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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Use of genome sequence data in the design and testing of SSR markers for <italic>Phytophthora </italic>species</title><author><name sortKey="Schena, Leonardo" sort="Schena, Leonardo" uniqKey="Schena L" first="Leonardo" last="Schena">Leonardo Schena</name><affiliation><nlm:aff id="I1">Department of "Gestione dei Sistemi Agrari e Forestali", Mediterranean University of Reggio Calabria, Località Feo di Vito, 89124 Reggio Calabria, Italy</nlm:aff></affiliation></author><author><name sortKey="Cardle, Linda" sort="Cardle, Linda" uniqKey="Cardle L" first="Linda" last="Cardle">Linda Cardle</name><affiliation><nlm:aff id="I2">Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK</nlm:aff></affiliation></author><author><name sortKey="Cooke, David El" sort="Cooke, David El" uniqKey="Cooke D" first="David El" last="Cooke">David El Cooke</name><affiliation><nlm:aff id="I2">Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19099584</idno><idno type="pmc">2647557</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2647557</idno><idno type="RBID">PMC:2647557</idno><idno type="doi">10.1186/1471-2164-9-620</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000097</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000097</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Use of genome sequence data in the design and testing of SSR markers for <italic>Phytophthora </italic>species</title><author><name sortKey="Schena, Leonardo" sort="Schena, Leonardo" uniqKey="Schena L" first="Leonardo" last="Schena">Leonardo Schena</name><affiliation><nlm:aff id="I1">Department of "Gestione dei Sistemi Agrari e Forestali", Mediterranean University of Reggio Calabria, Località Feo di Vito, 89124 Reggio Calabria, Italy</nlm:aff></affiliation></author><author><name sortKey="Cardle, Linda" sort="Cardle, Linda" uniqKey="Cardle L" first="Linda" last="Cardle">Linda Cardle</name><affiliation><nlm:aff id="I2">Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK</nlm:aff></affiliation></author><author><name sortKey="Cooke, David El" sort="Cooke, David El" uniqKey="Cooke D" first="David El" last="Cooke">David El Cooke</name><affiliation><nlm:aff id="I2">Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK</nlm:aff></affiliation></author></analytic><series><title level="j">BMC Genomics</title><idno type="eISSN">1471-2164</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Microsatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of <italic>Phytophthora </italic>population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of <italic>P. infestans</italic>, <italic>P. sojae </italic>and <italic>P. ramorum </italic>were mined for candidate SSR markers that could be applied to a wide range of <italic>Phytophthora </italic>species.</p></sec><sec><title>Results</title><p>A first approach, aimed at the identification of polymorphic SSR loci common to many <italic>Phytophthora </italic>species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to <italic>P. sojae </italic>(<italic>P. alni, P. cambivora, P. europaea </italic>and <italic>P. fragariae</italic>). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times.</p></sec><sec><title>Conclusion</title><p>Key studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of <italic>Phytophthora </italic>species was challenging. That said, many novel SSR loci for species other than the three 'source species' (<italic>P. infestans</italic>, <italic>P. sojae </italic>and <italic>P. ramorum</italic>) are reported, offering great potential for the investigation of <italic>Phytophthora </italic>populations. In addition to the presence of microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different <italic>Phytophthora </italic>species.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">BMC Genomics</journal-id><journal-title>BMC Genomics</journal-title><issn pub-type="epub">1471-2164</issn><publisher><publisher-name>BioMed Central</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19099584</article-id><article-id pub-id-type="pmc">2647557</article-id><article-id pub-id-type="publisher-id">1471-2164-9-620</article-id><article-id pub-id-type="doi">10.1186/1471-2164-9-620</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Use of genome sequence data in the design and testing of SSR markers for <italic>Phytophthora </italic>species</article-title></title-group><contrib-group><contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Schena</surname><given-names>Leonardo</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>lschena@unirc.it</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Cardle</surname><given-names>Linda</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>Linda.Milne@scri.ac.uk</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Cooke</surname><given-names>David EL</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>David.Cooke@scri.ac.uk</email></contrib></contrib-group><aff id="I1"><label>1</label>Department of "Gestione dei Sistemi Agrari e Forestali", Mediterranean University of Reggio Calabria, Località Feo di Vito, 89124 Reggio Calabria, Italy</aff><aff id="I2"><label>2</label>Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK</aff><pub-date pub-type="collection"><year>2008</year></pub-date><pub-date pub-type="epub"><day>19</day><month>12</month><year>2008</year></pub-date><volume>9</volume><fpage>620</fpage><lpage>620</lpage><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1471-2164/9/620"></ext-link><history><date date-type="received"><day>6</day><month>6</month><year>2008</year></date><date date-type="accepted"><day>19</day><month>12</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2008 Schena et al; licensee BioMed Central Ltd.</copyright-statement><copyright-year>2008</copyright-year><copyright-holder>Schena et al; licensee BioMed Central Ltd.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0"></ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p><pmc-comment>
Schena
Leonardo
lschena@unirc.it
Use of genome sequence data in the design and testing of SSR markers for Phytophthora species
2008 BMC Genomics 9(1): 620-. (2008) 1471-2164(2008)9:1<620> urn:ISSN:1471-2164
</license></permissions><abstract><sec><title>Background</title><p>Microsatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of <italic>Phytophthora </italic>population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of <italic>P. infestans</italic>, <italic>P. sojae </italic>and <italic>P. ramorum </italic>were mined for candidate SSR markers that could be applied to a wide range of <italic>Phytophthora </italic>species.</p></sec><sec><title>Results</title><p>A first approach, aimed at the identification of polymorphic SSR loci common to many <italic>Phytophthora </italic>species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to <italic>P. sojae </italic>(<italic>P. alni, P. cambivora, P. europaea </italic>and <italic>P. fragariae</italic>). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times.</p></sec><sec><title>Conclusion</title><p>Key studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of <italic>Phytophthora </italic>species was challenging. That said, many novel SSR loci for species other than the three 'source species' (<italic>P. infestans</italic>, <italic>P. sojae </italic>and <italic>P. ramorum</italic>) are reported, offering great potential for the investigation of <italic>Phytophthora </italic>populations. In addition to the presence of microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different <italic>Phytophthora </italic>species.</p></sec></abstract></article-meta></front><body><sec><title>Background</title><p>The genus <italic>Phytophthora</italic>, with other Oomycetes, fall within the kingdom Stramenopila, which also includes golden-brown algae, diatoms, and brown algae such as kelp [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B4">4</xref>]. This genus stands out among the plant pathogens since a significant number of the 80 or so described species continue to prove a threat to ecosystem stability and plant productivity on a global scale [<xref ref-type="bibr" rid="B5">5</xref>-<xref ref-type="bibr" rid="B8">8</xref>]. Despite the importance of <italic>Phytophthora </italic>species, studies of their molecular diversity have been limited by the power of the genetic markers and difficulties in comparing results among laboratories. Accurate studies based on the analysis of mitochondrial and nuclear DNA have resulted in a consensus of the phylogenetic relationships within the genus with a grouping into 10 genetically related clades now accepted [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B9">9</xref>]. However, these studies were based on genes commonly conserved within a species and therefore unsuitable to characterize intraspecific variability. Other approaches to study intraspecific variability among <italic>Phytophthora </italic>species including RAPD-PCR and AFLP have proved valuable within a particular study but comparing results from one laboratory to another has always proved challenging with such fingerprinting tools [<xref ref-type="bibr" rid="B10">10</xref>-<xref ref-type="bibr" rid="B13">13</xref>]. Although microsatellites or simple sequence repeats (SSRs) have been recognised as one of the most powerful choices of markers for molecular ecology they have only relatively recently been exploited in the study of <italic>Phytophthora </italic>populations. SSRs are tandemly repeated motifs of one to six bases which occur frequently and randomly in all eukaryotic genomes although their frequency varies significantly among different organisms [<xref ref-type="bibr" rid="B14">14</xref>]. They exhibit a high degree of length polymorphism among related organisms due to stepwise mutations affecting the number of repeat units and leading to polymorphism [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. Dinucleotide repeats account for the majority of microsatellites for many species whereas trinucleotide and hexanucleotide repeats are the most likely repeat classes to appear in coding regions because they do not cause a frameshift [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>]. Major advantages SSRs include: (i) multiple SSR alleles may be detected at a single locus using a simple PCR-based screen, (ii) SSRs are evenly distributed across the genome, (iii) they are co-dominant, (iv) very small quantities of DNA are required for screening, (v) analysis may be semi-automated, and (vi) results are objective compared to random amplification methods [<xref ref-type="bibr" rid="B18">18</xref>].</p><p>Microsatellites have been used to investigate genetic structure and reproductive biology of Oomycetes species including <italic>Plasmopara viticola</italic>, <italic>P. cinnamomi</italic>, <italic>P. infestans</italic>, and <italic>P. ramorum </italic>[<xref ref-type="bibr" rid="B19">19</xref>-<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B23">23</xref>-<xref ref-type="bibr" rid="B25">25</xref>]. However, a major limitation to their wider exploitation is the need for prior species-specific marker isolation that requires knowledge of the DNA sequence of the SSR flanking regions to which specific primers have to be designed. Such regions are usually conserved within a species but the likelihood of primers successfully working between species decreases with increasing genetic distance and, in practice, primers are usually developed anew for each species [<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B26">26</xref>]. Common methods for the discovery of SSR loci are based on constructing genomic DNA libraries enriched for SSR sequences. These methods were utilised for <italic>P. cinnamomi </italic>and <italic>P. ramorum</italic>, however they are time-consuming, and the specific sequencing of DNA libraries required is expensive [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B25">25</xref>]. Many commercial and academic laboratories specialise in microsatellite isolation services and can provide a set of polymorphic microsatellite loci for a new species in 3–6 months for a cost of approximately USD 1,500 per locus, or USD 10,000 for 10–15 loci [<xref ref-type="bibr" rid="B14">14</xref>].</p><p>The availability of entire genome sequences for an increasing number of species including <italic>P. infestans </italic><ext-link ext-link-type="uri" xlink:href="http://www.broad.mit.edu/"></ext-link>, <italic>P. ramorum </italic>and <italic>P. sojae </italic><ext-link ext-link-type="uri" xlink:href="http://genome.jgi-psf.org/"></ext-link> have proved novel opportunities to identify and evaluate potential SSR markers identified by computational tools (Abajian, 1994, <ext-link ext-link-type="uri" xlink:href="http://espressosoftware.com/pages/sputnik.jsp"></ext-link>) [<xref ref-type="bibr" rid="B27">27</xref>,<xref ref-type="bibr" rid="B28">28</xref>]. This approach has been utilised to identify SSRs for the study of European and USA populations of <italic>P. ramorum </italic>and for monitoring the genetic variation in populations of <italic>P. infestans </italic>across Europe and worldwide [<xref ref-type="bibr" rid="B23">23</xref>,<xref ref-type="bibr" rid="B24">24</xref>,<xref ref-type="bibr" rid="B29">29</xref>,<xref ref-type="bibr" rid="B30">30</xref>].</p><p>Recently, Garnica et al. used an <italic>in silico </italic>approach to survey and compare simple sequence repeats (SSRs) in transcript sequences from the genomes of <italic>P. sojae</italic>, <italic>P. ramorum </italic>and <italic>P. infestans </italic>[<xref ref-type="bibr" rid="B27">27</xref>]. They also evaluated <italic>in silico </italic>transferability of SSRs among the <italic>Phytophthora </italic>species and found that a proportion (7.5%) of primers could, in theory, be transferred between at least two of the three species. In the present study SSRs from <italic>P. infestans P. sojae </italic>and <italic>P. ramorum </italic>were analysed to identify useful loci common to many <italic>Phytophthora </italic>species (Approach 1) or to a restricted number of species closely related to <italic>P. sojae </italic>(Approach 2). Selected loci were amplified and sequenced from 16 (Approach 1) and 5 (Approach 2) different <italic>Phytophthora </italic>species and a comprehensive SSRs dataset was created.</p></sec><sec><title>Results</title><sec><title>Approach 1 – SSRs for many <italic>Phytophthora </italic>species</title><p>The aim of this approach was to identify loci containing SSRs common to a large number of <italic>Phytophthora </italic>species (Fig. <xref ref-type="fig" rid="F1">1A</xref>). The method was validated using 16 different species (Table <xref ref-type="table" rid="T1">1</xref>) representing the breadth of diversity across the genus [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>].</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Isolates of <italic>Phytophthora </italic>included in the study, their designations and origins.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><italic>Phytophthora </italic>species</td><td align="left">Isolate numbers</td><td align="left" colspan="3">Origin</td></tr><tr><td></td><td></td><td colspan="3"><hr></hr></td></tr><tr><td></td><td></td><td align="left">Host</td><td align="left">Country</td><td align="left">Year</td></tr></thead><tbody><tr><td align="left"><italic>P. alni </italic>subsp. <italic>alni</italic></td><td align="left">SCRP2</td><td align="left"><italic>Alnus </italic>sp.</td><td align="left">UK</td><td align="left">1995</td></tr><tr><td></td><td align="left">SCRP4<sup>(a)</sup></td><td align="left"><italic>Alnus </italic>sp.</td><td align="left">Germany</td><td align="left">1995</td></tr><tr><td></td><td align="left">SCRP8<sup>(a)</sup></td><td align="left"><italic>Alnus </italic>sp.</td><td align="left">France</td><td align="left">1996</td></tr><tr><td align="left"><italic>P. cambivora</italic></td><td align="left">SCRP67 (IMI 296831)</td><td align="left"><italic>Rubus idaeus</italic></td><td align="left">Scotland</td><td align="left">1985</td></tr><tr><td></td><td align="left">SCRP75<sup>(a)</sup></td><td align="left"><italic>Fagus </italic>sp.</td><td align="left">UK</td><td align="left">1995</td></tr><tr><td></td><td align="left">SCRP80<sup>(a)</sup></td><td align="left">Castanea sativa</td><td align="left">Italy</td><td align="left">1995</td></tr><tr><td></td><td align="left">SCRP82<sup>(a)</sup></td><td align="left">Eucalypt</td><td align="left">Australia</td><td></td></tr><tr><td align="left"><italic>P. cinnamomi</italic></td><td align="left">SCRP115 (CBS270.55)</td><td align="left"><italic>Chamaecyparis lawsoniana</italic></td><td align="left">Netherlands</td><td align="left">1993</td></tr><tr><td></td><td align="left">SCRP118 (CBS342.72)</td><td align="left"><italic>Persea gratissima</italic></td><td align="left">California</td><td align="left">1972</td></tr><tr><td></td><td align="left">SCRP121<sup>(a)</sup></td><td></td><td align="left">Australia</td><td></td></tr><tr><td align="left"><italic>P. citricola</italic></td><td align="left">SCRP130</td><td align="left"><italic>Rubus idaeus</italic></td><td align="left">Scotland</td><td align="left">1986</td></tr><tr><td></td><td align="left">SCRP136<sup>(a)</sup></td><td align="left">Soil</td><td align="left">UK</td><td align="left">1995</td></tr><tr><td></td><td align="left">SCRP140<sup>(a)</sup></td><td align="left"><italic>Taxus </italic>sp.</td><td align="left">UK</td><td align="left">1995</td></tr><tr><td></td><td align="left">SCRP143<sup>(a)</sup></td><td align="left"><italic>Quercus robur</italic></td><td align="left">Germany</td><td align="left">1994</td></tr><tr><td align="left"><italic>P. europaea</italic></td><td align="left">SCRP622</td><td align="left"><italic>Quercus robur</italic></td><td align="left">Switzerland</td><td align="left">1995</td></tr><tr><td align="left"><italic>P. fragariae var. rubi</italic></td><td align="left">SCRP333 (IMI355974)</td><td align="left"><italic>Rubus idaeus</italic></td><td align="left">Scotland</td><td align="left">1985</td></tr><tr><td align="left"><italic>P. ilicis</italic></td><td align="left">SCRP377</td><td align="left"><italic>Ilex aquilifolium</italic></td><td align="left">UK</td><td align="left">1995</td></tr><tr><td></td><td align="left">SCRP379<sup>(a)</sup></td><td align="left"><italic>Ilex aquilifolium</italic></td><td align="left">UK</td><td></td></tr><tr><td align="left"><italic>P. infestans</italic></td><td align="left">SC03.26.3.3</td><td align="left"><italic>Solanum tuberosum</italic></td><td align="left">Scotland</td><td align="left">2003</td></tr><tr><td align="left"><italic>P. inundata</italic></td><td align="left">SCRP644 (IMI389751)</td><td align="left"><italic>Salix </italic>sp.</td><td align="left">UK</td><td align="left">1972</td></tr><tr><td align="left"><italic>P. lateralis</italic></td><td align="left">SCRP390 (IMI 040503)</td><td align="left"><italic>Chamaecyparis lawsoniana</italic></td><td align="left">U.S.A.</td><td align="left">1942</td></tr><tr><td align="left"><italic>P. nemorosa</italic></td><td align="left">SCRP910</td><td></td><td></td><td></td></tr><tr><td align="left"><italic>P. pseudosyringae</italic></td><td align="left">SCRP674 (IMI390500)</td><td align="left"><italic>Malus pumila</italic></td><td align="left">Italy</td><td align="left">2001</td></tr><tr><td></td><td align="left">SCRP734<sup>(a)</sup></td><td align="left"><italic>Fagus sylvatica</italic></td><td align="left">Italy</td><td align="left">2003</td></tr><tr><td align="left"><italic>P. psychrophila</italic></td><td align="left">SCRP630</td><td align="left"><italic>Quercus ilex</italic></td><td align="left">France</td><td align="left">1996</td></tr><tr><td align="left"><italic>P. quercina</italic></td><td align="left">SCRP541</td><td align="left"><italic>Quercus robur</italic></td><td align="left">Germany</td><td align="left">1995</td></tr><tr><td align="left"><italic>P. ramorum</italic></td><td align="left">SCRP911</td><td align="left"><italic>Rhododendron </italic>sp.</td><td align="left">Scotland</td><td align="left">2004</td></tr><tr><td align="left"><italic>P. sojae</italic></td><td align="left">SCRP555</td><td align="left"><italic>Glycine max</italic></td><td align="left">USA</td><td></td></tr></tbody></table><table-wrap-foot><p><sup>(a) </sup>= Additional isolates utilised to evaluate intraspecific variability</p></table-wrap-foot></table-wrap><fig position="float" id="F1"><label>Figure 1</label><caption><p><bold>Schematic representation of two different approaches utilised to identify SSRs in a broad range of <italic>Phytophthora </italic>species (A) or in <italic>Phytophthora </italic>spp. Clade 7 (B)</bold>.</p></caption><graphic xlink:href="1471-2164-9-620-1"></graphic></fig><sec><title>Analysis of sequences from <italic>P. infestans</italic>, <italic>P. sojae </italic>and <italic>P. ramorum </italic>genome projects and scanning for homologous SSRs</title><p>Predicted gene datasets from <italic>P. infestans </italic><ext-link ext-link-type="uri" xlink:href="http://www.broad.mit.edu/"></ext-link><italic>P. sojae </italic>and <italic>P. ramorum </italic><ext-link ext-link-type="uri" xlink:href="http://genome.jgi-psf.org/"></ext-link> were scanned for the presence of microsatellites defined as short tandem repeat motifs (SSRs) of 2–6 bp. Both perfect and compound SSRs were selected with a minimal acceptable length of 10 bp (dinucelotide motifs) and 12 bp (tri- and tetranucletide motifs). SSRs with a minimum of three repeats were included in the analyses of penta-nucleotide repeats. This search yielded 9333 sequences containing SSRs (1465 from <italic>P. infestans</italic>, 5348 from <italic>P. sojae </italic>and 2520 from <italic>P. ramorum</italic>). The relative abundance of SSRs was 103, 183 and 114 per Mb of predicted gene sequence for <italic>P. infestans</italic>, <italic>P. sojae </italic>and <italic>P. ramorum</italic>, respectively.</p><p>Selected regions were compared by BLAST analysis to identify homologous regions flanking SSRs in at least two of the three species (<italic>P. sojae</italic>, <italic>P. ramorum </italic>and <italic>P. infestans</italic>). This analysis identified 4135 SSRs from <italic>P. infestans </italic>(688), <italic>P. ramorum </italic>(1470), and <italic>P. sojae </italic>(1977). A very limited number of loci containing SSRs were common to the three species; most loci were common to <italic>P. ramorum </italic>and <italic>P. sojae </italic>(81.6%), <italic>P. infestans </italic>and <italic>P. ramorum </italic>(7%) or <italic>P. infestans </italic>and <italic>P. sojae </italic>(11%). In most of the cases, homologous loci contained the same SSR motif in different Phytophthoras, however the number of repeats was consistently higher in the 'source' species than the other two.</p><p>Among the selected loci, the number of SSR repeats ranged from 3 to 13, from 3 to 12 and from 3 to 17 in <italic>P. infestans, P. ramorum </italic>and <italic>P. sojae </italic>respectively. Most SSRs showed seven repeats or less (98.2% <italic>P. infestans</italic>, 97.4% <italic>P. ramorum</italic>, 94.4% <italic>P. sojae</italic>), with a repeat number of four being the most common in all species.</p></sec><sec><title>Selection and amplification of target regions containing SSRs</title><p>The 4135 homologous regions previously identified were manually analysed to select those with the highest number of repeats and flanked by the most conserved sequences on both sides. The latter condition was necessary to design primers suitable for as many species as possible. Based on this analysis 6, 7 and 12 target regions were identified across the genome of <italic>P. infestans</italic>, <italic>P. ramorum </italic>and <italic>P. sojae </italic>respectively. These regions, containing 8, 17 and 33 SSRs respectively, were selected (Table <xref ref-type="table" rid="T2">2</xref>) for amplification from 16 different species of <italic>Phytophthora </italic>representing the breadth of diversity in the genus (Table <xref ref-type="table" rid="T1">1</xref>). To this aim, a total number of 62 different degenerate primers (12 for <italic>P. infestans</italic>, 18 for <italic>P. ramorum</italic>, and 32 for <italic>P. sojae</italic>) were designed (Table <xref ref-type="table" rid="T2">2</xref>). When target regions contained two or more SSRs and/or were too long to be amplified by a single amplification, a pool of different primers was designed (Table <xref ref-type="table" rid="T2">2</xref>). Considerable effort was made to obtain successful amplification from as many species as possible. This involved screening of several primer pairs for each genomic region and for each <italic>Phytophthora </italic>species (Table <xref ref-type="table" rid="T2">2</xref>) and adjustment of annealing temperatures and MgCl<sub>2 </sub>concentration for PCR reactions (Tables <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>, <xref ref-type="table" rid="T5">5</xref>).</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Set of primers designed with the 1<sup>st </sup>approach (Fig. <xref ref-type="fig" rid="F1">1A</xref>) to amplify genomic regions with candidate SSRs in a broad range of <italic>Phytophthora </italic>species (Table <xref ref-type="table" rid="T1">1</xref>).</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="left"><bold>Forward primers<sup>(a)</sup></bold></td><td align="left"><bold>Reverse primers<sup>(a)</sup></bold></td><td align="left"><bold>SSRs<sup>(a)</sup></bold></td><td align="left"><bold>Source<sup>(b)</sup></bold></td></tr></thead><tbody><tr><td align="center"><bold><italic>P. sojae</italic></bold></td><td align="left">S1F<break></break>ACGACGTGTCCAAGAACCAC</td><td align="left">S3R<break></break>ATGTTGACCGTGTTCTGCTG</td><td align="left">(CCG)<sub>7</sub>;(AGC)<sub>4</sub>; (AGC)<sub>14</sub></td><td align="left">scaffold_3:<break></break>1346836–1347814</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S4F<break></break>AARATGACGTGGACKGAGAG</td><td align="left">S5R<break></break>TGATSGTGGAGAARCTCATCT</td><td align="left">(AAC)<sub>14</sub></td><td align="left">scaffold_14:<break></break>861958–862585</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S6F<break></break>GGAGTTCGCCATCAACAACT</td><td align="left">S7R<break></break>TCAGCTTCTGTCGRTCGAC</td><td align="left">(AAG)<sub>14</sub></td><td align="left">scaffold_24:<break></break>159790–160289</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S8F<break></break>YGYGTCTCGCCCAYGAC</td><td align="left">S9R<break></break>GACGACACCGGSGAGAG</td><td align="left">(ACC)<sub>4</sub>;(AGC)<sub>4</sub>; (AGC)<sub>28</sub>; (AAC)<sub>4</sub></td><td align="left">scaffold_76:<break></break>171071–172890</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td></td></tr><tr><td></td><td align="left">S10F<break></break>GCGSTACGAGACCTGGAC</td><td align="left">S11R<break></break>GACTCRCCCTTCGACTCSTC</td><td align="left">(CAG)<sub>14</sub></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S12F<break></break>GGAGGCCGAGTCGGARTA</td><td align="left">S13R<break></break>TAYTCCGACTCGGCCTCC</td><td align="left">(AGC)<sub>14</sub></td><td align="left">scaffold_136:<break></break>56988–57479</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S14F<break></break>GACGCMSYYGAGTGGAAAG</td><td align="left">S15R<break></break>ATTTKGSACAGATACCGACG</td><td align="left">(AAG)<sub>15</sub></td><td align="left">scaffold_65:<break></break>332082–332852</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S16F<break></break>TCTACGTGAATGCCATGAGG</td><td align="left">S17R<break></break>CGTTCAGCTTCTGTCGATCR</td><td align="left">(AAG)<sub>15</sub></td><td align="left">scaffold_79:<break></break>50973–51428</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S18F<break></break>YACCATCTCCAACCTGCTG</td><td align="left">S19R<break></break>CACCACCTCGAGTAGCTCCC</td><td align="left">(AGC)<sub>7</sub>; (AGG)<sub>13</sub></td><td align="left">scaffold_2:<break></break>1159730–1160958</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td></td></tr><tr><td></td><td align="left">S19F<break></break>GGGAGCTACTCGAGGTGGTG</td><td align="left">S20R<break></break>TCGTCTCAATCTCKGACTGA</td><td align="left">(AGC)<sub>6</sub></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S21F<break></break>ATCTGGGCTTCCASGAGGT</td><td align="left">S22R<break></break>CTGATCCTCCGCCACAY</td><td align="left">(AAG)<sub>12</sub>; (ATC)<sub>6</sub>; (ATC)<sub>4</sub>; (AAG)<sub>5</sub></td><td align="left">scaffold_138:<break></break>18289–18854</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S23F<break></break>GACTCGGACTCGGACGAC</td><td align="left">S25R<break></break>CTCCTGCTCKTCTTTCAGGC</td><td align="left">(AGG)<sub>7</sub>; (AAG)<sub>10</sub>; (GAG)<sub>4</sub>; (AAG)<sub>12</sub>; (GAG)<sub>5</sub>; (AGA)<sub>6</sub></td><td align="left">scaffold_90:<break></break>249340–250137</td></tr><tr><td></td><td></td><td align="left">S37R<break></break>CTTRCCBTCCTTGTCCTTYT</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S27F<break></break>GAAGCGCGGGCGWGT</td><td align="left">S31R<break></break>TCCTCCTCTTCTTCTTCGTCW</td><td align="left">(AAG)<sub>4</sub>; (AGG)<sub>4</sub>; (ACG)<sub>4</sub>; (AGG)<sub>11</sub></td><td align="left">scaffold_16:<break></break>680126–681136</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td></td></tr><tr><td></td><td align="left">S31F<break></break>WGACGAAGAAGAAGAGGAGGA</td><td align="left">S28R<break></break>TCATTCATCAGCGTGTCRAT</td><td align="left">(GAG)<sub>4</sub></td><td></td></tr><tr><td></td><td align="left">S34F<break></break>ABGAWGACGABGAGGAVGAV</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">S29F<break></break>MGCAAGAAGGCGTCGTA</td><td align="left">S30R<break></break>CCTTCATCATGAGCTTCTGG</td><td align="left">(AGG)<sub>4 </sub>(AAG)<sub>11</sub></td><td align="left">scaffold_40:<break></break>353572–354360</td></tr><tr><td colspan="5"><hr></hr></td></tr><tr><td align="center"><bold><italic>P. ramorum</italic></bold></td><td align="left">R1F<break></break>GYGGCGGTGGCTACAGYG</td><td align="left">R3R<break></break>CTGCTGYTGCTGGTTGAAAG</td><td align="left">(ACC)<sub>4</sub>; (ACC)<sub>5</sub>; (ACC)<sub>4</sub></td><td align="left">scaffold_23:<break></break>349447–350425</td></tr><tr><td></td><td align="left">R2F<break></break>CTACTCSAGCCGCTACGC</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">R3F<break></break>CTTTCAACCAGCARCAGCAG</td><td align="left">R4R<break></break>GTTCATCATGCCWCCCATR</td><td align="left">(AGC)<sub>8</sub></td><td align="left">scaffold_23:<break></break>350406–351828</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td></td></tr><tr><td></td><td align="left">R4F<break></break>YATGGGWGGCATGATGAAC</td><td align="left">R5R<break></break>AGGACCAGGAGATGGAGGAC</td><td align="left">(AGC)<sub>4</sub>; (AGC)<sub>12</sub>; (AGC)<sub>4</sub></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">R7F<break></break>TGTTCCARACCCGCTTCC</td><td align="left">R8R<break></break>CACCAAGCAGCACKCGC</td><td align="left">(ACG)<sub>9</sub>; (AAC)<sub>5</sub>; (AGC)<sub>10</sub></td><td align="left">scaffold_10:<break></break>436897–437690</td></tr><tr><td></td><td></td><td align="left">R9R<break></break>GGAACGCACCAAAGACGC</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">R10F<break></break>GGAGATGACGGAAGATGACG</td><td align="left">R11R<break></break>CCATCGAARTACATSACACGA</td><td align="left">(AAGCC)<sub>4</sub>; (AGG)<sub>9</sub>; (AAG)<sub>7</sub></td><td align="left">scaffold_5:<break></break>750031–750612</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">R13F<break></break>AAGTCGAAGCTCGTGGTSAC</td><td align="left">R14R<break></break>GTATCCGCTGRAAGAGCGTC</td><td align="left">(AAG)<sub>10</sub></td><td align="left">scaffold_78:<break></break>40203–40815</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">R15F<break></break>CCGGAGCGCGTGGA</td><td align="left">R16R<break></break>GGTAGTTGAGCGGCTTCTTG</td><td align="left">(CCG)<sub>6</sub></td><td align="left">scaffold_2778:<break></break>35–305</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">R16F<break></break>CAAGAAGCCGCTCAACTACC</td><td align="left">R17R<break></break>TAACGGATCAGCTCTTGCTG</td><td align="left">(ATC)<sub>4</sub>; (AGG)<sub>8</sub></td><td align="left">scaffold_2778:<break></break>286–1134</td></tr><tr><td colspan="5"><hr></hr></td></tr><tr><td align="center"><bold><italic>P. infestans</italic></bold></td><td align="left">I3F<break></break>GCCTGTGGAYGAGAATGGYS</td><td align="left">I4R<break></break>CAGATCCACGACACCRGGY</td><td align="left">(AAG)<sub>8</sub></td><td align="left">Pi_002_41652<break></break>_Feb05</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">I5F<break></break>CATCAACAAGTGCTCGTWCS</td><td align="left">I6R<break></break>TAGTCRAYGTTCTTGTTGTTCA</td><td align="left">(AGC)<sub>5</sub>; (AGC)<sub>8</sub></td><td align="left">Supercont1.7<break></break>842678–843649</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">I7F<break></break>GHGTGGGCGAGTACTCCAAG</td><td align="left">I8R<break></break>AAGCTGGCTATRWACACTGCCG</td><td align="left">(AG)<sub>9</sub></td><td align="left">Supercont1.4<break></break>1481811–1482015</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">I9F<break></break>GCATYGGGTCGTTCCTGTA</td><td align="left">I10R<break></break>AGHGTGCAGTACAGACCCGC</td><td align="left">(AAG)<sub>11</sub></td><td align="left">Supercont1.5<break></break>1235771–1236143</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">I11F<break></break>TCGTCBGTGTCCTCBACGTC</td><td align="left">I12R<break></break>ACCAGCATCTTRTTCTGRGCAG</td><td align="left">(ACC)<sub>8</sub></td><td align="left">Supercont1.45<break></break>522394–522633</td></tr><tr><td></td><td></td><td></td><td colspan="1"><hr></hr></td><td colspan="1"><hr></hr></td></tr><tr><td></td><td align="left">I13F<break></break>GTCTGCGCTGTCGGAACT</td><td align="left">I14R<break></break>TRATGATGCGGTTCATCTCG</td><td align="left">(AAG)<sub>7</sub>; (AAG)<sub>4</sub></td><td align="left">Supercont1.220<break></break>167896–168460</td></tr></tbody></table><table-wrap-foot><p><sup>(a) </sup>= Primers are listed according to the localization in their respective genome projects (<italic>P. sojae</italic>, <italic>P. ramorum </italic>or <italic>P. infestans</italic>) and according to the flanked SSRs. In some circumstances, a pool of different primers was designed to amplify selected genomic regions from as many as possible <italic>Phytophthora </italic>species. Similarly, when target regions contained two or more SSRs and were too long to be amplified by a single amplification a pool of different primers was designed.</p><p><sup>(b) </sup>= Gene sequences available at <ext-link ext-link-type="uri" xlink:href="http://genome.jgi-psf.org/sojae1/sojae1.home.html"></ext-link> (<italic>P. sojae</italic>), <ext-link ext-link-type="uri" xlink:href="http://genome.jgi-psf.org/ramorum1/ramorum1.home.html"></ext-link> (<italic>P. ramorum</italic>) and <ext-link ext-link-type="uri" xlink:href="http://www.pfgd.org/"></ext-link> or <ext-link ext-link-type="uri" xlink:href="http://www.broad.mit.edu/annotation/genome/phytophthora_infestans/Home.html"></ext-link> (<italic>P. infestans</italic>).</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Accession numbers and SSRs for GenBank deposited sequences <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/"></ext-link> amplified from 16 <italic>Phytophthora </italic>species (Table <xref ref-type="table" rid="T1">1</xref>) using primers designed on <italic>P. sojae </italic>with the first approach (Fig.<xref ref-type="fig" rid="F1">1A</xref>).</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold><italic>Phytoph</italic></bold>.<break></break><bold>species</bold></td><td align="center" colspan="12"><bold>SELECTED PRIMERS<sup>(a)</sup></bold></td></tr><tr><td></td><td colspan="12"><hr></hr></td></tr><tr><td></td><td align="left"><bold>S1F-S3R</bold></td><td align="left"><bold>S4F-S5R</bold></td><td align="left"><bold>S6F-S7R</bold></td><td align="left"><bold>S10F-11R</bold></td><td align="left"><bold>S16F-17R</bold></td><td align="left"><bold>S18F-19R</bold></td><td align="left"><bold>S19F-20R</bold></td><td align="left"><bold>S21F-22R</bold></td><td align="left"><bold>S23F-S25R<sup>(1)</sup></bold></td><td align="left"><bold><sup>(2)</sup>S31F-28R</bold></td><td align="left"><bold>S27F-S31R</bold></td><td align="left"><bold>S29F-S30R</bold></td></tr></thead><tbody><tr><td align="left"><bold><italic>P. alni</italic></bold><break></break><bold>subsp. <italic>alni</italic></bold><break></break>SCRP2</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216617">EF216617</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216607">EF216607</ext-link><break></break>(aac)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216602">EF216602</ext-link><break></break>(agc)<sub>6</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216590">EF216590</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216580">EF216580</ext-link><break></break>(aag)<sub>5</sub><break></break>(agg)<sub>5</sub><break></break>(aag)<sub>4</sub><break></break>(aag)<sub>6</sub><break></break>(agg)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216565">EF216565</ext-link><break></break>(agg)<sub>7</sub><break></break>(aag)<sub>10</sub><break></break>(agg)<sub>16 </sub>(aag)<sub>12</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216555">EF216555</ext-link><break></break>(aag)<sub>8</sub><break></break>(agg)<sub>4</sub><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>5</sub><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>4</sub><break></break>(aag)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216552">EF216552</ext-link><break></break>(aag)<sub>8</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. cambiv</italic></bold>.<break></break>SCRP67</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216618">EF216618</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216606">EF216606</ext-link><break></break>(aac)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216601">EF216601</ext-link><break></break>(agc)<sub>4</sub><break></break>(cg)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216593">EF216593</ext-link><break></break>No SSR<break></break>55°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216581">EF216581</ext-link><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>5</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216569">EF216569</ext-link><break></break>(agg)<sub>10 </sub>(aag)<sub>10</sub><break></break>(agg)<sub>9</sub><break></break>(aag)<sub>12</sub><break></break>(agg)<sub>4</sub><break></break>(agg)<sub>7</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216551">EF216551</ext-link><break></break>(aag)<sub>8</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. cinnam</italic></bold>.<break></break>SCRP115</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF2165">EF2165</ext-link><break></break>(aag)<sub>8</sub><break></break>(agg)<sub>5</sub><break></break>(aag)<sub>9</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. citricola</italic></bold><break></break>SCRP130</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216619">EF216619</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216553">EF216553</ext-link><break></break>(aag)<sub>7</sub><break></break>(aag)<sub>7</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. europaea</italic></bold><break></break>SCRP622</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216616">EF216616</ext-link> (acg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216605">EF216605</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216600">EF216600</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216589">EF216589</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216576">EF216576</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216568">EF216568</ext-link><break></break>(agg)<sub>9</sub><break></break>(aag)<sub>10</sub><break></break>(aag)<sub>7</sub><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>6</sub><break></break>(agg)<sub>4</sub><break></break>(agg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF21655054">EF21655054</ext-link><break></break>(aag)<sub>9</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. fragariae</italic></bold><break></break><bold>var</bold>.<bold><italic>rubi</italic></bold><break></break>SCRP333</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216594">EF216594</ext-link> (aac)<sub>4</sub><break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216584">EF216584</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>60°C/0.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216542">EF216542</ext-link><break></break>(aag)<sub>7</sub><break></break>(aag)<sub>8</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. ilicis</italic></bold><break></break>SCRP377</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216608">EF216608</ext-link> (ccg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216575">EF216575</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>60°C/0.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216543">EF216543</ext-link><break></break>(aag)<sub>5</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. infestans</italic></bold><break></break>sc 03.26.3.3</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216615">EF216615</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216560">EF216560</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. inundata</italic></bold><break></break>SCRP644</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216624">EF216624</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216614">EF216614</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216599">EF216599</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216588">EF216588</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/0.7 mM*</td><td align="left">NS<sup>++</sup><break></break>60°C/0.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216549">EF216549</ext-link><break></break>(aag)<sub>8</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. lateralis</italic></bold><break></break>SCRP390</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216611">EF216611</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216604">EF216604</ext-link><break></break>(ac)<sub>5</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216598">EF216598</ext-link><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216592">EF216592</ext-link><break></break>(acg)<sub>4</sub><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216587">EF216587</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216579">EF216579</ext-link><break></break>(aag)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216564">EF216564</ext-link><break></break>(agg)<sub>5</sub><break></break>(aag)<sub>4</sub><break></break>(aag)<sub>6</sub><break></break>(agg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216556">EF216556</ext-link><break></break>(agg)<sub>4</sub><break></break>58°C/0.7 mM*</td><td align="left">NS<sup>++</sup><break></break>60°C/0.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216548">EF216548</ext-link><break></break>(aag)<sub>5</sub><break></break>(aag)<sub>9</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. nemor</italic></bold>.<break></break>SCRP910</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216613">EF216613</ext-link> (ccg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216597">EF216597</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216572">EF216572</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216559">EF216559</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216547">EF216547</ext-link><break></break>(aag)<sub>5</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. pseudos</italic></bold>.<break></break>SCRP674</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216622">EF216622</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216596">EF216596</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216573">EF216573</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216557">EF216557</ext-link><break></break>(agg)<sub>14</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>60°C/0.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216546">EF216546</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. psychro</italic></bold>.<break></break>SCRP630</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216623">EF216623</ext-link> (agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216612">EF216612</ext-link> (ccg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216574">EF216574</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216558">EF216558</ext-link><break></break>(agg)<sub>6</sub><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>4</sub><break></break>(agg)<sub>4</sub><break></break>58°C/0.7 mM*</td><td align="left">NS<sup>++</sup><break></break>60°C/0.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216545">EF216545</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. quercina</italic></bold><break></break>SCRP541</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216621">EF216621</ext-link><break></break>(agc)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216563">EF216563</ext-link><break></break>(aag)<sub>10 </sub>(aag)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216561">EF216561</ext-link><break></break>(agg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216544">EF216544</ext-link><break></break>(aag)<sub>7</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. ramorum</italic></bold><break></break>SCRP911</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216620">EF216620</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216610">EF216610</ext-link> (acc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216603">EF216603</ext-link><break></break>No SSR<break></break>55°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216595">EF216595</ext-link><break></break>No SSR<break></break>55°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216591">EF216591</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216586">EF216586</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216578">EF216578</ext-link><break></break>(aag)<sub>4</sub><break></break>(atc)<sub>4</sub><break></break>(aag)<sub>5</sub><break></break>(aag)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216562">EF216562</ext-link><break></break>(aag)<sub>4</sub><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>7</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/0.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216540">EF216540</ext-link> (aag)<sub>4</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. sojae</italic></bold><break></break>SCRP555</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216609">EF216609</ext-link><break></break>(aac)<sub>14</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382779">EF382779</ext-link><break></break>No SSR<break></break>55°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216585">EF216585</ext-link> (agc)<sub>6</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216577">EF216577</ext-link><break></break>(aag)<sub>12</sub><break></break>(atc)<sub>6</sub><break></break>(atc)<sub>4</sub><break></break>(aag)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/0.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/0.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216541">EF216541</ext-link><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>11</sub><break></break>58°C/1.0 mM*</td></tr></tbody></table><table-wrap-foot><p><sup>(a) </sup>= Primers listed in Table <xref ref-type="table" rid="T2">2</xref> and not reported in this table are those that did not produce any reliable sequence (see text).</p><p><sup>(1) </sup>= Primer S25R was replaced by primer S37R for <italic>P. nemorosa, P. pseudosyringae, P. psychrophila</italic>, and <italic>P. ilicis</italic>.</p><p><sup>(2) </sup>= Primer S31F was replaced by primer S34F for <italic>P. alni, P. citricola, P. infestans, P. nemorosa</italic>, and <italic>P. quercina</italic>.</p><p>NA<sup>+ </sup>= Isolate-primer combinations that did not produce any amplification or produced complex profiles (two or more fragments).</p><p>NS<sup>++ </sup>= Isolate-primer combinations for which single PCR bands were obtained but direct sequencing did not produce reliable results.</p><p>°C/mM* = Selected annealing temperature (°C) and MgCl<sub>2 </sub>concentration (mM) in PCR reactions.</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T4"><label>Table 4</label><caption><p>Accession numbers and SSRs for GenBank deposited sequences <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/"></ext-link> amplified from 16 <italic>Phytophthora </italic>species (Table <xref ref-type="table" rid="T1">1</xref>) using primers designed on <italic>P. ramorum </italic>with the first approach (Fig. <xref ref-type="fig" rid="F1">1A</xref>).</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold><italic>Phytophthora</italic></bold><break></break><bold>species</bold></td><td align="center" colspan="7"><bold>SELECTED PRIMERS<sup>(a)</sup></bold></td></tr><tr><td></td><td colspan="7"><hr></hr></td></tr><tr><td></td><td align="left"><sup>(1)</sup><bold>R1F-R3R</bold></td><td align="left"><bold>R3F-R4R</bold></td><td align="left"><bold>R4F-R5R</bold></td><td align="left"><bold>R7F-9R<sup>(2)</sup></bold></td><td align="left"><bold>R10F-R11R</bold></td><td align="left"><bold>R13F-R14R</bold></td><td align="left"><bold>R16F-R17R</bold></td></tr></thead><tbody><tr><td align="left"><bold><italic>P. alni </italic>subsp. <italic>alni</italic></bold><break></break>SCRP2</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216645">EF216645</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216671">EF216671</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216662">EF216662</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216651">EF216651</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216625">EF216625</ext-link><break></break>(acg)<sub>4</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. cambivora</italic></bold><break></break>SCRP67</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216673">EF216673</ext-link><break></break>(agc)<sub>8</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216663">EF216663</ext-link><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>4</sub><break></break>(agg)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216626">EF216626</ext-link><break></break>(acg)<sub>4</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. cinnamomi</italic></bold><break></break>SCRP115</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/<break></break>1.7 mM*</td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. citricola</italic></bold><break></break>SCRP130</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216644">EF216644</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216630">EF216630</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. europaea</italic></bold><break></break>SCRP622</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216643">EF216643</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216661">EF216661</ext-link><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>7</sub><break></break>(agg)<sub>5</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216655">EF216655</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. fragariae</italic></bold><break></break><bold><italic>var. rubi</italic></bold><break></break>SCRP333</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1 mM**</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216634">EF216634</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216672">EF216672</ext-link><break></break>(agc)<sub>7</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216657">EF216657</ext-link><break></break>(aag)<sub>4</sub><break></break>(agg)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216647">EF216647</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. ilicis</italic></bold><break></break>SCRP377</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216646">EF216646</ext-link><break></break>(agc)<sub>4</sub><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216635">EF216635</ext-link><break></break>(agc)<sub>4</sub><break></break>(accat)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216664">EF216664</ext-link><break></break>(actg)<sub>3</sub><break></break>(agc)<sub>6</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216648">EF216648</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. infestans</italic></bold><break></break>sc 03.26.3.3</td><td align="left">NS<sup>++</sup><break></break>58°C/<break></break>1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. inundata</italic></bold><break></break>SCRP644</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216633">EF216633</ext-link><break></break>No SSR<break></break>58°C/<break></break>1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216654">EF216654</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216629">EF216629</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. lateralis</italic></bold><break></break>SCRP390</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216631">EF216631</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216642">EF216642</ext-link><break></break>(aac)<sub>5</sub><break></break>(agc)<sub>5</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216669">EF216669</ext-link><break></break>(agc)<sub>5</sub><break></break>(aac)<sub>6</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216660">EF216660</ext-link><break></break>(agg)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216628">EF216628</ext-link><break></break>(agg)<sub>8</sub><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. nemorosa</italic></bold><break></break>SCRP910</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216639">EF216639</ext-link><break></break>(agc)<sub>4</sub><break></break>(accat)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216668">EF216668</ext-link><break></break>(actg)<sub>3</sub><break></break>(agc)<sub>6</sub><break></break>58°C/1.7 mM*</td><td align="left">NS++<break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216653">EF216653</ext-link><break></break>(agg)<sub>9</sub><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. pseudosyringae</italic></bold><break></break>SCRP674</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216632">EF216632</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216641">EF216641</ext-link><break></break>(accat)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216667">EF216667</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA+</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216652">EF216652</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. psychrophila</italic></bold><break></break>SCRP630</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216640">EF216640</ext-link><break></break>(accat)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216666">EF216666</ext-link><break></break>(actg)<sub>3</sub><break></break>(agc)<sub>6</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. quercina</italic></bold><break></break>SCRP541</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216638">EF216638</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216674">EF216674</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216651">EF216651</ext-link><break></break>(aag)<sub>8</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. ramorum</italic></bold><break></break>SCRP911</td><td align="left">NS<sup>++</sup><break></break>58°C/1 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216637">EF216637</ext-link><break></break>(agc)<sub>4</sub><break></break>(agc)<sub>10</sub><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216665">EF216665</ext-link><break></break>(agc)<sub>24</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216659">EF216659</ext-link><break></break>(aagcc)<sub>4</sub><break></break>(agg)<sub>9</sub><break></break>(aag)<sub>7</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216650">EF216650</ext-link><break></break>(aag)<sub>10</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216627">EF216627</ext-link><break></break>(atc)<sub>4</sub><break></break>(agg)<sub>8</sub><break></break>58°C/1.0 mM*</td></tr><tr><td align="left"><bold><italic>P. sojae</italic></bold><break></break>SCRP555</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216636">EF216636</ext-link><break></break>(agc)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216670">EF216670</ext-link><break></break>(agc)<sub>10</sub><break></break>(agcg)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216658">EF216658</ext-link><break></break>(agg)<sub>5</sub><break></break>(aag)<sub>6</sub><break></break>(agg)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216649">EF216649</ext-link><break></break>(acg)<sub>4</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td></tr></tbody></table><table-wrap-foot><p><sup>(a) </sup>= Primers listed in Table <xref ref-type="table" rid="T2">2</xref> and not reported in this table are those that did not produce any reliable sequence (see text).</p><p><sup>(1) </sup>= Primer R1F was replaced by primer R2F for <italic>P. cambivora</italic>, <italic>P. inundata, P. nemorosa, P. ilicis </italic>and <italic>P. fragaria</italic>.</p><p><sup>(2) </sup>= Primer R9R was replaced by primer R8R for <italic>P. quercina</italic>.</p><p>NA<sup>+ </sup>= Isolate-primer combinations that did not produce any amplification or produced complex profiles (two or more fragments).</p><p>NS<sup>++ </sup>= Isolate-primer combinations for which single PCR bands were obtained but direct sequencing did not produce reliable results.</p><p>°C/mM* = Selected annealing temperature (°C) and MgCl<sub>2 </sub>concentration (mM) in PCR reactions.</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T5"><label>Table 5</label><caption><p>Accession numbers and SSRs for GenBank deposited sequences <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/"></ext-link> amplified from 16 <italic>Phytophthora </italic>species (Table <xref ref-type="table" rid="T1">1</xref>) using primers designed on <italic>P. infestans </italic>with the first approach (Fig. <xref ref-type="fig" rid="F1">1A</xref>).</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold><italic>Phytophthora</italic></bold><break></break><bold>species</bold></td><td align="center" colspan="6"><bold>SELECTED PRIMERS<sup>(a)</sup></bold></td></tr><tr><td></td><td colspan="6"><hr></hr></td></tr><tr><td></td><td align="left"><bold>I3F-4R</bold></td><td align="left"><bold>I5F-I6R</bold></td><td align="left"><bold>I7F-I8R</bold></td><td align="left"><bold>I9F-I10R</bold></td><td align="left"><bold>I11F-I12R</bold></td><td align="left"><bold>I13F-I14R</bold></td></tr></thead><tbody><tr><td align="left"><bold><italic>P. alni </italic>subsp. <italic>alni</italic></bold><break></break>SCRP2</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216535">EF216535</ext-link><break></break>No SSR;<break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216513">EF216513</ext-link><break></break>(aag)<sub>4</sub><break></break>(agg)<sub>6</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216477">EF216477</ext-link><break></break>(agg)<sub>4</sub><break></break>(aag)<sub>5</sub><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. cambivora</italic></bold><break></break>SCRP67</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216516">EF216516</ext-link><break></break>(acg)<sub>4</sub><break></break>(aag)<sub>5</sub><break></break>(agg)<sub>6</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216478">EF216478</ext-link><break></break>(agg)<sub>5</sub><break></break>(aag)<sub>5</sub><break></break>58°C<break></break>1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. cinnamomi</italic></bold><break></break>SCRP115</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216509">EF216509</ext-link><break></break>(aag)<sub>14</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216494">EF216494</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. citricola</italic></bold><break></break>SCRP130</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216534">EF216534</ext-link><break></break>(agc)<sub>4</sub>; (agc)<sub>5</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216520">EF216520</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216498">EF216498</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216482">EF216482</ext-link><break></break>(agg)<sub>9</sub><break></break>(aag)<sub>5</sub><break></break>58°C/1.7 mM</td></tr><tr><td align="left"><bold><italic>P. europaea</italic></bold><break></break>SCRP622</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216512">EF216512</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216476">EF216476</ext-link><break></break>(agg)<sub>5</sub><break></break>(aag)<sub>5</sub><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. fragariae</italic></bold><break></break><bold>var. <italic>rubi</italic></bold><break></break>SCRP333</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216500">EF216500</ext-link><break></break>(acg)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. ilicis</italic></bold><break></break>SCRP377</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216532">EF216532</ext-link><break></break>(agc)<sub>9</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216501">EF216501</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216495">EF216495</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216483">EF216483</ext-link><break></break>(aag)<sub>4</sub><break></break>(agc)<sub>4</sub><break></break>58°C/1.7 mM</td></tr><tr><td align="left"><bold><italic>P. infestans</italic></bold><break></break>sc 03.26.3.3</td><td align="left">NS<sup>++</sup><break></break>58°C<break></break>1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216524">EF216524</ext-link><break></break>(agc)<sub>6</sub><break></break>(agc)<sub>5</sub><break></break>58°C/1.0 mM</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216499">EF216499</ext-link><break></break>(aag)<sub>11</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216487">EF216487</ext-link><break></break>(acc)<sub>8</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216474">EF216474</ext-link><break></break>(aag)<sub>7</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM</td></tr><tr><td align="left"><bold><italic>P. inundata</italic></bold><break></break>SCRP644</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216508">EF216508</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216497">EF216497</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td></tr><tr><td align="left"><bold><italic>P. lateralis</italic></bold><break></break>SCRP390</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216527">EF216527</ext-link><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216507">EF216507</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216493">EF216493</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216481">EF216481</ext-link><break></break>(agg)<sub>5</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. nemorosa</italic></bold><break></break>SCRP910</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216531">EF216531</ext-link><break></break>(agc)<sub>7</sub><break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216503">EF216503</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216492">EF216492</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216486">EF216486</ext-link><break></break>(acg)<sub>4</sub><break></break>(aag)<sub>4</sub><break></break>(agc)<sub>4</sub><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. pseudosyringae</italic></bold><break></break>SCRP674</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216529">EF216529</ext-link><break></break>(agc)<sub>7</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216502">EF216502</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216496">EF216496</ext-link><break></break>(aag)<sub>5</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216485">EF216485</ext-link><break></break>(acg)<sub>4</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. psychrophila</italic></bold><break></break>SCRP630</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216528">EF216528</ext-link><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216491">EF216491</ext-link><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216484">EF216484</ext-link><break></break>(aag)<sub>4</sub><break></break>(agc)<sub>4</sub><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. quercina</italic></bold><break></break>SCRP541</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216506">EF216506</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216490">EF216490</ext-link><break></break>(aag)<sub>5</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216480">EF216480</ext-link><break></break>(agg)<sub>6</sub><break></break>(aag)<sub>4</sub><break></break>58°C/1.7 mM*</td></tr><tr><td align="left"><bold><italic>P. ramorum</italic></bold><break></break>SCRP911</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216525">EF216525</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216505">EF216505</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216489">EF216489</ext-link><break></break>(acc)<sub>4</sub><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216479">EF216479</ext-link><break></break>(aac)<sub>7</sub><break></break>(agg)<sub>9</sub><break></break>58°C/1.7 mM</td></tr><tr><td align="left"><bold><italic>P. sojae</italic></bold><break></break>SCRP555</td><td align="left">NS<sup>++</sup><break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216526">EF216526</ext-link><break></break>No SSR<break></break>58°C/1.0 mM*</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216504">EF216504</ext-link><break></break>(agc)<sub>4</sub><break></break>58°C/1.0 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216488">EF216488</ext-link><break></break>No SSR<break></break>58°C/1.7 mM*</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216475">EF216475</ext-link><break></break>(agg)<sub>7</sub><break></break>(aag)<sub>5</sub><break></break>58°C/1.7 mM*</td></tr></tbody></table><table-wrap-foot><p><sup>(a) </sup>= Primers listed in Table <xref ref-type="table" rid="T2">2</xref> and not reported in this table are those that did not produce any reliable sequence (see text).</p><p>NA<sup>+ </sup>= Isolate-primer combinations that did not produce any amplification or produced complex profiles (two or more fragments).</p><p>NS<sup>++ </sup>= Isolate-primer combinations for which single PCR bands were obtained but direct sequencing did not produce reliable results.</p><p>°C/mM* = Selected annealing temperature (°C) and MgCl<sub>2 </sub>concentration (mM) in PCR reactions.</p></table-wrap-foot></table-wrap><p>The resultant primers enabled the amplification of 271 single PCR bands of the expected size (Fig. <xref ref-type="fig" rid="F2">2</xref>). In the remaining primer-species combinations, 193 amplifications did not produce any product or produced complex profiles (two or more PCR fragments) impeding direct sequencing (Fig. <xref ref-type="fig" rid="F2">2</xref>). Some primer combinations failed to amplify a product from any of the <italic>Phytophthora </italic>species whereas other combinations amplified single bands from all or most <italic>Phytophthora </italic>species (Tables <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>, <xref ref-type="table" rid="T5">5</xref>).</p><fig position="float" id="F2"><label>Figure 2</label><caption><p><bold>Amplification results obtained with 16 <italic>Phytophthora </italic>species (Table </bold><xref ref-type="table" rid="T1">1</xref><bold>) using primers designed against <italic>P. sojae, P. ramorum </italic>and <italic>P. infestans </italic>genomes using Approach 1 (Fig. </bold><xref ref-type="fig" rid="F1">1A</xref><bold>)</bold>. NA represents primer-species combinations in which amplification reactions did not produce any product or produced complex profiles (two or more PCR fragments) preventing direct sequencing. NS represents primer-species combinations in which amplification reactions produced single PCR bands, however direct sequencing did not yield reliable sequences. SQ represents primer-species combinations in which reliable sequences were obtained.</p></caption><graphic xlink:href="1471-2164-9-620-2"></graphic></fig></sec><sec><title>Sequencing of single PCR bands and scanning for SSRs</title><p>All single PCR bands (271) were purified to remove excess primers and nucleotides and sequenced in both directions using the same primers used for the amplification. When forward and/or reverse sequences were not identical, amplification, purification and sequencing were repeated twice and all unreliable sequences were discarded. Finally, 171 sequences were obtained with primers designed against <italic>P. sojae </italic>(70), <italic>P. ramorum </italic>(50) and <italic>P. infestans </italic>(51) genomes (Fig. <xref ref-type="fig" rid="F2">2</xref>) and scanned to identify SSRs by means of sputnik. Sequenced regions contained a total number of 211 SSRs distributed across the genome of the 16 target species with those of clade 7 (<italic>P. alni, P. cambivora, P. europaea</italic>, <italic>P. fragariae </italic>and <italic>P. sojae</italic>) and clade 8 (<italic>P. lateralis </italic>and <italic>P. ramorum</italic>) more highly represented (Fig. <xref ref-type="fig" rid="F3">3</xref>; Tables <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>, <xref ref-type="table" rid="T5">5</xref>). A single microsatellite was identified in <italic>P. inundata</italic>. All SSRs identified in <italic>P. infestans </italic>were amplified with primers designed against its own genome (Fig. <xref ref-type="fig" rid="F3">3</xref>). Identified SSRs ranged in the number of repeats from 4 to 16, from 3 to 16 and from 4 to 14 in <italic>P. sojae, P. ramorum </italic>and <italic>P. infestans </italic>respectively (Tables <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>, <xref ref-type="table" rid="T5">5</xref>). A single repeat of 24 was found in an SCRI isolate of <italic>P. ramorum </italic>(Table <xref ref-type="table" rid="T4">4</xref>). Most SSRs were of seven repeats or less (88.9% <italic>P. infestans</italic>, 82.8% <italic>P. ramorum</italic>, 76.8 <italic>P. sojae</italic>), with a repeat number of four being the most common in all species (Fig. <xref ref-type="fig" rid="F4">4</xref>). Overall, the most common motifs were (AAG)n, (AGG)n and (AGC)n representing 40.9%, 23.3% and 17.6% respectively of the total number of identified SSRs (Fig. <xref ref-type="fig" rid="F5">5</xref>). Trinucleotide repeats were the most common (94.7%) followed by pentanucleotide (2.4%), tetranucleotide (1.9%) and dinucleotide (1.0%) repeats.</p><fig position="float" id="F3"><label>Figure 3</label><caption><p><bold>Number of SSRs identified for each of the 16 <italic>Phytophthora </italic>species using primers designed against <italic>P. sojae, P. ramorum </italic>and <italic>P. infestans </italic>genomes (Approach 1, Fig. </bold><xref ref-type="fig" rid="F1">1A</xref><bold>)</bold>.</p></caption><graphic xlink:href="1471-2164-9-620-3"></graphic></fig><fig position="float" id="F4"><label>Figure 4</label><caption><p><bold>Number of repeated motifs identified in 16 target <italic>Phytophthora </italic>species (Table </bold><xref ref-type="table" rid="T1">1</xref><bold>) using primers designed against <italic>P. sojae, P. ramorum </italic>and <italic>P. infestans </italic>genomes according to Approach 1 (Fig. </bold><xref ref-type="fig" rid="F1">1A</xref><bold>)</bold>.</p></caption><graphic xlink:href="1471-2164-9-620-4"></graphic></fig><fig position="float" id="F5"><label>Figure 5</label><caption><p><bold>List and frequency of the different SSR motifs identified in 16 <italic>Phytophthora </italic>species (Table </bold><xref ref-type="table" rid="T1">1</xref><bold>) using primers designed on <italic>P. sojae, P. ramorum </italic>and <italic>P. infestans </italic>genomes according to Approach 1 (Fig. </bold><xref ref-type="fig" rid="F1">1A</xref><bold>).</bold></p></caption><graphic xlink:href="1471-2164-9-620-5"></graphic></fig><p>To evaluate intraspecific variability a few selected target regions amplified by primers S23F-S25R, S21F-S22R, I9-I10 and I5-6 (Table <xref ref-type="table" rid="T2">2</xref>) were examined and sequenced from additional isolates of <italic>P. alni</italic>, <italic>P. cambivora, P. cinnamomi</italic>, <italic>P. pseudosyringae </italic>and <italic>P. ilicis </italic>(Table <xref ref-type="table" rid="T1">1</xref>). The analysed target regions did not show intraspecific variability among analysed isolates of <italic>P. alni </italic>subsp. <italic>alni</italic>, <italic>P. pseudosyringae </italic>or <italic>P. ilicis</italic>, whereas <italic>P. cambivora </italic>and <italic>P. cinnamomi </italic>isolates were polymorphic in all the tested primer combinations. As an example, the target region amplified with primers I9-I10 from <italic>P. cinnamomi </italic>was characterised by 12, 14 and 18 repeated motifs (AGG) in three tested isolates (Table <xref ref-type="table" rid="T6">6</xref>).</p><table-wrap position="float" id="T6"><label>Table 6</label><caption><p>Accession numbers and SSRs for selected microsatellites amplified and sequenced from two or more isolates of the same species to evaluate intraspecific variability.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold><italic>Phytophthora </italic>species</bold></td><td align="left"><bold><italic>Phytophthora </italic>isolates</bold></td><td align="left"><bold>Primers</bold></td><td align="left"><bold>SSRs</bold></td><td align="left"><bold>Accession</bold><break></break><bold>number</bold></td></tr></thead><tbody><tr><td align="left"><bold><italic>P. alni </italic>subsp. <italic>alni</italic></bold></td><td align="left">SCRP2</td><td align="left">S23F-S25R</td><td align="left">(agg)<sub>7</sub>; (aag)<sub>10</sub>; (agg)<sub>16</sub>; (aag)<sub>12</sub>; (aag)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216565">EF216565</ext-link></td></tr><tr><td></td><td align="left">SCRP4</td><td></td><td align="left">(agg)<sub>7</sub>; (aag)<sub>10</sub>; (agg)<sub>16</sub>; (aag)<sub>12</sub>; (aag)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216567">EF216567</ext-link></td></tr><tr><td></td><td align="left">SCRP8</td><td></td><td align="left">(agg)<sub>7</sub>; (aag)<sub>10</sub>; (agg)<sub>16</sub>; (aag)<sub>12</sub>; (aag)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216566">EF216566</ext-link></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td align="left"><bold><italic>P. cambivora</italic></bold></td><td align="left">SCRP67</td><td align="left">S23F-S25R</td><td align="left">(agg)<sub>10</sub>; (aag)<sub>10</sub>; (agg)<sub>9</sub>; (aag)<sub>12</sub>; (agg)<sub>4</sub>; (agg)<sub>7</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216569">EF216569</ext-link></td></tr><tr><td></td><td align="left">SCRP75</td><td></td><td align="left">(agg)<sub>9</sub>; (aag)<sub>10</sub>; (agg)<sub>11</sub>; (aag)<sub>12</sub>; (agg)<sub>6</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216571">EF216571</ext-link></td></tr><tr><td></td><td align="left">SCRP82</td><td></td><td align="left">(agg)<sub>10</sub>; (aag)<sub>10</sub>; (agg)<sub>9</sub>; (aag)<sub>12</sub>; (agg)<sub>5</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216570">EF216570</ext-link></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td align="left"><bold><italic>P. cambivora</italic></bold></td><td align="left">SCRP67</td><td align="left">S21F-S22R</td><td align="left">(agg)<sub>4</sub>; (aag)<sub>5</sub>; (aag)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216581">EF216581</ext-link></td></tr><tr><td></td><td align="left">SCRP80</td><td></td><td align="left">(agg)<sub>4</sub>; (aag)<sub>5</sub>; (aag)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216582">EF216582</ext-link></td></tr><tr><td></td><td align="left">SCRP82</td><td></td><td align="left">(aag)<sub>4</sub>; (agg)<sub>6</sub>; (agg)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216583">EF216583</ext-link></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td align="left"><bold><italic>P. cambivora</italic></bold></td><td align="left">SCRP67</td><td align="left">I9F-I10R</td><td align="left">(acg)<sub>4</sub>; (aag)<sub>4</sub>; (agg)<sub>6</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216516">EF216516</ext-link></td></tr><tr><td></td><td align="left">SCRP75</td><td></td><td align="left">(acg)<sub>4</sub>; (aag)<sub>4</sub>; (agg)<sub>6</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216519">EF216519</ext-link></td></tr><tr><td></td><td align="left">SCRP80</td><td></td><td align="left">(acg)<sub>4</sub>; (aag)<sub>4</sub>; (agg)<sub>6</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216517">EF216517</ext-link></td></tr><tr><td></td><td align="left">SCRP82</td><td></td><td align="left">(aag)<sub>5</sub>; (agg)<sub>5</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216518">EF216518</ext-link></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td align="left"><bold><italic>P. cinnamomi</italic></bold></td><td align="left">SCRP115</td><td align="left">I9F-I10R</td><td align="left">(aag)<sub>14</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216509">EF216509</ext-link></td></tr><tr><td></td><td align="left">SCRP118</td><td></td><td align="left">(aag)<sub>18</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216511">EF216511</ext-link></td></tr><tr><td></td><td align="left">SCRP121</td><td></td><td align="left">(aag)<sub>12</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216510">EF216510</ext-link></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td align="left"><bold><italic>P. pseudosyringae</italic></bold></td><td align="left">SCRP674</td><td align="left">I5F-I6R</td><td align="left">(agc)<sub>7</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216529">EF216529</ext-link></td></tr><tr><td></td><td align="left">SCRP734</td><td></td><td align="left">(agc)<sub>7</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216530">EF216530</ext-link></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td align="left"><bold><italic>P. ilicis</italic></bold></td><td align="left">SCRP377</td><td align="left">I5F-I6R</td><td align="left">(agc)<sub>9</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216532">EF216532</ext-link></td></tr><tr><td></td><td align="left">SCRP379</td><td></td><td align="left">(agc)<sub>9</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF216533">EF216533</ext-link></td></tr></tbody></table></table-wrap></sec></sec><sec><title>Approach 2 – Identification of SSRs in <italic>Phytophthora </italic>spp. clade 7</title><p>The aim of this approach was to focus the search for SSR loci to a restricted range of four clade 7 <italic>Phytophthora </italic>species (<italic>P. alni, P. cambivora, P. europaea and P. fragariae</italic>) phylogenetically related to <italic>P. sojae </italic>(Fig <xref ref-type="fig" rid="F1">1B</xref>) [<xref ref-type="bibr" rid="B9">9</xref>].</p><sec><title>Identification of target regions</title><p>This approach was based on a detailed list of SSRs identified in the genome of <italic>P. sojae </italic>and provided by Dr. Niklaus Grunwald at the Agricultural Research Service, U.S. Department of Agriculture, Corvallis, Oregon. Among the list, sixty genomic regions (500–1000 bp) were manually selected on the basis of having the longest SSRs in exons (20), introns (20) and non coding regions (20). The selected regions (Table <xref ref-type="table" rid="T7">7</xref>) were screened using BLAST against the entire genomes of <italic>P. ramorum </italic>and <italic>P. infestans </italic>to search for homology irrespective of the SSR regions. None of these regions aligned with sequences from the <italic>P. infestans </italic>genome whereas 18 of the 60 regions were sufficiently conserved to match homologous genes in <italic>P. ramorum </italic>(6 were localised in exons and 12 in introns). Surprisingly none of these 18 regions contained SSRs in <italic>P. ramorum</italic>, however it was hypothesised that microsatellites could be present in homologous regions of other <italic>Phytophthora </italic>species more closely related to <italic>P. sojae</italic>. To verify this hypothesis, thirty-six primers (18 pairs) were designed in the conserved flanking regions and used to amplify the target regions from <italic>P. alni, P. cambivora, P. europaea, P. fragariae </italic>and an SCRI isolate of <italic>P. sojae </italic>(Table <xref ref-type="table" rid="T7">7</xref>). Degenerate primers were designed when necessary.</p><table-wrap position="float" id="T7"><label>Table 7</label><caption><p>Set of primers designed with the 2<sup>nd </sup>approach (Fig. <xref ref-type="fig" rid="F1">1B</xref>) to amplify genomic regions potentially containing SSRs in <italic>Phytophthora </italic>species of clade 7 [<xref ref-type="bibr" rid="B2">2</xref>].</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold>Forward primer</bold></td><td align="left"><bold>Reverse Primer</bold></td><td align="left"><bold>SSRs</bold></td><td align="left"><bold>Source<sup>(a)</sup></bold></td><td align="left"><bold>Gene<sup>(b)</sup></bold></td></tr></thead><tbody><tr><td align="left">S38F<break></break>TCGTSTTCTACGTGCTGGAY</td><td align="left">S39R<break></break>GTAGCACGCGAACATGAASA</td><td align="left">(AAC)<sub>18</sub></td><td align="left">scaffold_26:<break></break>667079–667377</td><td align="left">E</td></tr><tr><td align="left">S40F<break></break>TTCCTTAAGTGGGGGAGGAT</td><td align="left">S41R<break></break>TRTCGGCRTTCAGCTTCTGT</td><td align="left">(AAG)<sub>4</sub>; (AAG)<sub>22</sub></td><td align="left">scaffold_125:<break></break>153837–154222</td><td align="left">I</td></tr><tr><td align="left">S42F<break></break>GCTGCAAGAGTCSCTCGAGTA</td><td align="left">S43R<break></break>CTTGAGGATGTCRATGAGCA</td><td align="left">(AG)<sub>21</sub></td><td align="left">scaffold_89:<break></break>132819–133248</td><td align="left">I</td></tr><tr><td align="left">S44F<break></break>GTRGCTCCTTCCTTAAGTGG</td><td align="left">S45R<break></break>GTGCTGCASGTAYGGCTTC</td><td align="left">(AAG)<sub>17</sub></td><td align="left">scaffold_75:<break></break>344500–344901</td><td align="left">I</td></tr><tr><td align="left">S46F<break></break>GTTGCGCGTGAGGTTCTC</td><td align="left">S47R<break></break>CAAAAGCTCTGCGTCC</td><td align="left">(AG)<sub>22</sub></td><td align="left">scaffold_67:<break></break>282390–282656</td><td align="left">I</td></tr><tr><td align="left">S48F<break></break>YCGGGCSACGGTAGG</td><td align="left">S49R<break></break>AAGAGCGTRAGCAGGAACC</td><td align="left">(AG)<sub>18</sub></td><td align="left">scaffold_65:<break></break>225236–225440</td><td align="left">I</td></tr><tr><td align="left">S50F<break></break>GTGGCTTCCACTGYTGCTG</td><td align="left">S51R<break></break>YATCAAGGACGTCAACTCGA</td><td align="left">(AAC)<sub>9</sub>; (AACAGC)<sub>23</sub></td><td align="left">scaffold_48:<break></break>118994–119610</td><td align="left">E</td></tr><tr><td align="left">S52F<break></break>CGGGATTTRTCRGATCAGG</td><td align="left">S53R<break></break>CTGTYTGATCARCTCTCCGCT</td><td align="left">(AGG)<sub>19</sub></td><td align="left">scaffold_46:<break></break>153312–153646</td><td align="left">E</td></tr><tr><td align="left">S56F<break></break>CACGAGCTGCAGKCATAYCT</td><td align="left">S57R<break></break>AGAATKGAMGCGATCGAC</td><td align="left">(AGG)<sub>16</sub></td><td align="left">scaffold_21:<break></break>370731–371140</td><td align="left">E</td></tr><tr><td align="left">S58F<break></break>TCGATCRACAGAAGCTGCWA</td><td align="left">S59R<break></break>GGAGTTCGCCATCAACAACT</td><td align="left">(AAG)<sub>14</sub></td><td align="left">scaffold_19:<break></break>606139–606624</td><td align="left">I</td></tr><tr><td align="left">S60F<break></break>GGCGTTTAAAGGCGTTTAAA</td><td align="left">S61R<break></break>CGTCTTCTTCTTGACGCACA</td><td align="left">(AC)<sub>18</sub></td><td align="left">scaffold_52:<break></break>422559–422915</td><td align="left">I</td></tr><tr><td align="left">S64F<break></break>YTTGCGACTAGCAAAGTGG</td><td align="left">S65R<break></break>CGAACTCCTTGTACAGGATGG</td><td align="left">(AG)<sub>14</sub></td><td align="left">scaffold_56:<break></break>179585–179895</td><td align="left">E</td></tr><tr><td align="left">S66F<break></break>GCAGYAGGCCCGGCCT</td><td align="left">S67R<break></break>GGAGTTCGCCATCAACAACT</td><td align="left">(AAG)<sub>11</sub></td><td align="left">scaffold_12:<break></break>130593–130967</td><td align="left">E</td></tr><tr><td align="left">S68F<break></break>CGTCGGTGGAGTAAACATCA</td><td align="left">S69R<break></break>AAAGGCGTTCGGAGAGYTG</td><td align="left">(AG)<sub>14</sub></td><td align="left">scaffold_66:<break></break>83968:84423</td><td align="left">I</td></tr><tr><td align="left">S70F<break></break>ATGACGAGGCAGCAGTTGAC</td><td align="left">S71R<break></break>AAGAACWGCGTSTACCTGCG</td><td align="left">(ATC)<sub>13</sub></td><td align="left">scaffold_2:<break></break>564977–565285</td><td align="left">I</td></tr><tr><td align="left">S72F<break></break>GCARCAATCTTCTGCTTYTTC</td><td align="left">S73R<break></break>ACACCTSCGTACWTTCGTCA</td><td align="left">(AAC)<sub>12</sub></td><td align="left">scaffold_92:<break></break>221631–221935</td><td align="left">I</td></tr><tr><td align="left">S74F<break></break>CGGTGGTACTTGTCGTCCTC</td><td align="left">S75R<break></break>TSTCCGGCTACATCATCATC</td><td align="left">(ATT)<sub>12</sub></td><td align="left">scaffold_41:<break></break>327977–328190</td><td align="left">I</td></tr><tr><td align="left">S76F<break></break>GCATCTACGACCAGATCTACCC</td><td align="left">S77R<break></break>GTAGACSGAGATGATGGCGT</td><td align="left">(AC)<sub>8</sub></td><td align="left">scaffold_127:<break></break>112530–112930</td><td align="left">I</td></tr></tbody></table><table-wrap-foot><p><sup>(a) </sup>= Gene sequences available at <ext-link ext-link-type="uri" xlink:href="http://genome.jgi-psf.org/sojae1/sojae1.home.html"></ext-link>.</p><p><sup>(b) </sup>E = exons; I = Introns</p></table-wrap-foot></table-wrap></sec><sec><title>Amplification, sequencing and SSR scoring</title><p>Most primer-species combinations produced single PCR bands of the expected size (Table <xref ref-type="table" rid="T8">8</xref>). Purification and direct sequencing of these PCR fragments produced 54 reliable sequences which were analysed as previously described for Approach 1. Twelve different microsatellites were identified: 2 in <italic>P. europaea</italic>, 3 in <italic>P. fragariae </italic>and <italic>P. alni </italic>and 4 in <italic>P. cambivora </italic>(Table <xref ref-type="table" rid="T8">8</xref>). Among these, 10 were trinucleotides and 2 were tetranucleotides repeated 4, 5 or 6 times. All regions sequenced from the SCRI isolate of <italic>P. sojae </italic>contained the predicted/expected SSR (Table <xref ref-type="table" rid="T8">8</xref>).</p><table-wrap position="float" id="T8"><label>Table 8</label><caption><p>Accession numbers and SSRs for GenBank deposited sequences amplified using primers designed with the second approach (Fig. <xref ref-type="fig" rid="F1">1B</xref>).</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold>Selected</bold><break></break><bold>primers<sup>(a)</sup></bold></td><td align="left" colspan="5"><bold><italic>Phytophthora </italic>species</bold></td></tr><tr><td></td><td colspan="5"><hr></hr></td></tr><tr><td></td><td align="left"><bold><italic>P. alni </italic>subsp. alni</bold><break></break>SCRP2</td><td align="left"><bold><italic>P. cambivora</italic></bold><break></break>SCRP67</td><td align="left"><bold><italic>P. europaea</italic></bold><break></break>SCRP622</td><td align="left"><bold><italic>P. fragariae</italic></bold><break></break>SCRP333</td><td align="left"><bold><italic>P. sojae</italic></bold><break></break>SCRP555</td></tr></thead><tbody><tr><td align="left"><bold>S38–39</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382833">EF382833</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382832">EF382832</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382831">EF382831</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382830">EF382830</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382829">EF382829</ext-link><break></break>(aac)<sub>18</sub></td></tr><tr><td align="left"><bold>S40–41</bold></td><td align="left">NS<sup>++</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382801">EF382801</ext-link><break></break>(aac)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382800">EF382800</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382799">EF382799</ext-link><break></break>(aac)<sub>4</sub></td><td align="left">NS<sup>++</sup></td></tr><tr><td align="left"><bold>S42–43</bold></td><td align="left">NS<sup>++</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382798">EF382798</ext-link><break></break>(ACG)<sub>6</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382797">EF382797</ext-link><break></break>(acg)<sub>6</sub>; (agg)<sub>5</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382796">EF382796</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382795">EF382795</ext-link><break></break>(ag)<sub>21</sub></td></tr><tr><td align="left"><bold>S44–45</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382793">EF382793</ext-link><break></break>(aac)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382792">EF382792</ext-link><break></break>(aac)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382794">EF382794</ext-link><break></break>No SSR</td><td align="left">NS<sup>++</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382791">EF382791</ext-link><break></break>(aag)<sub>5</sub>; (aag)<sub>5</sub></td></tr><tr><td align="left"><bold>S50–51</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382790">EF382790</ext-link><break></break>Any SSR</td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382789">EF382789</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382788">EF382788</ext-link><break></break>No SSR</td><td align="left">NS<sup>++</sup></td></tr><tr><td align="left"><bold>S52–53</bold></td><td align="left">NA<sup>+</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382786">EF382786</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382787">EF382787</ext-link><break></break>No SSR</td><td align="left">NS<sup>++</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382785">EF382785</ext-link><break></break>(agg)<sub>19</sub></td></tr><tr><td align="left"><bold>S58–59</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382784">EF382784</ext-link><break></break>(aac)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382783">EF382783</ext-link><break></break>(aac)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382782">EF382782</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382781">EF382781</ext-link><break></break>(aac)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382780">EF382780</ext-link><break></break>(aag)<sub>5</sub>; (aag)<sub>5</sub></td></tr><tr><td align="left"><bold>S64–65</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382828">EF382828</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382827">EF382827</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382826">EF382826</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382825">EF382825</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382824">EF382824</ext-link><break></break>(ag)<sub>14</sub></td></tr><tr><td align="left"><bold>S68–69</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382820">EF382820</ext-link><break></break>(aagg)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382821">EF382821</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382822">EF382822</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382831">EF382831</ext-link><break></break>(aac)<sub>4</sub>; (aagg)<sub>4</sub></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382819">EF382819</ext-link><break></break>(ag)<sub>14</sub></td></tr><tr><td align="left"><bold>S70–71</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382815">EF382815</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382816">EF382816</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382817">EF382817</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382818">EF382818</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382814">EF382814</ext-link><break></break>(act)<sub>13</sub></td></tr><tr><td align="left"><bold>S72–73</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382810">EF382810</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382811">EF382811</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382812">EF382812</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382813">EF382813</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382809">EF382809</ext-link><break></break>(aac)<sub>12</sub></td></tr><tr><td align="left"><bold>S74–75</bold></td><td align="left">NS<sup>++</sup></td><td align="left">NS<sup>++</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382807">EF382807</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382808">EF382808</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382806">EF382806</ext-link><break></break>(aat)<sub>12</sub></td></tr><tr><td align="left"><bold>S76–77</bold></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382802">EF382802</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382803">EF382803</ext-link><break></break>No SSR</td><td align="left">NS<sup>++</sup></td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382805">EF382805</ext-link><break></break>No SSR</td><td align="left"><ext-link ext-link-type="gen" xlink:href="EF382804">EF382804</ext-link><break></break>(ac)<sub>8</sub></td></tr></tbody></table><table-wrap-foot><p><sup>(a) </sup>= Primers listed in Table <xref ref-type="table" rid="T7">7</xref> and not reported in this table are those that did not produce any reliable sequence.</p><p>NA<sup>+ </sup>= Isolate-primer combinations that did not produce any amplification or produced complex profiles (two or more fragments).</p><p>NS<sup>++ </sup>= Isolate-primer combinations for which single PCR bands were obtained but direct sequencing did not produced reliable results.</p></table-wrap-foot></table-wrap></sec></sec></sec><sec><title>Discussion</title><p>The present study was undertaken to develop a method to rapidly identify loci containing SSRs and to create a pool of microsatellite markers for species of the genus <italic>Phytophthora </italic>taking advantage of publicly available sequences for <italic>P. sojae</italic>, <italic>P. ramorum </italic>and <italic>P. infestans</italic>. Recently, Garnica et al. explored the transferability of microsatellites across <italic>P. sojae</italic>, <italic>P. ramorum </italic>and <italic>P. infestans </italic>via an <italic>in silico </italic>virtual PCR approach [<xref ref-type="bibr" rid="B27">27</xref>]. In the present study, such an analysis on the same three species was conducted but also followed up with a comprehensive screening and validation process on multiple species to provide a practical evaluation of the procedure as a means of accelerating the search for new SSR markers in the genus <italic>Phytophthora</italic>.</p><p>The first approach was aimed at the identification of informative SSR loci common to many <italic>Phytophthora </italic>species. This approach was based on the hypothesis that among the large number of microsatellites distributed across the genome of species of the genus there may be a proportion in genes common to many species with sufficient sequence conservation in flanking regions to allow the design and use of universal SSR primers. Our search of the predicted gene sets yielded approximately 10% fewer SSRs and a corresponding lower abundance of SSRs per Mb of sequence than that of Garnica et al [<xref ref-type="bibr" rid="B27">27</xref>]. Preliminary analyses revealed a very limited number of loci containing SSRs that were common to the three <italic>Phytophthora </italic>species tested. The majority of the identified loci (81.6%) were common to <italic>P. sojae </italic>and <italic>P. ramorum </italic>only which is consistent with their closer phylogenetic relationship in clades 7 and 8 than to <italic>P. infestans </italic>in clade 1 [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>]. Similarly, Garnica et al. found in their <italic>in silico </italic>analysis that 7.5% of their primers were, in theory, transferable between at least two species (mainly <italic>P. ramorum </italic>and <italic>P. sojae</italic>) and only 1.0% transferable between the three species [<xref ref-type="bibr" rid="B27">27</xref>]. Among the selected sequences satisfying the above conditions, the number of repeats ranged from 3 to 17 and most SSRs showed seven repeats or less, with a repeat number of four being the most common in all species. The abundance of different repeat motifs differed slightly between species however, on average, (AAG)n, (AGG)n and (AGC)n were the most abundant triplets in all three Phytophthoras (Fig. <xref ref-type="fig" rid="F5">5</xref>). These results differ from those reported by Garnica et al. in which (AGC)n, (ACG)n and (AGG)n were the most abundant triplets amongst all the screened EST sequences [<xref ref-type="bibr" rid="B27">27</xref>]. It should, however be considered that unlike the study of Garnica our data are confined to SSR sequences for which it was possible to identify a homologue in at least one of the other two species. Therefore it could be hypothesised that motifs (AAG)n and (AGG)n are more abundant in more conserved genes. The dominance of trinucleotide SSRs compared to dinucleotide SSRs was not surprising considering that trinucleotides are abundant in coding regions of all higher eukaryotic genomes [<xref ref-type="bibr" rid="B31">31</xref>-<xref ref-type="bibr" rid="B33">33</xref>]. Dinucleotide repeats, in contrast, are characterised by higher mutation rates which may explain their abundance in introns and non-coding regions and lower frequency in coding regions, which cannot tolerate frame-shift mutations [<xref ref-type="bibr" rid="B34">34</xref>,<xref ref-type="bibr" rid="B35">35</xref>].</p><p>Primers designed in the present study with the first approach were tested against a panel of 16 different <italic>Phytophthora </italic>species representing the breadth of diversity across the genus to amplify <italic>P. sojae</italic>, <italic>P. ramorum </italic>and <italic>P. infestans </italic>target regions containing 33, 17 and 8 SSRs respectively. Overall, these primers enabled the sequencing of 171 target regions which contained 211 SSRs ranging in repeat number from 3 to 16. Most of these SSRs showed seven repeats or less with four the most common repeat number and (AAG)n, (AGG)n and (AGC)n the most common motifs. Trinucleotide repeats were dominant followed by pentanucleotide, tetranucleotide and dinucleotide repeats. This data indicate that such an approach can be useful to identify cross-specific SSR loci in the genus <italic>Phytophthora</italic>. As further genome sequences become available, for example, <italic>P. capsici </italic><ext-link ext-link-type="uri" xlink:href="http://www.jgi.doe.gov/sequencing/why/CSP2006/Pcapsici.html"></ext-link>, the process can be refined to specific subsets of the genus. The mutation rates and, consequently, the practical utility of the identified SSRs in the study of the specific <italic>Phytophthora </italic>species need to be examined further. Undoubtedly, a risk of this approach is that the selection is biased towards more conserved sequences which may subsequently have a lower mutation rate that reduces their utility as polymorphic markers. Furthermore, the fact that <italic>P. infestans </italic>SSRs were all identified using primers designed on its own genome (Fig. <xref ref-type="fig" rid="F3">3</xref>) may indicate that this approach is less appropriate for distant relatives considering that, as stated above, <italic>P. infestans </italic>is phylogenetically distant from <italic>P. sojae </italic>and <italic>P. ramorum</italic>. However, the identification of intraspecific polymorphisms in some selected SSRs is encouraging and demonstrates that at least some of the selected SSRs are valuable for immediate practical applications (Table <xref ref-type="table" rid="T6">6</xref>). This is consistent with the reported applicability of EST-SSRs across closely related taxa in other organisms as well as <italic>Phytophthora </italic>[<xref ref-type="bibr" rid="B23">23</xref>,<xref ref-type="bibr" rid="B36">36</xref>-<xref ref-type="bibr" rid="B38">38</xref>]. In the present study, the focus on the breadth of species (16) prevented the analyses of a wider number of target regions. However, the same method could be easily applied to the study of more regions from one or a few species.</p><p>The application of the first method enabled the identification of novel SSRs from all the 16 target species with those of clade 7 and 8 more highly represented (Fig. <xref ref-type="fig" rid="F3">3</xref>). A higher proportion of SSRs from species of the clade 7 and 8 was expected considering that <italic>P. sojae </italic>and <italic>P. ramorum </italic>belong to these two clades [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>]. In light of this fact, a second approach to identify a greater number of polymorphic SSRs from within a more limited range of clade 7 taxa more closely related to <italic>P. sojae </italic>was investigated. Sixty <italic>P. sojae </italic>SSR candidates were compared by BLAST analysis against the complete genome sequence of the other two species yielding 18 SSR candidates which could be aligned with homologous regions in <italic>P. ramorum</italic>. However in none of these 18 candidates (6 exons and 12 introns) was the SSR maintained in <italic>P. ramorum</italic>. In four of the more closely related species (<italic>P. alni, P. cambivora, P. europaea </italic>and <italic>P. fragariae</italic>), however, some of the SSR regions were conserved (Table <xref ref-type="table" rid="T8">8</xref>). In this study the focus was on discovery of SSRs in invasive forest <italic>Phytophthora </italic>species within the clade 7a, perhaps a higher success rate in marker discovery would have followed a search amongst the closest related species in clade 7b (<italic>P. sinensis</italic>, <italic>P. melonis</italic>, <italic>P. cajanae </italic>and <italic>P. vignae</italic>) [<xref ref-type="bibr" rid="B2">2</xref>]. Although a few SSR markers with potential were discovered using this approach, it was not a highly efficient means of identifying new polymorphic SSR loci and highlights the lack of conservation of SSR loci, even amongst coding regions within a single ITS clade of <italic>Phytophthora</italic>. Some degree of cross-species amplification has been observed between SSRs in <italic>P. infestans </italic>with other Clade 1c taxa and it is therefore likely that a wider application of this method concentrated on the closest relatives would be more productive [<xref ref-type="bibr" rid="B23">23</xref>].</p></sec><sec><title>Conclusion</title><p>The present study has tested two different methods to generate SSR markers that can be utilised across a broad range of <italic>Phytophthora </italic>species. The final number of identified loci for any single species may not be sufficient to run a complete population genetics analysis and key studies on the inter- and intraspecific variation remain. A comprehensive dataset of candidate SSRs from a range of species has been created (Table <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>, <xref ref-type="table" rid="T5">5</xref>). The detailed groundwork needed to amplify these regions from such a diverse collection of species and target regions has been completed which moves beyond the previous <italic>in silico </italic>approach to improve our understanding of the range and sequence conservation of SSR loci amongst species [<xref ref-type="bibr" rid="B27">27</xref>]. In general, the level of interspecific SSR sequence conservation, even amongst more closely related species within a single clade, was low and the method may not be the most efficient means of identifying novel SSR loci. Apart from their application as molecular markers, determining the abundance and density of SSRs in Oomycetes may help understand whether these sequences have any functional and evolutionary significance [<xref ref-type="bibr" rid="B17">17</xref>]. Furthermore, irrespective of the microsatellites, some of the amplified regions represent valuable marker regions for a number of applications [<xref ref-type="bibr" rid="B39">39</xref>]. A single optimal target gene for all <italic>Phytophthora </italic>species and assay requirements is unlikely to exist, therefore the continued identification and characterization of new target genes offers new opportunities for detection and phylogenetic studies [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B40">40</xref>,<xref ref-type="bibr" rid="B41">41</xref>].</p></sec><sec sec-type="methods"><title>Methods</title><sec><title><italic>Phytophthora </italic>isolates and DNA extractions</title><p>Twenty-eight isolates (16 <italic>Phytophthora </italic>species) sourced from the SCRI culture collection were used in this study (Table <xref ref-type="table" rid="T1">1</xref>). Isolates and species were selected to represent taxa most relevant to European forestry that also represented the breadth of <italic>Phytophthora </italic>diversity defined according to clades based on ITS sequence analysis [<xref ref-type="bibr" rid="B2">2</xref>]. Isolates were stored on oatmeal agar at 5°C and grown on French bean agar for routine stock cultures.</p><p>Total DNA was extracted from pure cultures of <italic>Phytophthora </italic>according to Schena and Cooke, diluted to 10 ng/μl and maintained at 5°C for routine amplifications and at -20°C for long term storage [<xref ref-type="bibr" rid="B42">42</xref>].</p></sec><sec><title>Analysis of sequences from <italic>P. infestans</italic>, <italic>P. sojae </italic>and <italic>P. ramorum </italic>genome projects and scanning for homologous SSRs</title><p>The predicted protein datasets of <italic>P. infestans </italic>(from the NCGR XGI database that was available prior to the Broad genome sequencing project) and <italic>P. ramorum </italic>and <italic>P. sojae </italic><ext-link ext-link-type="uri" xlink:href="http://genome.jgi-psf.org/"></ext-link> were screened for SSR loci using Sputnik (Chris Abaijan <ext-link ext-link-type="uri" xlink:href="http://espressosoftware.com/pages/sputnik.jsp"></ext-link>). Pairwise BLAST analysis using the default parameters was used to select loci conserved in different species combinations [<xref ref-type="bibr" rid="B43">43</xref>]. Manual screening of these loci on the basis of SSR and flanking region DNA sequence conservation yielded a short-list for further analysis.</p></sec><sec><title>Primer design and amplification conditions</title><p>All primers (Table <xref ref-type="table" rid="T2">2</xref> and <xref ref-type="table" rid="T7">7</xref>) were designed with the Primer3 Software set up to generate a Tm of 60°C ± 2, a GC% between 20 and 80% and a length of 18–26 bp [<xref ref-type="bibr" rid="B44">44</xref>]. Primers were purchased from Eurogentec ltd. (Belgium). Considerable effort was made to obtain successful amplification of single PCR bands from as many species as possible. This involved adjustment of MgCl<sub>2 </sub>concentration (0.7, 1.0 or 1.7 mM) and annealing temperatures (55 or 58°C) for PCR reactions (Table <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>, <xref ref-type="table" rid="T5">5</xref>). Furthermore in some circumstances alternative primers were designed and tested to amplify the target regions from as many taxa as possible (Table <xref ref-type="table" rid="T2">2</xref>). PCR reactions were performed in a total volume of 15 μl containing 10 ng of genomic DNA, 1.5 μl of 10× Reaction Buffer (Promega Corporation, WI, USA), 100 μM dNTPs, 0.7, 1 or 1.7 mM MgCl<sub>2</sub>, 15 μg BSA, 2 unit of <italic>Taq </italic>polymerase (Taq DNA polymerase, Promega Corporation) and 1 μM of primers. PCR amplification conditions consisted of: 1 cycle of 95°C for 2 min; 40 cycles of 94°C for 30 s, 55 or 58°C for 30 s, 72°C for 60 s; and a final cycle of 72°C for 5 min.</p></sec><sec><title>DNA sequencing</title><p>The best primers and amplification conditions were identified for all primer-species combinations and target DNA was re-amplified in a total volume of 50 μl to provide sufficient amplicon for direct sequencing. Single PCR bands were purified with the MinElute PCR Purification Kit (Qiagen Ltd. West Sussex, UK) to remove excess primers and nucleotides. Sequencing was carried out with the same primers utilized for the amplification in a dye-terminator cycle-sequencing reaction (FS sequencing kit, Applied Biosystems, Warrington, UK) and run on an ABI373 automated sequencer (Applied Biosystems). All selected PCR fragments were sequenced using both the forward and the reverse primers.</p></sec><sec><title>Sequence analysis and SSRs scanning</title><p>The "Sequence Navigator" software (Applied Biosystems) was utilised to evaluate reliability of sequences and to compare forward and reverse sequences to create a consensus sequence. Non-reliable sequences in which both forward and reverse sequences contained doubtful bases were discarded. All sequences obtained in the present study were also parsed to a web version of SPUTNIK <ext-link ext-link-type="uri" xlink:href="http://cbi.labri.fr/outils/Pise/sputnik.html"></ext-link>, which uses a recursive algorithm to search for repeated patterns of nucleotides of length between 2 and 5.</p></sec></sec><sec><title>Authors' contributions</title><p>LS conceived the study, carried out the molecular analyses, optimized the described method, performed data acquisition, data analysis and data interpretation and wrote the manuscript.</p><p>LM performed analysis of sequences from genome projects and scanned for homologous SSRs.</p><p>DELC coordinated, supervised and contributed to the design of the study, provided intellectual input to optimize the method and revised the manuscript critically.</p></sec></body><back><ack><sec><title>Acknowledgements</title><p>This work was funded by the European Union with a Marie Curie Intra-European Fellowship (Project: MEIFCT-2003-502327) and by the Scottish Government Rural and Environment Research and Analysis Directorate (RERAD). 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