2.2.30
THE USE OF MITOCHONDRIAL DNA FOR ISOLATE IDENTIFICATION AND PHYLOGENETIC PURPOSES IN THE GENUS PYTHIUM

FN MARTIN

USDA-ARS, 1636 East Alisal Street, Salinas, CA 93905, USA

Background and objectives
With over 120 described species, some of which are separated by only minor morphological differences, isolate identification in the genus Pythium can be a challenging task. Having a rapid and reliable method to accomplish this would facilitate research on this economically important genus as well as provide a means for classification of isolates that are taxonomically difficult (isolates that do not form a sexual stage or have morphological features intermediate between described species). Analysis of mtDNA is useful for this purpose owing to the high degree of intraspecific conservation and interspecific variation of this genome [1]. The mtDNA of this genus has a circular map containing a large inverted repeat (IR) separated by a small and large unique single-copy region. It is the large size of the IR (as much as 80% of the genome, depending on the species) that is believed to provide the level of observed intraspecifc stability. Digestion with restriction enzymes such as HindIII provides RFLP data that is useful for species identification [1]. Additional analysis of this genome was performed to clarify the phylogeny of this genus and to develop PCR-based procedures for isolate identification.

Results and conclusions
Restriction maps of mtDNA were constructed for 14 species and coding regions for five genes placed. Differences among species, even morphologically similar ones, were readily apparent. The IR and two unique regions varied in size and location of restriction sites depending on the species, with some species having linear rather than circular restriction maps. Variation in the location of coding regions also was observed. Although the L-rRNA and S-rRNA genes were in the same location in all species (these genes were cleaved at the SalI and SstII restriction sites, respectively, which are conserved in all species), it was variable for cytochrome c oxidase subunits I and II and ATP9. For species with filamentous or lobate sporangia the COI and COII genes were located in the small unique region or adjacent IR, while for species with spherical sporangia they were located in the large unique region. ATP9 was found in the small unique region or adjacent IR.

In addition to gene order differences, sequence analysis of a particular region of the mtDNA also was useful for determining species relationships. Over 50 isolates, encompassing a geographic representation of 20 species, have been examined and phylogenetic inferences determined with PAUP. Species groupings were somewhat similar to sporangial types, with heterothallism and oogonial ornamentation polyphyletic and not associated with any single clade.

To develop a rapid means of isolate identification a region of the mtDNA was identified that was conserved within a species but variable among species. PCR primers capable of amplifying this region from all Pythium spp. were constructed and used to amplify a 1.1 kb region. Digestion of the amplified product with four- base recognizing restriction enzymes provided a means of isolate identification. Although no single restriction enzyme was successful for species discrimination, comparisons of RFLP groupings for three restriction enzymes generally provided the resolution necessary for accurate classification.

In an attempt to construct species-specific markers, sequences from the most variable region of the amplified products were compared and a primer constructed from species unique sequences. This species-secific primer is used in conjunction with a primer conserved in all species for the amplification of DNA fragments that are diagnostic for the particular species. Trials with several species are in progress and will be discussed in the poster. The use of the unique species primer as a species-specific oligonucleotide probe also is being explored.

References
1. Martin FM, Kistler HC 1990. Experimental Mycology 14, 32-46