ITS region of the rDNA for developing species-specific diagnostic molecular markers   

Historically the Internal Transcribed Spacer (ITS) region of the ribosomal DNA (rDNA) has been commonly used for developing species-specific diagnostic molecular markers (Table 1).  One reason for this is the sequences in this region tend to be relatively conserved on an intra-specific basis but variable in comparisons among many species in the genus (the exception being some of the phylogenetically closely related species).  The coding regions for the various subunits of the ribosomal RNAs are arranged as indicated in the figure below with each of these repeat units present in multiple copies per genome.  The copy number has not been determined for a Phytophthora spp., but in Eumycotan fungi it has been reported to range from 60 to 220 copies per haploid genome (Cassidy et al. 1984; Russell et al. 1976).  Based on Southern analysis it is believed that the rDNA copy number in Phytophthora is high as well.  The comparatively high copy number of the rDNA in Phytophthora spp. is an advantage for molecular diagnostics because the sensitivity of detection increases along with copy number.  The early development of PCR primers for amplification of this region (White et al. 1990) and the large number of sequences for this region deposited in GenBank has contributed to the use of this region for diagnostic purposes as well.

A PCR-ELISA detection procedure has been described for a variety of Pythium and Phytophthora spp. but the capture probes have not been widely tested for species specificity (Bailey et al. 2002).  During PCR amplification of the ITS 1 region the amplicon was labeled with digoxygenin – 11 – UTP (DIG).  A capture probe designed from variable regions of the ITS1 region was labeled with biotin and immobilized on a streptavidin coated microtiter plate.  The DIG labeled amplicon was hybridized with the capture probe and the DIG detected immunologically in an ELISA assay.

In addition to species-specific molecular markers, genus-specific primer pairs have been developed as well (Table 1).

Primers and conditions for amplification and sequencing this region can be found in the Genetic Markers section of the database.


Sequence alignment that can be used for development of diagnostics markers (.msf, .nex, .fastA)


 References

Bailey AM, Mitchell DJ, Manjunath KL, Nolasco G, Niblett CL. 2002 Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA. FEMS Microbiol. Lett. 207:153-158

Jeane Rhodes Cassidy, David Moore, Benjamin C. Lu, Patricia J. Pukkila 1984 Unusual organization and lack of recombination in the ribosomal RNA genes of Coprinus cinereus Current Genetics 8:607-613

Russell PJ, Hammett JR, Selker EU. 1976 Neurospora crassa cytoplasmic ribosomes; ribosomal ribonucleic acid synthesis in the wild type. J. Bacteriol. 127:785-793

White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds. Academic Press, Inc., San Diego, CA.