did not have the intron. By analyzing the intron sequence in the remaining isolates, we could distinguish five polymorphic groups, one of them corresponding to the sequence published by Fulton and Brown (1997)Go. The intron sequence published by Snyder and Jones (1999)Go and not found in this study was classified into a sixth polymorphic group.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Isolates. – M. fructicola isolates used and their sources are listed in TABLE I. Potato-dextrose agar in Petri dishes (39 g/L) was inoculated with rehydrated lyophilized cultures. To limit potato DNA contamination, DNA extraction was done from mycelia that had grown over glass cover slips that had been deposited on the culture medium surface. All isolates were confirmed to be M. fructicola by random amplified polymorphic DNA (RAPD) technique (Förster and Adaskaveg 2000Go).


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  		TABLE I. List of the Monilinia fructicola isolates, their origin and the molecular characterization of the SSU rDNA intron polymorphism
 
DNA extraction. – DNA was extracted following the method of Möller et al (1992)Go with the following modifications. Mycelium was rinsed from the glass cover slip with 550 µL of extraction buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 2% SDS) into a 1.5 mL microcentrifuge tube; 100 µg of proteinase K (Roche, Laval, Québec, Canada) was added and the mixture was incubated at 57 C for 60 min. The mixture was vortexed twice during incubation. After incubation, 140 µL of 5 N NaCl was added, followed by 65 µL of preheated 10% CTAB (hexadecyltrimethylammonium bromide). After 10 min at 65 C, 700 µL of chloroform : isoamyl alcohol (24:1) was added and the mixture was placed on ice for 30 min. After 10 min centrifugation at 14 000 g at 4 C, 600 µL of the supernatant were transferred to microcentrifuge tubes containing 225 µL of 5 M ammonium acetate and placed on ice for 30 min. After 10 min centrifugation at 14 000 g at 4 C, the supernatant was added to 0.55 volume of isopropanol, mixed well and incubated on ice at least 10 min. The mixture was centrifuged 10 min at 14 000 g at 4 C, and the supernatant was discarded. DNA pellets were washed with 70% ethanol, dried and resuspended in 100 µL of sterile water. DNase-free RNase (Roche, Laval, Québec, Canada) was added (1 µg), and the DNA extracts were incubated at 37 C for 20 min.

PCR amplification (RAPD and sequence-specific). – All PCR reactions were performed with Invitrogen Canada reagents (Burlington, Ontario). All sequence-specific primers were synthesized by Invitrogen Canada (Burlington, Ontario). The random primer 353 (5'-TGG GCT CGC T-3') used for RAPD analysis was synthesized by the University of British Columbia, Canada (Dr. John Hobbs Nucleic Acid-Protein Service [NAPS] Unit Biotechnology Laboratory). PCR amplifications using specific primers were performed with 1 µL of a 1/10 or 1/100 dilution of mycelial DNA (1–20 {eta}g/ µL) in a solution of 20 mM Tris-HCl pH 8.4, 50 mM KCl, 200 µM of each dNTP, 2.0 mM MgCl2 and 0.025 units/µL Taq polymerase. One type of reaction included primers NS3 (5'-GCA AGT CTG GTG CCA GCA GCC-3'; White et al 1990Go) and NS6 (5'-GCA TCA CAG ACC TGT TAT TGC CTC-3'; White et al 1990Go) at 0.2 µM each. Another set of reactions employed primer: NS5 (5'-AAC TTA AAG GAA TTG ACG GAA G-3'; White et al 1990Go) paired with mfs-3 (5'-CAC TCG AAA GCA TTG AGT TG-3'; Fulton and Brown 1997Go), nu-SSI(943)-251-3'-Mf (Nomenclature: Gargas and DePriest 1996Go) (5'-CCA TTC CCA TTT AGT CTC TG-3') or NS6 at 0.4 µM each. Amplification reactions were carried out in a GeneAmp 9600 PCR System thermocycler (Applied Biosystems, Foster City, California) for primer pair NS3–NS6, with an initial denaturation of 94 C for 2 min, followed by 35 cycles of 94 C for 15 s, annealing at 58 C for 30 s, extension at 72 C for 1 min and, after the last cycle, a final extension at 72 C for 3 min; or in a PTC-200 DNA engine thermocycler (MJ Research, Watertown, Massachusetts) for primer pairs NS5-mfs-3 and NS5-nu-SSI(943)-251-3'-Mf using essentially the same program with a modified annealing step of 60 C for 15 s. Reactions using the primer pair NS5-NS6 differed by using 30 cycles of 95 C for 15 s, annealing at 35 C for 15 s and extension at 72 C for 1 min. The relative positions for primers are shown in FIG. 1, except for primer NS3, which is 575 bp upstream of NS5, and primer NS6, which is 270 bp downstream the intron sequence. For RAPD amplifications approximately 1 µL of a 1/20 dilution of DNA (1–20 {eta}g/µL) was amplified in 10 mM Tris-HCl pH 8.3, 50 mM KCl, 200 µM of each dNTP, 2.5 mM MgCl2, 0.95 µM 10-mer random primer and 0.5 unit of Taq polymerase (Invitrogen Canada, Burlington, Ontario, Canada) for a total volume of 20 µL. The reactions were carried out in a Perkin-Elmer 9600 thermocycler (Applied Biosystems, Foster City, California) with an initial denaturation at 94 C for 3 min, followed by 35 cycles of 94 C for 5 s, 35 C for 30 s, a 2 min ramping to 72 C (0.3 C/sec) and an extension at 72 C for 2.5 min and a final extension at 72 C for 3 min. Amplified products were visualized under ultraviolet light after electrophoresis in 1.5% agarose gels and staining with ethidium bromide.



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  		FIG. 1. Sequence alignment of M. fructicola ribosomal SSU intron and the nucleotide insertion/substitutions for each intron type characterized. The nucleotides showed are the intron sequence originally characterized by Fulton and Brown (1997)Go; type A. The boxes are the group I intron internal elements, P, Q, R, S (Waring and Davies 1984Go). Nucleotide variations from the original sequence are indicated for each polymorphic type. Identical nucleotides are represented by dots, and absent nucleotides are shown by hyphens. Primers used for PCR assays and sequence analysis in this study are indicated in the sequence by bold letters. The primers are designated by both the original published names (NS5, White et al 1990Go; mfs-3, Fulton and Brown 1997Go) and following the nomenclature of Gargas and DePriest (1996)Go.

 
Sequencing of PCR products. – Selected amplified DNA products were treated with the PCR product presequencing kit (USB Corp., Cleveland, Ohio) following the manufacturer’s instructions. The treated PCR products were sequenced with radioactively labeled (33P-{gamma}ATP) primers NS5, NS6, nu-SSI(943)-251-3'-Mf and nu-SSI(943)-231-5'-Mf (5'-CGC ATC CTT TCC CTT CAT ACG C-3', see FIG. 1 for relative position) using the Thermo Sequenase Cycle Sequencing Kit (USB Corp., Cleveland, Ohio), following the manufacturer’s instructions. Sequencing reactions were fragmented on 6% polyacrylamide-8M urea gel, and after autoradiography the sequences were read with a Hitachi Tablet Digitizer and analyzed with DNASIS® Sequencing Analysis software for Windows (Helixx Technologies Inc., Toronto, Ontario, Canada).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
RAPD amplification to confirm the identification of M. fructicola strains. – Random amplified polymorphic DNA was used to confirm the identification of M. fructicola strains used in this study. All M. fructicola strains showed identical or nearly identical patterns when compared to M. laxa strains (FIG. 2).



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  		FIG. 2. RAPD amplification of M. laxa and M. fructicola isolates. 1. 100 bp ladder (Invitrogen Canada, Burlington, Ontario). 2. M. laxa; ATCC 9961. 3. M. laxa; ATCC 62881. 4. M. fructicola; CBS 301.31. 5. M. fructicola; NRRL A-28151. 6. M. fructicola; DAOM 186890. 7. M. fructicola; CPQP CD9. 8. M. fructicola; CPQP CD11. 9. M. fructicola; DAOM 110195. 10. M. fructicola; JN AN3-6. 11. M. fructicola; JN WC3-8. 12. M. fructicola; JN NB3-1. 13. M. fructicola; DAOM 144721. 14. M. fructicola; ATCC 42248. 15. M. fructicola; CBS 203.25. 16. M. fructicola; ATCC 46606. 17. PCR negative. The sizes (bp) of some of the DNA ladder are indicated on the left.

 
PCR amplification of the ribosomal SSU region containing the group I intron. – The 13 isolates of M. fructicola were screened using NS5 and mfs-3 primers. These primers specifically should amplify a 444 bp band corresponding to 26 bp of the ribosomal sequence upstream of the intron and most of the intron sequence in M. fructicola isolates. The 444 bp band (FIG. 3A) was amplified in only four of the 13 M. fructicola isolates. An amplification using ribosomal SSU primers NS5 and NS6 then was performed. In the absence of the intron, there should be amplification of a PCR product of 307 bp, while in the presence of the intron there should be an amplification of a 725 bp product (FIG. 3B). All isolates that showed an amplified product using the NS5 and mfs-3 primers showed the expected 725 bp amplified product, indicating the presence of the intron. However, five isolates gave a 726 bp amplified product indicating the presence of the intron, even if previously there was no amplification using the NS5 and mfs-3 primers. Four isolates that did not give an amplified product using NS5 and mfs-3 primers gave a 307 bp amplified product expected in the absence of the intron. To confirm the presence or absence of the intron, a primer (nu-SSI(943)-251-3'-Mf) that would amplify in a very conserved region of group I introns, the R internal element (Waring and Davies 1984Go), was designed and used with the NS5 primer. FIGURE 3C shows that all nine isolates that gave an amplified 725 or 726 bp band using primers NS5-NS6 also gave an amplified band using NS5-nu-SSI(943)-251-3'-Mf, confirming the presence of an intron. The four isolates that gave the 307 bp band using NS5–NS6 primers pairs did not give an amplified product using NS5-nu-SSI(943)-251-3'-Mf primers. TABLE I presents a summary of the PCR results.



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  		FIG. 3. PCR assay on M. fructicola isolates using, panel A, NS5-mfs-3; panel B, NS5–NS6 and panel C, NS5-MO1343. 1. CBS 301.31. 2. NRRL A-28151. 3. DAOM 186890. 4. CPQP CD9. 5. CPQP CD11. 6. DAOM 110195. 7. JN AN3-6. 8. JN WC3-8. 9. JN NB3-1. 10. DAOM 144721. 11. ATCC 42248. 12. CBS 203.25. 13. ATCC 46606. 14. PCR negative. 15. 100 bp ladder (Invitrogen Canada, Burlington, Ontario). The sizes (bp) of the amplified fragments are indicated on the right of each panel.

 
Sequence analysis of M. fructicola isolates ribosomal group I introns. – Primers NS3 and NS6 were used to amplify a PCR product from the ribosomal region containing the intron from nine isolates (TABLE I). NS5 and NS6 primers were used to sequence from both directions. Two of the four isolates that failed to amplify a product using specific group I intron primers and produced a smaller band using NS5–NS6 primers were sequenced as well (TABLE I). The ribosomal sequences obtained for the 10 M. fructicola isolates were derived from two independent PCR reactions, and each PCR product was sequenced in both directions; GenBank accession numbers for M. fructicola strains sequenced across the SSU intron insertion site are: AF257086–AF257094, inclusive. The sequence analysis confirmed the absence of the intron for M. fructicola isolates JN WC3-8 (Ontario, Canada) and CBS 301.31 (Australia). The remaining eight isolates contained intron sequences with some nucleotide variations or insertions regrouping the sequences into five polymorphic types (FIG. 1). Isolate CBS 203.25 (United States) intron sequence is identical to the sequence of Fulton and Brown (1997)Go New Zealand isolate intron (type A, FIG. 1). The intron sequences of isolates ATCC 42248 (New Zealand) and JN AN3-6 (Ontario, Canada) are similar (type B and C, FIG. 1) to the published sequence (Fulton and Brown 1997Go). Therefore, isolates CBS 203.25, ATCC 42248 and JN AN3-6 all gave an amplified PCR product using NS5 and mfs-3 primers (FIG. 3A). Isolates NRRL A-28151, CPQP CD9, DAOM 110195, DAOM 144721 (all from Canada) and isolate ATCC 46606 (United States) that contained an intron but did not yield an amplified product using NS5-mfs-3 primers correspond to types E and F on FIG. 1. The M. fructicola intron sequence published by Snyder and Jones (1999)Go (type D, FIG. 1) differs from all sequences reported in this study and was reported by the same authors to amplify with NS5-mfs-3 primers.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The species identity of the M. fructicola strains used in this study was supported by the fact that isolates possessed very similar RAPD patterns when compared to M. laxa (FIG. 2), with only slight intraspecific variation.

A group I intron has been characterized in the ribosomal SSU of M. fructicola (Fulton and Brown 1997Go). PCR primers designed to amplify a region containing 26 bp of the ribosomal SSU rDNA upstream region and most of the intron sequence failed to produce an amplified product from some M. fructicola isolates (this study, Förster and Adaskaveg 2000Go, Hughes et al 2000Go). Furthermore, some Japanese isolates of M. fructicola were shown to lack the intron (Fulton et al 1999Go). The present study showed that the absence of amplification can result not only from the absence of an intron but also from nucleotide variations in the intron mfs-3 priming site (FIG. 1).

The sequence of the polymorphic type A intron (Fulton and Brown 1997Go) was folded into its presumed secondary structure following the structural conventions for group I introns of Burke et al (1984) (FIG. 4). Nucleotide variations observed among the sequences of the different polymorphic types of the M. fructicola introns are indicated on the secondary structure shown in FIG. 4. It was observed that none of the nucleotide variations occur in the conserved elements P, Q, R, S, and most of the variations do not occur in the pairing (P) segments responsible for the secondary structure of the group I intron (Waring and Davies 1984Go) (FIG. 4). When a nucleotide substitution occurs in a pairing segment, it is expected that there will be either a compensating change in the pairing nucleotide or the variation will have a minor effect on the integrity of the pairing segment (FIG. 4). The optional presence of group I introns in SSU rDNA within a fungal species, and also within fungal populations, has been well documented (DePriest and Been 1992Go, DePriest 1993Go). Optional group I introns in SSU rDNA, at position 1165 relative to Saccharomyces cerevisiae SSU rDNA, in some or most isolates of the same species of fungi have been reported for Beauveria bassiana (Coates et al 2002Go), Histoplasma capsulatum (Lasker et al 1998Go), Hymenoscyphus ericae (Perotto et al 2000Go) and Ophiosphaerella narmari (Wetzel et al 1999Go). All of the above ribosomal SSU introns are located at the same position as the M. fructicola ribosomal SSU intron and their sequences aligned properly. It was demonstrated that the presence of the Ophiosphaerella narmari intron was not evenly distributed among all ribosomal tandem repeats (Wetzel et al 1999Go). Therefore, it was possible that the presence of one copy of the M. fructicola intron remained undetected in a PCR reaction using primers NS5–NS6 by simply having been out-competed by the intronless PCR product that would be favored by its size and number of copies. This possibility was assessed by using a reverse primer complementary to the conserved R internal element (Waring and Davies 1984Go) paired with NS5 so it would amplify any polymorphic type of intron present at low copy number. From our experiment, we conclude that the absence of the ribosomal SSU intron in the four M. fructicola isolates would be uniform throughout the ribosomal tandem repeats.



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  		FIG. 4. Secondary structure of the polymorphic type A intron (Fulton and Brown 1997Go) located in the SSU rDNA of M. fructicola. Exon sequences are in lower case and the intron sequences in upper case. Putative 5' and 3' splice sites are indicated by arrows. The pairing segments P1 to P9 (Burke et al 1987Go) are indicated. The nucleotide substitutions occurring in the other polymorphic types (FIG. 1) are circled and linked to an arrow pointing to the substituting nucleotide. Nucleotides inserted in the sequence are linked to an arrow pointing to the insertion site within the intron sequence.

 
Among the isolates we studied, there was no correlation between the dates of sampling and intron type. As for geographical origin, all intron types were found in isolates originating from North America. The type D intron has been found only in Michigan (Snyder and Jones 1999Go); none were found in this study, and therefore this type may be related to isolate origin. However, in each of the provinces of Ontario and British Columbia, Canada, there were combinations of isolates without introns, isolates with introns similar to those reported by Fulton and Brown (1997Go; types A and C) and isolates with type F introns, which differ the most from those previously reported. Sequence polymorphisms have been reported in the ribosomal SSU introns from isolates of Beauveria bassiana (Coates et al 2002Go), Histoplasma capsulatum (Lasker et al 1998Go) and Ophiosphaerella narmari (Wetzel et al 1999Go), all introns located at the same position as the M. fructicola intron.

The accumulation of nucleotide variation among introns from isolates of M. fructicola, together with the broad distribution of isolates possessing introns, would suggest a long association between the intron at position 1165 and this species. If so, the occurrence of intron-containing and intronless isolates from the same locations in Ontario and British Columbia therefore might reflect random intron deletion events. On the other hand, it is possible that the intron has an evolutionary history apart from that of M. fructicola and that the current intron distribution in M. fructicola represents multiple insertion events. To address this question fully, more isolates of M. fructicola from different locations will have to be investigated and relationships among isolates will have to be compared with relationships among introns.

In summary, PCR and sequence analysis of the ribosomal SSU of M. fructicola isolates confirmed the absence of the previously characterized group I intron in some of the isolates. Furthermore, the analysis demonstrated that across isolates the intron displays nucleotide substitutions and insertions. Comparisons among the intron sequences obtained in this study and those published previously (Fulton and Brown 1997Go, Snyder and Jones 1999Go) resulted in the identification of six polymorphic types (FIG. 1) that may relate in part to isolate origin.


   ACKNOWLEDGMENTS
 
We thank GP White for providing morphological identification. We also thank J Northover for supplying isolates. We are grateful to RC Hamelin, CA Lévesque and R Phillippe for reviewing the manuscript.


   FOOTNOTES
 
Accepted for publication August 26, 2003.

1 Corresponding author. E-mail: cotemj{at}inspection.gc.ca


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Batra LR. 1991. World species of Monilinia (fungi): their ecology, biosystematics and control. Mycologia Mem 16:1–246.

Bhattacharya D, Friedl DT, Damberger S. 1996. Nuclear-encoded rDNA group I introns: origin and phylogenetic relationships of insertion site lineages in the green algae. Mol Biol Evol 13:978–989.[Abstract]

Burke JM, Belfort M, Cech TR, Davies RW, Schweyen RJ, Shub DA, Szostak JW, Tabak HF. 1987. Structural conventions for group I introns. Nucleic Acids Res 15: 7217–7221.[Abstract/Free Full Text]

Byrde RJW, Willetts HJ. 1977. The brown rot fungi of fruit. Their biology and control. Oxford: Pergamon Press. 246 p.

Cech TR, Herschlag D. 1996. Group I ribozymes: substrate recognition, catalytic strategies, and comparative mechanistic analysis. In: Eckstein F, Lilley DMJ, eds. Catalytic RNA. Berlin Heidelberg: Springer-Verlag. p 1–18.

Coates BS, Hellmich RL, Lewis LC. 2002. Nuclear small subunit rRNA group I intron variation among Beauveria spp provide tools for strain identification and evidence of horizontal transfer. Curr Genet 41:414–424.[Medline]

DePriest PT, Been MD. 1992. Numerous group I introns with variable distributions in the ribosomal DNA of a lichen fungus. J Mol Biol 228:315–321.[Medline]

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Förster H, Adaskaveg JE. 2000. Early brown rot infections in sweet cherry fruit are detected by Monilinia-specific DNA primers. Phytopathology 90(2):171–178.[Medline]

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Fulton CF, van Leeuwen GCM, Brown AE. 1999. Genetic variation among and within Monilinia species causing brown rot of stone and pome fruit. Eur J Plant Pathol 105:495–500.

Gargas A, DePriest PT, Taylor JW. 1995. Positions of multiple insertions in SSU rDNA of lichen-forming fungi. Mol Biol Evol 12(2):208–218.[Abstract]

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Hibbett DS. 1996. Phylogenetic evidence for horizontal transmission of group I introns in the nuclear ribosomal DNA of mushroom-forming fungi. Mol Biol Evol 13(7):903–917.[Abstract]

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