3.3.7
FAMILIES OF REPEATED DNA IN PHYTOPHTHORA INFESTANS,THEIR DISTRIBUTION WITHIN THE GENUS AND THEIR USE AS TARGETS FOR PCR-BASED DIAGNOSTIC ASSAYS

HS JUDELSON and TA RANDALL

Department of Plant Pathology, University of California, Riverside, California 92521, USA

Background and objectives
The genomes of Phytophthora species are relatively large as they contain a substantial amount of repetitive DNA sequences. We have investigated the composition of the nuclear genome of Phytophthora infestans, cause of late blight diseases of potato and tomato, with the goals of (i) understanding the nature and distribution of repetitive elements; (ii) illuminating how genomes have evolved within the genus; (iii) developing sets of primers for polymerase chain reaction-based assays for identifying late blight; and (iv) assembling tools useful for physical reconstructions of Phytophthora genomes, chromosome walking and genomic sequencing.

Materials and methods
Repetitive clones were obtained from genomic libraries of P. infestans DNA and assembled into families based on cross-hybridization and DNA blot analysis. Southern blots and dot-blot assays were used to indicate the abundance of each sequence in P. infestans and their distribution throughout the genus. Selected clones were sequenced to indicate their structure. PCR primers were developed against high-copy elements specific to P. infestans.

Results and conclusions
33 distinct families of repetitive DNA were identified. Some repeat elements were generally specific to P. infestans while others were present in most species, based on analyses of 32 members of the genus The distribution of the families did not conform to the traditional taxonomic groups used for the genus. Characterization of the repeated sequences in P. infestans indicated that they included both dispersed and tandemly repeated elements, with copy numbers ranging from 70 to 8400 per haploid genome. In total, these repeats were estimated to represent 51% of the nuclear genome of P. infestans. Reverse transcriptase motifs were detected in seven of the repeat families and these were widely distributed throughout the genus.

Five sets of PCR primers based on the repetitive sequences were designed that enabled the sensitive and species-specific detection of P. infestans. These proved capable of detecting as little as 50 fg of purified P. infestans DNA (equivalent to 5-10% of a single nucleus). No cross-reaction was observed against DNA from each of 32 species of Phytophthora tested (with the exception of P. mirabilis and P. phaseoli) or from bacteria, higher fungi and plants. The sensitivities and specificities revealed in these assays exceed those described for other published sets of diagnostic primers, such as those based on ribosomal DNA.

References
1. Judelson HS, Randall TA, 1998. Genome, in press.