Harvard University Herbaria

Molecular systematics

The Co-PI, along with postdoctoral associate Dr. David Hibbett, has conducted molecular phylogenetic studies in basidiomycetes (Polyporaceae: Hibbett and Donoghue, 1995; Lentinula, Tricholomataceae: Hibbett et al., in review), based on both mitochondrial and nuclear small- and large-subunit ribosomal DNA sequences, and sequences of the Internal Transcribed Spacer (ITS) region of nuclear ribosomal DNA (see Baldwin, 1992; Baldwin et al., 1995). Based on the success of these studies, and similar analyses by other workers (e.g., Bruns et al., 1991; Bruns and Szaro, 1992; Hibbett, 1992; Karbone and Kohn, 1993; O'Donnell, 1992), we intend to extend such work to filamentous ascomycetes in connection with the monographic studies proposed here. DNAs will be obtained primarily from cultured material (see above), and will be extracted using an SDS mini-prep method (Raeder and Broda, 1985; Hibbett and Vilgalys, 1993), followed by digestion with RNAase, chloroform extraction, and ethanol precipitation. PCR will be carried out using standard primers (see White et al., 1990), although these may need to be redesigned if there are significant mismatches (e.g., one ITS primer was redesigned in Lentinula; Hibbett et al., in review). Double-stranded PCR products will be cleaned with GeneClean II (Bio 101), to remove primers and unincorporated nucleotides. 100-500 ng of template are required for each sequencing reaction, which generally can be obtained from 2-5 ul of robust amplification product.

Sequencing will be performed using PRISM Ready Reaction Dye Terminator Cycle Sequencing kits (Applied Biosystems), following protocols suggested by the manufacturer. Unincorporated nucleotides and primers are removed from sequencing reactions using Centri-Sep columns (Princeton Separations), and the cleaned sequencing reactions are ethanol precipitated, re-suspended in formamide-EDTA and electrophoresed in 6% Sequagel (National Diagnostics) acrylamide gels on either the Applied Biosystems 370A or 373A automated fluorescent DNA sequencer (see "Facilities, Equipment and Other Resources"). In general, following a 10-hour run we can read up to 400 bases, and two runs are possible in a twenty-four hour period on each machine. Sequence and gel image files are exported via Ethernet from the DNA sequencer to computers in the Donoghue lab for proofreading and sequence editing (SeqEd ver. 1.0.1, Applied Biosystems), and these files are routinely archived on 128 MB optical discs. Applied Biosystems software has been used for automated base calling. Both strands will be sequenced as a means of eliminating artifacts and ambiguities. Multiple sequence alignments will be facilitated using Clustal V (Higgins et al., 1992) and Malign (Wheeler and Gladstein, 1993).

In light of our previous experience, we anticipate little difficulty in obtaining sequences from the DNA regions of interest. Mitochondrial rDNA may yield ca 600-850 bp, nuclear rDNA ca. 1.2 kilobases, and ITS ca. 600 bp. Despite high copy number (e.g., of the nuclear ITS region) it appears in many cases that sequences are effectively homogenized, which allows direct sequencing of pooled PCR products. However, divergent paralogues have been discovered in some cases (e.g., Ritland et al., 1993; Suh et al., 1993) and may be common in some groups (Baldwin et al., 1995). The presence of different repeat types has in some cases been discovered through direct sequencing of PCR products, i.e., by observing more than one band/peak per site, and cloning is required for accurate characterization of such variation (Baldwin et al., 1995).