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Amplification
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AMPLIFICATION PRIMERS
  • Forward primer - FM 75 (dCCTTGGCAATTAGGATTTCAAGAT)
    •  Located in the 5' end of the cox II gene (starting at base 55 of the gene in P. infestans, NC002387)
  • Reverse primers -.FM 77 (d CACCAATAAAGAATAACCAAAAATG) and FM 83 (dCTCCAATAAAAAATAACCAAAAATG)
    • Located in the 3' end of the cox I gene (starting at base 1261 of the gene in P. infestans, NC002387)
    • While FM 75 and FM 77 amplify most species, P. capsici, P. cinnamomi, P. citricola, and P. colocasiae were amplified better using FM 83 instead of FM 77
      • A mixture of both primers is generally used in the amplification.
  • Amplicon is approximately 2.2 kb in size depending on the species
    • the spacer between the cox I and II genes can be variable in length among species
  • Amplifies best with 3 mM MgCl2 and annealing temperature of 60?C
    • Amplification reactions were done in 50 ?l and contained approximately 20 to50 ng of DNA, a final concentration of 1 ?M of the forward and reverse primers, 5 ?l of 10X buffer, 100 ?M of each dNTP, 3 mM MgCl2, and 3 units of Taq DNA polymerase. 
    • Amplifications were done with a 3?C/s ramping time using the following parameters: one cycle at 95? C for 3 min; 35 cycles of 1 min annealing at 60?C, 1 min extension at 72? C, and 1 min denaturation at 94?C; followed by one extension cycle at 72?C for 10 min.   
    • More recent work has identified isolates that do not amplify well under these conditions.  While reducing the annealing temperature can alleviate this problem for some isolates, new primers are under evaluation to broaden the number of species that will be amplified and will be posted when the work has been completed.
  • 4 ?l of the amplification reaction was run on a 1.5% agarose gel to confirm the quality of the amplification and quantification of the amplicon.
  • These primers do not amplify bands from a range of different plant species, but they do amplify bands from the related genus Pythium

 

RESTRICTION ANALYSIS

  • Digestions with restriction enzymes were conducted overnight in a total volume of 17 ?l in accordance with the manufacturer's recommendations (New England Biolabs, Beverly, MA)
  • Digested fragments were separated in 3% NuSieve 3:1 agarose in 0.5X TBE buffer at 45 V for 6 h or until the bromophenol blue dye in the loading buffer had migrated 8 cm from the well. 
    • A 100-bp ladder mixed with a nondigested amplicon from P. infestans (isolate 580; approximately 2.2 kb based on sequence analysis) was used as size markers. 
  • The gel was stained in ethidium bromide (0.5 ?g/ml) for 30 min, destained in deionized water for 30 min, and photographed under short wave UV using either Polaroid Type 55 film or a digital imaging system .
  • Scanned digital images of the Polaroid negatives or the digital images were imported into the computer program BioNumerics (ver. 2.5, Applied Maths, Sint-Martens-Latem, Belgium).  This computer program automatically determines the molecular size of the RFLP bands relative to molecular size standards included on each agarose gel. 
    • Bands less than 100 bp in size were excluded from the analysis due to the diffuse nature of the bands when using a 3% NuSieve 3:1 agarose gel. 
  • The images were processed using standard procedures and molecular size determinations of digested bands done automatically with manual confirmation. 
    • To optimize band matching between isolates, the positional tolerance of each band (the maximum shift between two bands that is allowed to consider the bands matching, expressed as a percentage of the length of the gel run) and optimization of the data (this allows for a shift between any two patterns to optimize comparisons of banding patterns, expressed as a percentage of the run length) was determined from the data using the tolerance and optimization options of the program. 
      • Positional tolerances of 1.06%, 1.0%, 1.16%, and 1.0% were obtained for AluI, MspI, RsaI, and TaqI, respectively. 
      • Optimization was determined to be 0 except for RsaI, which was set at 0.54%. 

 

RESULTS

  • Digests with AluI alone could generate a species-specific diagnostic banding profiles able to differentiate most species evaluated in this investigation (see table below).
    •  A total of four restriction enzymes were used to increase the level of variation observed among species and improve the resolution of the technique (Fig. 1a, b, c for gel pictures or Fig. 2 for cluster analysis).

 Sizes of restriction fragments for Phytophthora spp. amplified with primers FM 75 and FM 77/83 and digested with the indicated restriction enzymea

 

Phytophthora sp.

Isolate #

AluI

MspI

RsaI

TaqI

P. arecae

441

648, 460, 238, 227, 203, 143, 117

2190

663, 550, 332, 300, 226, 182

2184

P. boehmeriae

325

565, 355, 315, 238, 227, 201, 179, 164

1305, 1016

621, 550, 332, 290, 182

1870, 437

P. cactorum

311

944, 327, 227, 201, 153, 132

1145

684, 550, 332, 300, 182, 148

2184

 

385

944, 327, 227, 201, 153, 132

1145, 564

684, 550, 332, 300, 182, 148

2184

P. capsici

302

697, 451, 227, 203, 179, 132

2190

712, 550, 332, 300, 226, 182

1573, 743

 

 

307

697, 451, 227, 203, 179, 132

1415, 901

712, 550, 332, 300, 226, 182

1573, 743

 

 

Cp-1

697, 451, 227, 203, 179, 132

1140, 1080

712, 550, 332, 300, 226, 182

1187, 834

P. cinnamomi

448

697, 421, 267, 202, 179, 132

1145, 755, 412

712, 586, 405, 332, 300

2004, 319

P. citricola

SB2078

697, 267, 227, 203, 179, 132

2190

783, 586, 332, 300, 182, 148

2184

 

 

Cr-4

697, 289, 267, 227, 203, 179, 132

2190

783, 586, 332, 300, 182, 148

2184

P. citrophthora

414

697, 267, 227, 203, 179, 132

2190

712, 550, 332, 300, 226, 182

2184

P. colocasiae

350

565, 354, 315, 203

1533, 532, 218

712, 550, 332, 290, 226, 182

1870, 457

P. cryptogea

438

697, 466, 298, 227, 179

1679, 564

712, 550, 332, 300, 226, 182

1619, 743

P. drechsleri

439

697, 298, 227, 201, 153, 143

1333, 564, 364

712, 550, 332, 300, 226, 182

2004, 373

P. erythroseptica

368

697, 466, 298, 227, 179

1679, 564

712, 550, 332, 300, 226, 182

1619, 743

 

365

697, 298, 267, 245, 227, 201

1679, 564

712, 550, 332, 300, 226, 182

1619, 743

P. fragariae              var. fragariae

393

665, 354, 327, 227, 203, 179, 153, 117

1145, 673, 490

712, 332, 290, 262, 226, 178, 125

2184

P. fragariae var. rubi

397

665, 421, 354, 227, 203, 179, 153, 117

1533, 603, 155

684, 332, 290, 262, 226, 178, 125

2184

P. gonapodyides

393

697, 466, 227, 203, 153

2101, 211

684, 550, 332, 300, 116

1278, 591, 437

P. heveae

Hv-2

665, 466, 298, 227, 179, 132, 117

2190

712, 550, 332, 300, 226, 182

1801, 284, 205

P. hibernalis

378

697, 466, 298, 227, 143

1533, 445, 327

621, 550, 332, 299, 182, 148

1120, 825, 373

P. ilicis

353

851, 354, 327, 227, 201, 143

1679, 673

663, 550, 332, 300, 182, 153

2004, 278

P. infestans

550

944, 327, 187, 153

2190

684, 351, 332, 300, 243, 226, 178

1503, 591, 205

P. lateralis

440

666, 267, 201, 179, 143, 117

1533, 755

621, 550, 332, 170, 134, 125

914, 743, 373, 205

P. megasperma

336

697, 298, 267, 227, 201, 164, 143

1969, 211, 122

712, 550, 332, 300, 226, 182

2184

 

309

697, 466, 354, 179, 143

1016, 564, 445, 327

684, 550, 332, 300, 182, 153

1870, 392

 

437

697, 466, 370, 164

1016, 755, 532

823, 550, 332, 300, 182

1278, 591, 392

 

335

697, 451, 298, 250, 209, 179, 132

1533, 532, 211

663, 550, 332, 300, 182

1870, 437

P. megakarya

327

697, 665, 209, 153

1071, 673, 564

663, 550, 405, 332, 300, 148

2004, 548, 205

 

328

648, 327, 201, 187, 153

1785, 532

663, 550, 332, 300, 226, 182

2004, 550, 205

P. mirabilis

354

944, 421, 201, 153

2190

684, 351, 332, 300, 226, 182

1703, 591

P. nemorosa

P-13

962, 356, 229, 202, 178, 149

2190

648, 553, 332, 226, 181

2184

P. nicotianae

361

648, 466, 315, 297, 203, 179, 153

2190

684, 550, 332, 226, 170, 125

2184

P. palmivora

329

648, 466, 238, 227, 201, 143, 117

1679, 564

663, 550, 332, 300, 226, 182

2184

 

Pl-14

648, 466, 238, 227,201, 143, 117

2190

663, 550, 332, 300, 226, 182

2184

P. phaseoli

373

944, 327, 315, 201

1305, 532, 490

684, 351, 332, 300, 243, 226, 182

1703, 591

P. pseudosyringae

470

944, 327, 250, 238, 209, 179

2190

823, 550, 332, 300, 182

2184

P. pseudotsugae

308

944, 354, 227, 201, 132

2190

684, 550, 332, 300, 226, 182

2184

P. sojae

312

697, 451, 298, 250, 203, 179, 132

2190

684, 550, 406, 332, 300

2184

P. ramorum

 

697, 466, 298, 187, 132

1305, 954

621, 550, 332, 226, 178, 125

2184

P. syringae

442

852, 315, 267, 227, 203

2190

1101, 550, 332, 300

1187, 1120

 

468

852, 315, 267, 227, 210, 152

2190

1101, 550, 332, 300

1187, 1019, 113

 

469

944, 327, 250, 209, 179

2190

762, 550, 332, 300

1187, 1120

 a Amplified products were digested with the indicated restriction enzyme and separated in a 3% NuSieve 3:1 agarose gel.  Fragment sizes were determined and the database managed using the computer program BioNumerics (ver. 2.5).  While fragment sizes are reported to the base pair by BioNumerics, this level of accuracy is artificial and not supported by the agarose electrophoresis method used for estimation.  Doublet bands and fragment sizes smaller than 100 bp are not reported or included in the cluster analysis.