Identification of subpopulations   

There are a number of different approaches that can be used to identify subpopulations within a Phytophthora spp.

1. Sequence Analysis
2. Single Nucleotide Polymorphisms (SNPs)
3. RFLP analysis
4. RAPDs and AFLP analysis
5. Microsatellites
6. Mitochondrial haplotypes


Sequence Analysis 

Schena and Cooke (2006) have identified 4 intergenic regions from the mitochondrial DNA, a portion of the intergenic region of the rDNA, and spacer regions of the ras-related protein Ypt1 that can be useful for developing species-specific markers and may be useful for identification of subpopulations within a species (overview).

Schena L, Cooke DE. 2006 Assessing the potential of regions of the nuclear and mitochondrial genome to develop a J. Microbiological Methods 67:70-85. (pdf of reprint)



Single Nucleotide Polymorphisms (SNPs) 

Single base changes at specific loci can also be used to identify pathogen subopulations (as well as for develop of diagnostic molecular markers for their detection). For example, Bilodeau et al. (2007) identified SNPs in the ő≤-tubulin and CBEL (cellulose binding elicitor lectin) genes that differentiated the North American from the European populations of P. ramorum. Likewise, Kroon et al. (2004) identified a SNP in the mitochondrially encoded cox1 gene that also differentiated these populations.

G.J. Bilodeau, C.A. Lévesque, A.W.A.M. de Cock, S.C. Briére, and R.C. Hamelin 2007 Differentiation of European and North American genotypes of P. ramorum by real time polymerase chain reaction primer extension. Can. J. Plant Pathol. 29:408-420  (pdf of reprint)

Kroon LP, Verstappen EC, Kox LF, Flier WG, Bonants PJ. 2004 A Rapid Diagnostic Test to Distinguish Between American and European Populations of Phytophthora ramorum. Phytopathology 94:613-620 (pdf of reprint)



RFLP analysis 

Southern analysis of digested genomic DNA with repetitive elements can be useful for identification of subpopulations (see probe RG57 in Goodwin et al. 1992). RFLP analysis of regions such as the ITS of the rDNA repeat unit or a portion of the cox1 and cox2 gene cluster can also identify some subpopulations (review)

Stephen B. Goodwin, André Drenth, William E. Fry 1992 Cloning and genetic analyses of two highly polymorphic, moderately repetitive nuclear DNAs from Phytophthora infestans Current Genetics 22:107-115



RAPDs and AFLP analysis 

There have been a number of studies using random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) analysis to look at the population structure of different species (the references below are just a sampling).  Both AFLPs and RAPDs are dominant markers providing only presence or absence of an allele in the diploid Phytopththora. They are often chosen because they sample the whole genome and many genetic loci in a single assay. An important consideration when using RAPD markers is the portability of the markers and the ability of other labs to generate the same data set (to help accomplish this only robust bands should be used in data analysis).  To reduce this problem with portability between labs the desired amplicons can be cloned and sequenced with a second set of regular PCR primers designed to amplify the target sequences (sequence characterized amplified regions, of SCARs).

AFLP is more reproducible than RAPDs and is the marker system of choice for species were microsatellites have not been developed. AFLP protocols can be developed quite rapidly by selecting the right selective primer combination to sample a large number of loci.

With both methods inclusion of appropriate positive controls and replication of assays (from DNA extraction to PCR) are suggested.

SAMEN F. M. Abu-El (1) ; SECOR G. A. (1) ; GUDMESTAD N. C. (1) ; 2003 Genetic variation among asexual progeny of Phytophthora infestans detected with RAPD and AFLP markers Plant Pathology 52:314-325

Bowers JH, Martin FN, Tooley PW, Luz ED. 2007 Genetic and Morphological Diversity of Temperate and Tropical Isolates of Phytophthora capsici. Phytopathology 97:492-503

COOKE D. E. L. (1) ; KENNEDY D. M. (1) ; GUY D. C. (1) ; RUSSELL J. (1) ; UNKLES S. E. (1) ; DUNCAN J. M. 1996 Relatedness of Group I species of Phytophthora as assessed by randomly amplified polymorphic DNA (RAPDs) and sequences of ribosomal DNA Mycological research 100:297-303

IVORS Kelly L. ; HAYDEN Katherine J. ; BONANTS Peter J. M. ; RIZZO David M. ; GARBELOTTO Matteo 2004 AFLP and phylogenetic analyses of North American and European populations of Phytophthora ramorum Mycological research 108:378-392

Lamour, K.H., Hausbeck, M.K. 2001 Investigating the Spatiotemporal Genetic Structure of Phytophthora capsici in Michigan. Phytopathology 91:973-980

 

Thanks to Nik Grunwald for contributions on this section



Microsatellites 

Microsatellites have become the marker system of choice for analysis of populations because they are codominant, very repeatable, and provide many alleles per locus. They are costly to develop but very affordable once developed. Microsatellites have been used to characterize populations of P. ramorum (Prospero et al. 2004, 2007, Ivors et al. 2006), P. cinnamomi (Dobrowolski et al. 2002), and P. infestans (Lees et al. 2006).  The markers reported by Lees et al. (2006) also were reported to amplify microsatellite loci for other species as well.  Garnica et al. (2006) have mined microsatellite loci from the genomes of P. sojae and P. ramorum. This work is a good starting point for development of novel microsatellite markers. Note, however, that most microsatellite loci are only conserved within a species or among closely related species. Rarely, are microsatellite loci transferable across species. As with AFLP and RAPD inclusion of positive controls is suggested. In addition, verification of a novel alleles by sequencing is needed to assure that PCR is amplifying target regions.

Dobrowolski MP, Tommerup IC, Shearer BL, O'Brien PA. 2003 Three Clonal Lineages of Phytophthora cinnamomi in Australia Revealed by Microsatellites. Phytopathology 93:695-704

Garnica, D.P., Pinzon, A.M., Quesada-Ocampo, L.M., Bernal, A.J., Barreto, E., Grunwald, N.J., and Restrepo, S. 2006 Survey and analysis of microsatellites from transcript sequences in Phytophthora species: frequency, distribution, and potential as markers for the genus. BMC Genomics 7:245-

Ivors K, Garbelotto M, Vries ID, Ruyter-Spira C, Te Hekkert B, Rosenzweig N, Bonants P. 2006 Microsatellite markers identify three lineages of Phytophthora ramorum in US nurseries, yet single lineages in US forest and European nursery populations. Molecular Ecology 15:1493-1505

LEES A. K. (1) ; WATTIER R. (2) ; SHAW D. S. (2) ; SULLIVAN L. (1) ; WILLIAMS N. A. (1) ; COOKE D. E. L. (1) ; 2006 Novel microsatellite markers for the analysis of Phytophthora infestans populations Plant Pathology 55:311-319

S. PROSPERO , J. A. BLACK and L. M. WINTON 2007 Isolation and characterization of microsatellite markers in Phytophthora ramorum, the causal agent of sudden oak death Molecular Ecology Notes 4:672-674

S. PROSPERO*, E. M. HANSEN*, N. J. GRÜNWALD*Ę” and L. M. WINTONĘ‘ 2007 Population dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon from 2001 to 2004 Molecular Ecology 16:2958-2973

Thanks to Nik Grunwald for contributions on this section



Mitochondrial haplotypes 

Mitochondrial haplotypes can also be useful markers for monitoring subpopulations of Phytophthora spp., especially when they are clonally reproducing populations (mitochondria are maternally inherited). Perhaps the most widely used haplotype determination in the genus is with P. infestans, where 4 haplotypes have been identified and their classification by RFLP analysis has been used to characterize isolates from around the world (Griffiths and Shaw 1998, Gavino and Fry 2002). Representative mitochondrial genomes for all 4 haplotypes have been sequenced (Paquin et al. 1997, Avila-Adame et al. 2005).

Historically RFLP analysis of purified mitochondrial DNA has been used for haplotype determination for a number of species (for examples see Erwin and Ribeiro 1996), but analysis using PCR amplified regions has been useful as well (Griffiths and Shaw 1998). More recently sequence based analysis has been used to differentiate haplotypes. The mitochondrial genomes from the European and North American genotypes of P. ramorum have been sequenced and PCR primers were developed for amplification and sequencing of polymorphic regions from a range of isolates for identification of haplotypes. A total of 28 SNPs were characterized that identified 4 mitochondrial haplotypes in the 41 isolates examined (Martin 2008). In an effort to reduce the need for sequencing to identify haplotypes and to shorten the time commitment for sample processing, the primers used to identify haplotypes for this pathogen have been modified to give smaller amplicons and melt curve analysis is used to differentiate haplotypes (Martin, unpublished).

The genus-specific primers FMPhy-8b and FMPhy-10b have annealing sites in the terminal regions of the cox1 and cox2 genes and generate an amplicon that spans the spacer region between the genes (Martin et al. 2004). This primer pair is used for the first round amplification of field samples and is followed by a nested amplification using species-specific primers designed from polymorphic sequences in the spacer region. This region recently has been sequenced in 600+ isolates representing 91 species and 38 species were found to have intraspecific sequence polymorphisms in this region. Examination of the alignments has identified regions that can be used for development of specie-specific TaqMan probes as well as for mitochondrial haplotype classification.  This data is being puller together and will be added to this section at a later time (please contact Frank Martin if you have any questions).

The mitochondrial genomes of 25 Phytophthora species are currently being sequenced; once completed this work should provide background information that should help facilitate haplotype evaluation for a number of additional species (Martin, unpublished). Their comparison with the mitochondrial genomes of 14 Pythium spp. that have also been sequenced will provide an opportunity to evaluate gene order differences between the genera and sequence data that will be useful for development of genus and species-specific diagnostic markers.  This data will be added to the web site once the analysis has been completed.

Avila-Adame, C., Gomez-Alpizar, L., Zismann, V., Jones, K.M., Buell, C.R., Beagle Ristaino, J. 2006 Mitochondrial genome sequences and molecular evolution of the Irish potato famine pathogen, Phytophthora infestans. Current Genetics 49:39-46

Erwin, D. C. and Ribeiro, O. K 1996 Phytophthora Disease Worldwide APS Press 0:-

Pia D. Gavino 2002 Diversity in and evidence for selection on the mitochondrial genome of Phytophthora infestans Mycologia 94:781-793

GARETH W. GRIFFITH* AND DAVID S. SHAW 1998 Polymorphisms in P. infestans: four mitochondrial haplotypes are deteced after PCR amplification of DNA from pure cultures or from host lesions. Appl. Environ. Microbiol. 64:4007-4014

Martin FN. 2008 Mitochondrial haplotype determination in the oomycete plant pathogen Phytophthora ramorum. Current Genetics 54:23-34 (pdf of reprint)

Paquin B, Laforest MJ, Forget L, Roewer I, Wang Z, Longcore J, Lang BF. 1997 The fungal mitochondrial genome project: evolution of fungal mitochondrial genomes and their gene expression. Current Genetics 31:380-395