PARENT SESSION
Poster Session 23: Soil Ecology
Wednesday, August 10, 5:00 PM - 6:30 PM, Exhibit Hall 220 A-E, Level 2, Palais des congrès de Montréal
ITS2 rDNA secondary structure defines species-specific probe regions for fungi.
Gargas, Andrea*,1, Landis, Frank *,2, 1 University of Wisconsin-Madison, Madison, WI2 University of Akron, Akron, OH
ABSTRACT- We designed prototype DNA microarrays including 183 ITS2 rDNA sequences from 162 fungal species known to inhabit soil, with representative species from each of the five Eumycota phyla plus Oomycota. Probes were based on 20 nt segments of each ITS2 sequence, with microarrays containing all possible 20 bp segments. Arrays were designed to detect multiple fungal species from soil samples. Initial results provided substantial insight into designing eukaryotic rDNA microarray detectors, as these microarrays were compromised by two design limitations: 1) 55% of the probes were shared among two or more species. This is understandable, as ITS2 regions contain substantial phylogenetic information, suggesting that a number of 20-nt oligonucleotide segments should be similar or identical among species. 2) Most seriously, prototype design also proved susceptible to spoofing, responding to fungi not represented on the array. For example, one test species hybridized with single-copy probes from seven different species, although supposedly it was not represented in the array. Since most soil fungal species are unidentified, probes designed to detect known taxa must also have safeguards against responding to unknown taxa. Fortunately, one significant result potentially overcomes both limitations. Analysis of probe duplications against 119 of the array sequences showed that unique sequences tended to occur most often around nt position 40 of ITS 2, where 67% of probes were unique, and duplicates were confined to near relatives. Position 40 corresponds precisely with loop 2 of the ITS2 folding structure, a folding structure repeated in all 119 sequences. Our findings suggest that probes based on loop 2 sequences would a priori be close to taxon-specific and therefore resistant to spoofing. More generally, analysis of secondary structure folding patterns in rapidly evolving sequences holds promise for the design of taxon-specific oligonucleotide probes.
Key words: fungi, ITS2 rDNA, microarray, probes