Nematology 2005 Vol 7(2) 285-293

Multiple displacement amplification (MDA) of total genomicDNA from Meloidogyne spp and comparison to crude DNA

extracts in PCR of ITS1 28S D2-D3 rDNA and Hsp90Andrea M SKANTAR lowast and Lynn K CARTA

USDA-ARS Nematology Laboratory 10300 Baltimore Avenue Beltsville MD 20705 USA

Received 4 January 2005 revised 10 March 2005Accepted for publication 10 March 2005

Summary ndash Because the quantity of nematode specimens available for molecular analysis is often limited the number of analysespossible is constrained by the availability of DNA Multiple displacement amplification (MDA) was assessed for whole genomeamplification of crude DNA from several Meloidogyne species MDA produced microgram quantities of template that resulted insuccessful amplification of the ribosomal internal transcribed spacer (ITS1) and 28S D2-D3 expansion regions producing PCR resultsthat were comparable to template generated by the single nematode smash method MDA greatly improved degenerate primer PCR ofsingle-copy Hsp90 a gene which is more sensitive than multi-copy ribosomal genes to limited DNA template MDA should expand thenumber of molecular analyses possible for single nematodes MDA will also be useful for archiving DNA from valuable specimens andprovide a way for laboratories to share identical genetic material for nematode diagnosis

Keywords ndash diagnosis Meloidogyne arenaria M chitwoodi M floridensis M hapla M incognita M javanica molecularidentification phylogenetics

With international trade on the increase and the height-ened awareness of global pathogen spread there comesan urgent need for ways to monitor quickly and accu-rately the movement of nematodes of agricultural con-cern Root-knot nematodes (Meloidogyne species) attacka wide range of crops and are known to cause seriouseconomic damage throughout the world The absence ofaccurate diagnostic tests sensitive enough to distinguishroot-knot nematodes from one another hinders efforts tocontrol the spread of highly damaging or invasive species

Morphological identification of nematodes (see reviewby Eisenback and Triantaphyllou 1991) provides a criti-cal line of defence in preventing the introduction of newnematode pests but a great deal of skill and expertise is re-quired for effective interpretation of the subtle variationsthat often occur among individuals in a population Mole-cular methods useful for distinguishing Meloidogyne sppinclude isozyme analysis (Esbenshade amp Triantaphyllou1985) RAPDs (Cenis 1993 Baum et al 1994 Bloket al 1997a) PCR-RFLPs (Harris et al 1990 Powersamp Harris 1993 Hugall et al 1994) and sequence differ-

lowast Corresponding author e-mail skantarabaarsusdagov

ences within ribosomal (Zijlstra et al 1995 1997 Bloket al 1997b Wishart et al 2002 Chen et al 2003) ormitochondrial DNA (Blok et al 2002) Some moleculardiagnostic tests are straightforward to perform but limitedin capacity to discriminate species others are specific butmore time consuming to perform While new assays basedupon single copy genes would be welcome additions toplant-parasitic nematode diagnostics and phylogeny onlya few have been introduced including RNA polymerase II(Baldwin et al 1997) major sperm protein (MSP) (Set-terquist et al 1996) and Hsp70 (Beckenbach et al 1992Hashmi et al 1997) A technical barrier to such develop-ments is imposed by the small size of many plant-parasiticnematode juveniles which severely limits the amount ofgenetic material available for testing DNA extracted fromthe popular lsquosingle nematode smashrsquo procedure (Powersamp Harris 1993) is often consumed entirely by a singlePCR reaction preventing the replication of results or theability to perform more than one assay upon an individualUsing pools of nematodes alleviates the limit on availableDNA and allows PCR reactions to be replicated but con-

copy Koninklijke Brill NV Leiden 2005 285Also available online - wwwbrillnl

AM Skantar amp LK Carta

strains the ability to address questions involving intraspe-cific variation

Within the past decade a number of methods have beendeveloped for whole genome amplification (WGA) fromlimited amounts of starting material (reviewed in Laskenand Egholm 2003) These advances promise by produc-ing vast quantities of genomic DNA from minimal start-ing material to revolutionise the development of novelmolecular diagnostics for rare biological specimens Mul-tiple displacement amplification (MDA) is a non-PCRmethod developed by Dean et al (2001 2002) whichincludes the bacteriophage φ29 DNA polymerase andexonuclease-resistant thiophosphate-modified degener-ate hexamers to amplify genomic DNA from crude or puresources Unlike PCR methods that employ thermostableDNA polymerases φ29 DNA polymerase replicates DNAat a constant temperature of 30C The major advantagesattributed to MDA compared to PCR-based methods in-clude more complete genome coverage and unbiased am-plification (Dean et al 2002) The high fidelity and ac-curacy of MDA has been established in genotyping andSSCP mutation assays of human DNA samples that variedin quality and GC content (Yan et al 2004) Evaluationof single nucleotide polymorphisms (SNPs) performedon MDA from clinical specimens revealed 9982 com-plete genome representation and a direct sequencing errorrate similar to that of unamplified genomic DNA (Paezet al 2004) While MDA has recently been applied toother microbes (Gorrochotegui-Escalante amp Black 2003Jeyaprakash amp Hoy 2004) it has not yet been tested fornematodes

Recently we introduced Hsp90 as a new molecule fornematode phylogeny (Skantar amp Carta 2005) BecauseHsp90 is single copy in nematodes (Birnby et al 2000Skantar amp Carta 2005 Scholl amp Bird pers comm) suc-cessful amplification of this gene from uncharacterisedspecimens is highly dependent upon the quantity andquality of DNA template The aim of this study wasto facilitate Hsp90 amplification from a limited num-ber of specimens available for nematode molecular diag-nostics Our first objective was to assess the PCR per-formance of MDA DNA template relative to DNA ob-tained by a crude extraction method widely used in plant-parasitic nematology (Powers amp Harris 1993) The sec-ond objective was to employ MDA DNA for the im-provement of Hsp90 amplification from root-knot nema-todes

Materials and methods

NEMATODE ISOLATES

The present study consisted of seven root-knot nema-tode species that had previously been verified by morphol-ogy morphometrics and molecular characters The originof nematodes were Meloidogyne arenaria (North Car-olina State population 156) Eastern VA USA M incog-nita Salisbury MD USA M javanica Tifton GA USAM hapla Maui Hawaii M chitwoodi Parma ID USAM floridensis Gainesville FL USA and M mayaguen-sis Palm Beach County FL USA Groups of nine ne-matode juveniles were mechanically disrupted in 50 microltwo juveniles or single Meloidogyne chitwoodi femaleswere disrupted in 20 microl extraction buffer as described inThomas et al (1997) and then stored in PCR tubes atminus80C Extracts were prepared from thawed pools by in-cubating the tubes at 60C for 60 min followed by 95Cfor 15 min to deactivate the proteinase K Extracts werestored at minus20C when not in use

MULTIPLE DISPLACEMENT AMPLIFICATIONS

The Genomiphi (MDA) kit (Amersham PiscatawayNJ USA) was used for multiple displacement amplifica-tion of nematode DNA according to the manufacturerrsquosinstructions Genomiphi reactions were performed usingcrude extract (CE) from single or pooled nematodes as de-scribed above In each case reactions containing 1 microl ne-matode CE was combined with 9 microl sample buffer heatedto 95C for 3 min then chilled at 4C To each tube 9 microlof reaction buffer and 1 microl Genomiphi enzyme mix wereadded and the reactions incubated at 30C for 16 h Theamplified genomic DNA was ethanol precipitated accord-ing to the kit manufacturerrsquos recommendations and resus-pended in 20 microl 10 mM Tris pH 80 DNA preparationswere stored at minus20C and used for PCR reactions as de-scribed below

PCR REACTIONS

All PCR reactions contained Eppendorf HotMasterTaq(Brinkmann Westbury NY USA) and the buffer suppliedby the manufacturer all other components were added inthe proportions described in the protocols for each geneDNA templates for each experiment included 5 2 1 05or 01 microl CE or 2 1 05 01 or 001 microl MDA DNAwith volumes less than 1 microl generated by serial dilu-tion In addition no-template control reactions were rou-tinely performed All PCR primers were synthesised by

286 Nematology

MDA in Meloidogyne PCR

Sigma-Genosys (The Woodlands TX USA) The nuclearribosomal internal transcribed spacer (ITS1) segment wasamplified with the primers rDNA2 5prime-TTGATTACGTC-CCTGCCCTTT-3prime (Vrain et al 1992) and rDNA158S5prime-ACGAGCCGAGTGATCCACCG-3prime as described pre-viously (Cherry et al 1997) The ribosomal LSU D2-D3expansion segment was amplified with primers D2A5prime-ACAAGTACCGTGAGGGAAAGTTG-3prime and D3B5prime-TCGGAAGGAACCAGCTACTA-3prime (Courtright et al2000) as previously described (Al-Banna et al 1997)The Hsp90 primers M3F 5prime-GCGTCGYATTCGTGAA-GTGG-3prime and M2R 5prime-ATGGGCTTTGTCTTGTTMAR-CTCTT-3prime were based upon an alignment of root-knot ne-matode Hsp90 sequences (Skantar unpubl) Each 25 microlHsp90 PCR reaction contained 500 microM of primers M3Fand M2R 200 microM each dNTP plus 1 unit Taq poly-merase and the indicated amount of template DNAPCR reactions were incubated at 94C for 2 min followedby 45 cycles of 94C for 20 s 58C for 30 s and 68C for30 s with a final step of 68C for 5 min The second setof Hsp90 primers U831B 5prime-AAYAARACMAAGCCNA-TYTGGAC-3prime and L1110B 5prime-TCGCARTTCTCCATR-ATCAA-3prime were similar to primers described previously(Skantar amp Carta 2000) which were based upon an align-ment of Caenorhabditis elegans Brugia pahangi zebra-fish salmon mouse and human sequences Each 25 microlPCR reaction contained 500 microM of each of U831B andL1110B 200 microM dNTPs 1 unit Taq polymerase and theindicated amount of template DNA PCR reactions wereincubated using a modified ramped annealing PCR (Skan-tar amp Carta 2000) 94C for 2 min followed by 45 cyclesof 94C for 20 s 65C for 5 s 60C for 5 s 55C for5 s 50C for 5 s and 68C for 1 min and finishing witha 15 min step at 68C For all genes examined 125 microlof each PCR reaction was separated on 1 agarose gelsin Tris-Acetate-EDTA (TAE) stained with ethidium bro-mide and visualised with UV illumination

Results

The initial goal was to assess the PCR performance ofMDA DNA compared to DNA prepared by the lsquonematodesmashrsquo method that is commonly used for identificationand phylogenetic studies Successful amplification ofHsp90 is more sensitive to the amount of input genomicDNA than amplification of the ribosomal genes whichare present in the genome in multiple copies Thereforeit was necessary to use extracts from pools of nematodesin order to raise the level of Hsp90 target template so

Fig 1 Size of typical multiple displacement amplificationproducts from nematodes The 1 kb ladder (MW) was from NewEngland Biolabs (Beverly MA USA) and includes fragments of10 8 6 5 4 3 2 15 10 and 05 kb MDA reactions contained1 microl (150th volume) of a crude extract from a pool of ninenematodes Each MDA lane contained 5 microl out of the 20 microlMDA reaction mixed with 1 microl electrophoresis sample bufferMDA products ranged in size from approximately 05 kb to 12 kbMf = Meloidogyne floridensis Mh = Meloidogyne hapla

that side-by-side comparisons CE and MDA PCR resultscould be performed To simplify the overall analysisand comparisons of results with different genes pooledextracts of nine J2 nematodes were employed for all ofthe genes tested

One microl of the pooled extract was subjected to multipledisplacement amplification and then purified by ethanolprecipitation MDA typically produced microgram quan-tities of genomic DNA ranging in size from 05-12 kb(Fig 1) A wide range of template concentrations was ex-amined in order to compare the quantity and quality ofPCR products produced CE DNA was tested over a 50-fold range of DNA concentrations and MDA DNA wastested over a 200-fold range (Fig 2) For M arenariaM incognita and M javanica the expected ca 750 bpITS1 PCR products were observed at or above 05 microl CEtemplate and at all levels of MDA DNA For M hapla1-5 microl CE gave detectable PCR products but lower levelsdid not The M hapla MDA reactions gave rise to the ex-pected product at all DNA amounts through 01 microl exceptfor the lowest amount 001 microl However two other largerproducts were also observed

Another experiment using the same template prepara-tions involved amplification of the LSU D2-D3 expansionsegments of rDNA (Fig 3) For M arenaria M incog-nita and M javanica the expected ca 800 bp product wasgenerated at most CE levels except the lowest although

Vol 7(2) 2005 287

AM Skantar amp LK Carta

Fig 2 Comparison of ribosomal ITS1 region amplified from four different root-knot nematodes DNA templates were either crudeextracts (CE) from pools of nine nematodes or MDA DNA generated from 1 microl (150th volume) of the same pooled CE Agarosegel electrophoresis of PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbers belowrepresent the amount of template DNA in microl added to 25 microl PCR reactions Arrows denote position of expected products M = 100 bpladder (New England Biolabs)

Fig 3 Comparison of ribosomal 28S D2-D3 expansion segments amplified from four different root-knot nematodes DNA templateswere either crude extracts (CE) from pools of nine nematodes or MDA DNA generated from 1 microl (150th volume) of the same pooled CEAgarose gel electrophoresis of PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbersbelow represent the amount of template DNA added to 25 microl PCR reactions Arrows denote position of expected PCR products M =100 bp ladder with fragment sizes 1517 1200 1000 900 800 700 600 517500 (comigrating bands) 400 300 200 and 100 bp(New England Biolabs)

the reactions at each DNA concentration did not generateas much D2-D3 product as ITS1 product The MDA reac-tions were stronger over the range of DNA concentrationstested than those containing CE For M hapla the CE re-actions failed to generate any products The major MDAD2-D3 PCR product was smaller than for the other root-knot nematodes (ca 650 bp) an additional band at ca 400bp was also observed

A similar side-by-side comparison of CE and MDAdilution series using the U831L1110 primer pair wasnot possible due to the poor amplification of Hsp90 withless than 10 microl CE (not shown) Thus the root-knotnematode-specific Hsp90 primers M3F and M2R wereused for this purpose (Fig 4) With these primers CEtemplate from most of the root-knot nematodes showedstrong amplification of the expected ca 300 bp Hsp90

288 Nematology

MDA in Meloidogyne PCR

Fig 4 Schematic diagram of Hsp90 with positions of PCR primers M3F M2R U831B and L1110B marked by arrows The conservedamino acid domains corresponding to the primer sequences are indicated in parentheses

Fig 5 Comparison of Hsp90 amplified from four different root-knot nematodes PCR reactions of 25 microl included primers M3F and M2Rand DNA template from either crude nematode extracts (CE) or multiple displacement amplification (MDA) Agarose gel electrophoresisof PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbers below represent the amountof template DNA added to 25 microl PCR reactions Arrows denote position of expected PCR products M = 100 bp ladder (New EnglandBiolabs)

PCR products However the M hapla CE reactions wererelatively weaker than the other species reactions at lowlevels of DNA (Fig 5) MDA DNA from all four speciesproduced strong PCR bands over the entire 200-fold rangeof template DNA added

The next experiment involved an examination of Hsp90fragments produced with MDA DNA and the U831B andL1110B primers MDA DNA was generated from CE pre-pared from one two or nine J2 of several root-knot nema-tode species The number of nematodes in a given speciespool was purely incidental reflecting the sometimes lim-ited material obtained as interceptions Meloidogyne are-naria M incognita M javanica and M hapla were allincluded because of their worldwide distribution and im-portance to agriculture Three other species of regulatoryimportance M mayaguensis M floridensis and M chit-woodi were also tested Strong Hsp90 amplification wasobserved in all of the MDA reactions (Fig 6) Two Hsp90PCR products of slightly different size were detected for

M incognita but single bands were visible for all otherspecies with small differences in migration distance ap-parent for some of them

Discussion

We have demonstrated that MDA is an effective methodof WGA for several species of root-knot nematode whichfurther suggests that this method will have broad applica-bility in nematode molecular systematics and identifica-tion MDA produced robust amounts of genomic DNA re-gardless of the number of nematodes in the crude extractand worked equally well with mechanically disrupted ju-veniles and females MDA from single M chitwoodi fe-males produced micrograms of genomic DNA from as lit-tle as 5 of the crude extracts and similar yields were ob-tained from pools of two to nine juveniles Because suc-cessful PCR was achieved with as low as 001 microl MDADNA we estimate that crude extracts subjected to MDA

Vol 7(2) 2005 289

AM Skantar amp LK Carta

Fig 6 Agarose gel electrophoresis of Hsp90 PCR productsproduced with degenerate primers U831B and L1110B andMDA DNA generated from 1 microl of lsquonematode smashrsquo crudeextracts Extracts from Ma Mi Mj and Mh were prepared frompools of nine J2 Mm and Mh were from pools of two J2Mc was from a single female Ma = Meloidogyne arenariaMi = M incognita Mj = M javanica Mh = M hapla Mm= M mayaguensis Mf = M floridensis Mc = M chitwoodiMW = 100 bp ladder (New England Biolabs)

can produce sufficient template for as many as 10 000 to40 000 PCR reactions depending upon the number sizeand stage of nematodes in the starting material and thegene being amplified We did not attempt MDA with lessthan 1 microl CE but it is possible that using lower amountsof input CE could extend this estimate even further Thepresence of non-DNA contaminants within crude nema-tode extracts prevented accurate measurements of theirDNA concentrations so it was not possible to directlyequate concentrations of CE and MDA genomic DNANevertheless these experiments provide a practical com-parison of PCR results using MDA-produced genomicDNA and the nematode CE volumes typically employed

The ribosomal internal transcribed spacer ITS1 and theLSU D2-D3 expansion regions are two readily amplifiedribosomal genes that have been previously examinedfrom root-knot nematodes (Powers amp Harris 1993 Chenet al 2003) thus providing an established experimentalcontext in which to evaluate template produced by MDAThe sizes of ITS1 PCR products amplified from MDADNA were consistent with those reported previously(Powers et al 1997) For most root-knot species bothCE and MDA templates produced strong bands at allDNA concentrations tested (Fig 2) As low as 001 microlMDA DNA produced strong ITS1 products that werecomparable to or greater than the signals resulting from05 microl nematode CE The exception was M hapla whichproduced a relatively strong ITS1 signal from 5 microl CE

DNA that gradually disappeared at lower levels of CEWhile high levels of MDA DNA from M hapla producedstrong bands at the expected size (ca 450 bp) loweramounts of template produced additional bands at ca 600and ca 800 bp The identity of these products is unclearthey could be an artefact of MDA amplification resultfrom mispriming in the rDNA or may indicate that thecrude DNA preparation used for MDA of M hapla was oflesser quality than for the other nematodes

The results from the D2-D3 comparison generally gavesimilar results to the ITS1 although the signal strengths ofthe D2-D3 products were lower overall (Fig 3) Some ofthe CE PCR reactions were higher or lower than expectedfor serial dilutions of the DNA It is likely these datapoints resulted from pipetting errors (Fig 3 M incognitaand M javanica 1 microl CE) Again the M hapla CE andMDA results were not as robust as those from the othernematodes CE reactions gave no products and MDAtemplates produced a smaller band in addition to the ca600 bp D2-D3 band These reactions were performed withthe same CE and MDA DNA as for ITS1 indicatingthat both genes in M hapla were affected similarly Aswith any method involving DNA polymerase activity thatreplicates input DNA without bias MDA results can beaffected by contamination Therefore nematodes used forMDA should be prepared with care to ensure they are freefrom microbial contamination The precise reason for thelower quality PCR results with M hapla is not clear butas a cautionary measure fresh nematode CE and MDADNA were prepared for the next set of experiments

The M3FM2R Hsp90 reactions were successful for allconcentrations of CE and MDA for all nematodes (Fig 5)with the exception of the lowest CE levels for M haplaand the 05 microl CE point for M javanica M3F (two-fold degenerate) and M2R (four-fold) were designed toamplify Hsp90 within the genus Meloidogyne with theinitial aim of developing this fragment as a diagnosticmarker While the PCR procedure was straightforwardwe discovered no apparent size variation in this fragmentamong the root-knot nematodes (Fig 5) and a low level ofsequence variation among the mitotic parthenogens (datanot shown) thus limiting its diagnostic potential for thegenus However increasing the degeneracy of primersM3F and M2R may yet prove useful for examiningvariation across a broader range of nematode taxa

The U831BL1110B Hsp90 fragment was of interestbecause significant sequence variation in this region hadbeen previously detected among several root-knot nema-tode species (Skantar amp Carta 2005) This variability is

290 Nematology

MDA in Meloidogyne PCR

apparent in the slightly different sizes of PCR products(Fig 6) such as the slower-migrating band for M incog-nita and the slightly faster migrating bands for M are-naria and M hapla U831B and L1110B are less degen-erate modifications (24-fold and four-fold respectively)of a previous primer set (Skantar amp Carta 2000) How-ever even with increased specificity these primers some-times required two cycles of amplification to producestrong PCR products from the extracts of single nema-todes (Skantar amp Carta 2005) The benefit of MDA foramplification of Hsp90 with these degenerate primers wasreflected in the strong products obtained from a singleround of PCR It is likely that adding more genomic DNAincreased the number of productive interactions betweenthe degenerate primers and the target gene leading tohigher product yield Raising the level of crude nema-tode extracts in Hsp90 PCR reactions generally does notachieve the same advantage (data not shown) as high lev-els of impure extract may contribute an excess of PCR-inhibiting contaminants While genomic DNA prepara-tions from bulk quantities of nematodes also give strongHsp90 PCR products (data not shown) preparing themrequires significantly more nematodes time and labourthan MDA

The technical improvements shown here should signifi-cantly broaden the appeal of Hsp90 and open the door foranalysis of other single-copy genes for nematode identifi-cation and phylogenetics The combination of MDA anddegenerate primers will facilitate future surveys of Hsp90across the phylum Nematoda On a finer scale with MDAit will be possible to address questions about Hsp90 al-lelic variation within and among individuals in a popula-tion Whether Hsp90 sequences can be used to developreliable diagnostics for the major root-knot nematodes orfor species of regulatory importance remains to be vali-dated through the examination of multiple populations ofthese species

The potential applications of MDA to nematode iden-tification and systematics are far reaching For instancewith MDA it should be possible to archive the geneticmaterial from individual nematodes thereby eliminatingthe need for more labour-intensive culture methods suchas propagating single egg-mass cultures (Hugall et al1999) MDA could facilitate the development and produc-tion of DNA lsquotype specimensrsquo that may be shared amongscientists Thus it would be possible to generate a set ofDNA standards that could be widely distributed provid-ing a practical tool for greater uniformity in nematodemolecular diagnostics Another possible advance result-

ing from MDA is the ability to archive large quantitiesof genetic material from rare difficult-to-maintain or his-torically valuable specimens It will now be feasible toperform morphological analysis on a specimen catalogueits unique features in a digital archive (De Ley amp Bert2002) and with MDA produce a corresponding geneticarchive Also MDA provides an attractive alternative tothe international or intrastate exchange of live specimensa practice that presents a number of regulatory challengesand restrictions

Acknowledgements

We thank Maria Hult Sharon Ochs and Donna Elling-ton for technical assistance We thank the following peo-ple for providing nematode isolates Saad Hafez M chit-woodi Janete Brito M mayaguensis Andy NyzcepirM floridensis Don Schmitt M hapla

Mention of trade names or commercial products inthis publication is solely for the purpose of providingspecific information and does not imply recommendationor endorsement by the US Department of Agriculture

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmental evolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

BAUM TJ GRESSHOFF PM LEWIS SA amp DEAN RA(1994) Characterization and phylogenetic analysis of fourroot-knot nematode species using DNA amplification finger-printing and automated polyacrylamide gel electrophoresisMolecular Plant-Microbe Interactions 7 39-47

BECKENBACH K SMITH MJ amp WEBSTER JM (1992)Taxonomic affinities and intra- and interspecific variation inBursaphelenchus spp as determined by polymerase chainreaction Journal of Nematology 24 140-147

BIRNBY DA LINK EM VOWELS JJ TIAN H CO-LACURCIO PL amp THOMAS JH (2000) A transmembraneguanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate acommon set of chemosensory behaviors in Caenorhabditiselegans Genetics 155 85-104

BLOK VC PHILLIPS MS MCNICOL JW amp FARGETTEM (1997a) Genetic variation in tropical Meloidogyne spp

Vol 7(2) 2005 291

MDA in Meloidogyne PCR

Sigma-Genosys (The Woodlands TX USA) The nuclearribosomal internal transcribed spacer (ITS1) segment wasamplified with the primers rDNA2 5prime-TTGATTACGTC-CCTGCCCTTT-3prime (Vrain et al 1992) and rDNA158S5prime-ACGAGCCGAGTGATCCACCG-3prime as described pre-viously (Cherry et al 1997) The ribosomal LSU D2-D3expansion segment was amplified with primers D2A5prime-ACAAGTACCGTGAGGGAAAGTTG-3prime and D3B5prime-TCGGAAGGAACCAGCTACTA-3prime (Courtright et al2000) as previously described (Al-Banna et al 1997)The Hsp90 primers M3F 5prime-GCGTCGYATTCGTGAA-GTGG-3prime and M2R 5prime-ATGGGCTTTGTCTTGTTMAR-CTCTT-3prime were based upon an alignment of root-knot ne-matode Hsp90 sequences (Skantar unpubl) Each 25 microlHsp90 PCR reaction contained 500 microM of primers M3Fand M2R 200 microM each dNTP plus 1 unit Taq poly-merase and the indicated amount of template DNAPCR reactions were incubated at 94C for 2 min followedby 45 cycles of 94C for 20 s 58C for 30 s and 68C for30 s with a final step of 68C for 5 min The second setof Hsp90 primers U831B 5prime-AAYAARACMAAGCCNA-TYTGGAC-3prime and L1110B 5prime-TCGCARTTCTCCATR-ATCAA-3prime were similar to primers described previously(Skantar amp Carta 2000) which were based upon an align-ment of Caenorhabditis elegans Brugia pahangi zebra-fish salmon mouse and human sequences Each 25 microlPCR reaction contained 500 microM of each of U831B andL1110B 200 microM dNTPs 1 unit Taq polymerase and theindicated amount of template DNA PCR reactions wereincubated using a modified ramped annealing PCR (Skan-tar amp Carta 2000) 94C for 2 min followed by 45 cyclesof 94C for 20 s 65C for 5 s 60C for 5 s 55C for5 s 50C for 5 s and 68C for 1 min and finishing witha 15 min step at 68C For all genes examined 125 microlof each PCR reaction was separated on 1 agarose gelsin Tris-Acetate-EDTA (TAE) stained with ethidium bro-mide and visualised with UV illumination

Results

The initial goal was to assess the PCR performance ofMDA DNA compared to DNA prepared by the lsquonematodesmashrsquo method that is commonly used for identificationand phylogenetic studies Successful amplification ofHsp90 is more sensitive to the amount of input genomicDNA than amplification of the ribosomal genes whichare present in the genome in multiple copies Thereforeit was necessary to use extracts from pools of nematodesin order to raise the level of Hsp90 target template so

Fig 1 Size of typical multiple displacement amplificationproducts from nematodes The 1 kb ladder (MW) was from NewEngland Biolabs (Beverly MA USA) and includes fragments of10 8 6 5 4 3 2 15 10 and 05 kb MDA reactions contained1 microl (150th volume) of a crude extract from a pool of ninenematodes Each MDA lane contained 5 microl out of the 20 microlMDA reaction mixed with 1 microl electrophoresis sample bufferMDA products ranged in size from approximately 05 kb to 12 kbMf = Meloidogyne floridensis Mh = Meloidogyne hapla

that side-by-side comparisons CE and MDA PCR resultscould be performed To simplify the overall analysisand comparisons of results with different genes pooledextracts of nine J2 nematodes were employed for all ofthe genes tested

One microl of the pooled extract was subjected to multipledisplacement amplification and then purified by ethanolprecipitation MDA typically produced microgram quan-tities of genomic DNA ranging in size from 05-12 kb(Fig 1) A wide range of template concentrations was ex-amined in order to compare the quantity and quality ofPCR products produced CE DNA was tested over a 50-fold range of DNA concentrations and MDA DNA wastested over a 200-fold range (Fig 2) For M arenariaM incognita and M javanica the expected ca 750 bpITS1 PCR products were observed at or above 05 microl CEtemplate and at all levels of MDA DNA For M hapla1-5 microl CE gave detectable PCR products but lower levelsdid not The M hapla MDA reactions gave rise to the ex-pected product at all DNA amounts through 01 microl exceptfor the lowest amount 001 microl However two other largerproducts were also observed

Another experiment using the same template prepara-tions involved amplification of the LSU D2-D3 expansionsegments of rDNA (Fig 3) For M arenaria M incog-nita and M javanica the expected ca 800 bp product wasgenerated at most CE levels except the lowest although

Vol 7(2) 2005 287

AM Skantar amp LK Carta

Fig 2 Comparison of ribosomal ITS1 region amplified from four different root-knot nematodes DNA templates were either crudeextracts (CE) from pools of nine nematodes or MDA DNA generated from 1 microl (150th volume) of the same pooled CE Agarosegel electrophoresis of PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbers belowrepresent the amount of template DNA in microl added to 25 microl PCR reactions Arrows denote position of expected products M = 100 bpladder (New England Biolabs)

Fig 3 Comparison of ribosomal 28S D2-D3 expansion segments amplified from four different root-knot nematodes DNA templateswere either crude extracts (CE) from pools of nine nematodes or MDA DNA generated from 1 microl (150th volume) of the same pooled CEAgarose gel electrophoresis of PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbersbelow represent the amount of template DNA added to 25 microl PCR reactions Arrows denote position of expected PCR products M =100 bp ladder with fragment sizes 1517 1200 1000 900 800 700 600 517500 (comigrating bands) 400 300 200 and 100 bp(New England Biolabs)

the reactions at each DNA concentration did not generateas much D2-D3 product as ITS1 product The MDA reac-tions were stronger over the range of DNA concentrationstested than those containing CE For M hapla the CE re-actions failed to generate any products The major MDAD2-D3 PCR product was smaller than for the other root-knot nematodes (ca 650 bp) an additional band at ca 400bp was also observed

A similar side-by-side comparison of CE and MDAdilution series using the U831L1110 primer pair wasnot possible due to the poor amplification of Hsp90 withless than 10 microl CE (not shown) Thus the root-knotnematode-specific Hsp90 primers M3F and M2R wereused for this purpose (Fig 4) With these primers CEtemplate from most of the root-knot nematodes showedstrong amplification of the expected ca 300 bp Hsp90

288 Nematology

MDA in Meloidogyne PCR

Fig 4 Schematic diagram of Hsp90 with positions of PCR primers M3F M2R U831B and L1110B marked by arrows The conservedamino acid domains corresponding to the primer sequences are indicated in parentheses

Fig 5 Comparison of Hsp90 amplified from four different root-knot nematodes PCR reactions of 25 microl included primers M3F and M2Rand DNA template from either crude nematode extracts (CE) or multiple displacement amplification (MDA) Agarose gel electrophoresisof PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbers below represent the amountof template DNA added to 25 microl PCR reactions Arrows denote position of expected PCR products M = 100 bp ladder (New EnglandBiolabs)

PCR products However the M hapla CE reactions wererelatively weaker than the other species reactions at lowlevels of DNA (Fig 5) MDA DNA from all four speciesproduced strong PCR bands over the entire 200-fold rangeof template DNA added

The next experiment involved an examination of Hsp90fragments produced with MDA DNA and the U831B andL1110B primers MDA DNA was generated from CE pre-pared from one two or nine J2 of several root-knot nema-tode species The number of nematodes in a given speciespool was purely incidental reflecting the sometimes lim-ited material obtained as interceptions Meloidogyne are-naria M incognita M javanica and M hapla were allincluded because of their worldwide distribution and im-portance to agriculture Three other species of regulatoryimportance M mayaguensis M floridensis and M chit-woodi were also tested Strong Hsp90 amplification wasobserved in all of the MDA reactions (Fig 6) Two Hsp90PCR products of slightly different size were detected for

M incognita but single bands were visible for all otherspecies with small differences in migration distance ap-parent for some of them

Discussion

We have demonstrated that MDA is an effective methodof WGA for several species of root-knot nematode whichfurther suggests that this method will have broad applica-bility in nematode molecular systematics and identifica-tion MDA produced robust amounts of genomic DNA re-gardless of the number of nematodes in the crude extractand worked equally well with mechanically disrupted ju-veniles and females MDA from single M chitwoodi fe-males produced micrograms of genomic DNA from as lit-tle as 5 of the crude extracts and similar yields were ob-tained from pools of two to nine juveniles Because suc-cessful PCR was achieved with as low as 001 microl MDADNA we estimate that crude extracts subjected to MDA

Vol 7(2) 2005 289

AM Skantar amp LK Carta

Fig 6 Agarose gel electrophoresis of Hsp90 PCR productsproduced with degenerate primers U831B and L1110B andMDA DNA generated from 1 microl of lsquonematode smashrsquo crudeextracts Extracts from Ma Mi Mj and Mh were prepared frompools of nine J2 Mm and Mh were from pools of two J2Mc was from a single female Ma = Meloidogyne arenariaMi = M incognita Mj = M javanica Mh = M hapla Mm= M mayaguensis Mf = M floridensis Mc = M chitwoodiMW = 100 bp ladder (New England Biolabs)

can produce sufficient template for as many as 10 000 to40 000 PCR reactions depending upon the number sizeand stage of nematodes in the starting material and thegene being amplified We did not attempt MDA with lessthan 1 microl CE but it is possible that using lower amountsof input CE could extend this estimate even further Thepresence of non-DNA contaminants within crude nema-tode extracts prevented accurate measurements of theirDNA concentrations so it was not possible to directlyequate concentrations of CE and MDA genomic DNANevertheless these experiments provide a practical com-parison of PCR results using MDA-produced genomicDNA and the nematode CE volumes typically employed

The ribosomal internal transcribed spacer ITS1 and theLSU D2-D3 expansion regions are two readily amplifiedribosomal genes that have been previously examinedfrom root-knot nematodes (Powers amp Harris 1993 Chenet al 2003) thus providing an established experimentalcontext in which to evaluate template produced by MDAThe sizes of ITS1 PCR products amplified from MDADNA were consistent with those reported previously(Powers et al 1997) For most root-knot species bothCE and MDA templates produced strong bands at allDNA concentrations tested (Fig 2) As low as 001 microlMDA DNA produced strong ITS1 products that werecomparable to or greater than the signals resulting from05 microl nematode CE The exception was M hapla whichproduced a relatively strong ITS1 signal from 5 microl CE

DNA that gradually disappeared at lower levels of CEWhile high levels of MDA DNA from M hapla producedstrong bands at the expected size (ca 450 bp) loweramounts of template produced additional bands at ca 600and ca 800 bp The identity of these products is unclearthey could be an artefact of MDA amplification resultfrom mispriming in the rDNA or may indicate that thecrude DNA preparation used for MDA of M hapla was oflesser quality than for the other nematodes

The results from the D2-D3 comparison generally gavesimilar results to the ITS1 although the signal strengths ofthe D2-D3 products were lower overall (Fig 3) Some ofthe CE PCR reactions were higher or lower than expectedfor serial dilutions of the DNA It is likely these datapoints resulted from pipetting errors (Fig 3 M incognitaand M javanica 1 microl CE) Again the M hapla CE andMDA results were not as robust as those from the othernematodes CE reactions gave no products and MDAtemplates produced a smaller band in addition to the ca600 bp D2-D3 band These reactions were performed withthe same CE and MDA DNA as for ITS1 indicatingthat both genes in M hapla were affected similarly Aswith any method involving DNA polymerase activity thatreplicates input DNA without bias MDA results can beaffected by contamination Therefore nematodes used forMDA should be prepared with care to ensure they are freefrom microbial contamination The precise reason for thelower quality PCR results with M hapla is not clear butas a cautionary measure fresh nematode CE and MDADNA were prepared for the next set of experiments

The M3FM2R Hsp90 reactions were successful for allconcentrations of CE and MDA for all nematodes (Fig 5)with the exception of the lowest CE levels for M haplaand the 05 microl CE point for M javanica M3F (two-fold degenerate) and M2R (four-fold) were designed toamplify Hsp90 within the genus Meloidogyne with theinitial aim of developing this fragment as a diagnosticmarker While the PCR procedure was straightforwardwe discovered no apparent size variation in this fragmentamong the root-knot nematodes (Fig 5) and a low level ofsequence variation among the mitotic parthenogens (datanot shown) thus limiting its diagnostic potential for thegenus However increasing the degeneracy of primersM3F and M2R may yet prove useful for examiningvariation across a broader range of nematode taxa

The U831BL1110B Hsp90 fragment was of interestbecause significant sequence variation in this region hadbeen previously detected among several root-knot nema-tode species (Skantar amp Carta 2005) This variability is

290 Nematology

MDA in Meloidogyne PCR

apparent in the slightly different sizes of PCR products(Fig 6) such as the slower-migrating band for M incog-nita and the slightly faster migrating bands for M are-naria and M hapla U831B and L1110B are less degen-erate modifications (24-fold and four-fold respectively)of a previous primer set (Skantar amp Carta 2000) How-ever even with increased specificity these primers some-times required two cycles of amplification to producestrong PCR products from the extracts of single nema-todes (Skantar amp Carta 2005) The benefit of MDA foramplification of Hsp90 with these degenerate primers wasreflected in the strong products obtained from a singleround of PCR It is likely that adding more genomic DNAincreased the number of productive interactions betweenthe degenerate primers and the target gene leading tohigher product yield Raising the level of crude nema-tode extracts in Hsp90 PCR reactions generally does notachieve the same advantage (data not shown) as high lev-els of impure extract may contribute an excess of PCR-inhibiting contaminants While genomic DNA prepara-tions from bulk quantities of nematodes also give strongHsp90 PCR products (data not shown) preparing themrequires significantly more nematodes time and labourthan MDA

The technical improvements shown here should signifi-cantly broaden the appeal of Hsp90 and open the door foranalysis of other single-copy genes for nematode identifi-cation and phylogenetics The combination of MDA anddegenerate primers will facilitate future surveys of Hsp90across the phylum Nematoda On a finer scale with MDAit will be possible to address questions about Hsp90 al-lelic variation within and among individuals in a popula-tion Whether Hsp90 sequences can be used to developreliable diagnostics for the major root-knot nematodes orfor species of regulatory importance remains to be vali-dated through the examination of multiple populations ofthese species

The potential applications of MDA to nematode iden-tification and systematics are far reaching For instancewith MDA it should be possible to archive the geneticmaterial from individual nematodes thereby eliminatingthe need for more labour-intensive culture methods suchas propagating single egg-mass cultures (Hugall et al1999) MDA could facilitate the development and produc-tion of DNA lsquotype specimensrsquo that may be shared amongscientists Thus it would be possible to generate a set ofDNA standards that could be widely distributed provid-ing a practical tool for greater uniformity in nematodemolecular diagnostics Another possible advance result-

ing from MDA is the ability to archive large quantitiesof genetic material from rare difficult-to-maintain or his-torically valuable specimens It will now be feasible toperform morphological analysis on a specimen catalogueits unique features in a digital archive (De Ley amp Bert2002) and with MDA produce a corresponding geneticarchive Also MDA provides an attractive alternative tothe international or intrastate exchange of live specimensa practice that presents a number of regulatory challengesand restrictions

Acknowledgements

We thank Maria Hult Sharon Ochs and Donna Elling-ton for technical assistance We thank the following peo-ple for providing nematode isolates Saad Hafez M chit-woodi Janete Brito M mayaguensis Andy NyzcepirM floridensis Don Schmitt M hapla

Mention of trade names or commercial products inthis publication is solely for the purpose of providingspecific information and does not imply recommendationor endorsement by the US Department of Agriculture

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmental evolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

BAUM TJ GRESSHOFF PM LEWIS SA amp DEAN RA(1994) Characterization and phylogenetic analysis of fourroot-knot nematode species using DNA amplification finger-printing and automated polyacrylamide gel electrophoresisMolecular Plant-Microbe Interactions 7 39-47

BECKENBACH K SMITH MJ amp WEBSTER JM (1992)Taxonomic affinities and intra- and interspecific variation inBursaphelenchus spp as determined by polymerase chainreaction Journal of Nematology 24 140-147

BIRNBY DA LINK EM VOWELS JJ TIAN H CO-LACURCIO PL amp THOMAS JH (2000) A transmembraneguanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate acommon set of chemosensory behaviors in Caenorhabditiselegans Genetics 155 85-104

BLOK VC PHILLIPS MS MCNICOL JW amp FARGETTEM (1997a) Genetic variation in tropical Meloidogyne spp

Vol 7(2) 2005 291

AM Skantar amp LK Carta

Fig 2 Comparison of ribosomal ITS1 region amplified from four different root-knot nematodes DNA templates were either crudeextracts (CE) from pools of nine nematodes or MDA DNA generated from 1 microl (150th volume) of the same pooled CE Agarosegel electrophoresis of PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbers belowrepresent the amount of template DNA in microl added to 25 microl PCR reactions Arrows denote position of expected products M = 100 bpladder (New England Biolabs)

Fig 3 Comparison of ribosomal 28S D2-D3 expansion segments amplified from four different root-knot nematodes DNA templateswere either crude extracts (CE) from pools of nine nematodes or MDA DNA generated from 1 microl (150th volume) of the same pooled CEAgarose gel electrophoresis of PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbersbelow represent the amount of template DNA added to 25 microl PCR reactions Arrows denote position of expected PCR products M =100 bp ladder with fragment sizes 1517 1200 1000 900 800 700 600 517500 (comigrating bands) 400 300 200 and 100 bp(New England Biolabs)

the reactions at each DNA concentration did not generateas much D2-D3 product as ITS1 product The MDA reac-tions were stronger over the range of DNA concentrationstested than those containing CE For M hapla the CE re-actions failed to generate any products The major MDAD2-D3 PCR product was smaller than for the other root-knot nematodes (ca 650 bp) an additional band at ca 400bp was also observed

A similar side-by-side comparison of CE and MDAdilution series using the U831L1110 primer pair wasnot possible due to the poor amplification of Hsp90 withless than 10 microl CE (not shown) Thus the root-knotnematode-specific Hsp90 primers M3F and M2R wereused for this purpose (Fig 4) With these primers CEtemplate from most of the root-knot nematodes showedstrong amplification of the expected ca 300 bp Hsp90

288 Nematology

MDA in Meloidogyne PCR

Fig 4 Schematic diagram of Hsp90 with positions of PCR primers M3F M2R U831B and L1110B marked by arrows The conservedamino acid domains corresponding to the primer sequences are indicated in parentheses

Fig 5 Comparison of Hsp90 amplified from four different root-knot nematodes PCR reactions of 25 microl included primers M3F and M2Rand DNA template from either crude nematode extracts (CE) or multiple displacement amplification (MDA) Agarose gel electrophoresisof PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbers below represent the amountof template DNA added to 25 microl PCR reactions Arrows denote position of expected PCR products M = 100 bp ladder (New EnglandBiolabs)

PCR products However the M hapla CE reactions wererelatively weaker than the other species reactions at lowlevels of DNA (Fig 5) MDA DNA from all four speciesproduced strong PCR bands over the entire 200-fold rangeof template DNA added

The next experiment involved an examination of Hsp90fragments produced with MDA DNA and the U831B andL1110B primers MDA DNA was generated from CE pre-pared from one two or nine J2 of several root-knot nema-tode species The number of nematodes in a given speciespool was purely incidental reflecting the sometimes lim-ited material obtained as interceptions Meloidogyne are-naria M incognita M javanica and M hapla were allincluded because of their worldwide distribution and im-portance to agriculture Three other species of regulatoryimportance M mayaguensis M floridensis and M chit-woodi were also tested Strong Hsp90 amplification wasobserved in all of the MDA reactions (Fig 6) Two Hsp90PCR products of slightly different size were detected for

M incognita but single bands were visible for all otherspecies with small differences in migration distance ap-parent for some of them

Discussion

We have demonstrated that MDA is an effective methodof WGA for several species of root-knot nematode whichfurther suggests that this method will have broad applica-bility in nematode molecular systematics and identifica-tion MDA produced robust amounts of genomic DNA re-gardless of the number of nematodes in the crude extractand worked equally well with mechanically disrupted ju-veniles and females MDA from single M chitwoodi fe-males produced micrograms of genomic DNA from as lit-tle as 5 of the crude extracts and similar yields were ob-tained from pools of two to nine juveniles Because suc-cessful PCR was achieved with as low as 001 microl MDADNA we estimate that crude extracts subjected to MDA

Vol 7(2) 2005 289

AM Skantar amp LK Carta

Fig 6 Agarose gel electrophoresis of Hsp90 PCR productsproduced with degenerate primers U831B and L1110B andMDA DNA generated from 1 microl of lsquonematode smashrsquo crudeextracts Extracts from Ma Mi Mj and Mh were prepared frompools of nine J2 Mm and Mh were from pools of two J2Mc was from a single female Ma = Meloidogyne arenariaMi = M incognita Mj = M javanica Mh = M hapla Mm= M mayaguensis Mf = M floridensis Mc = M chitwoodiMW = 100 bp ladder (New England Biolabs)

can produce sufficient template for as many as 10 000 to40 000 PCR reactions depending upon the number sizeand stage of nematodes in the starting material and thegene being amplified We did not attempt MDA with lessthan 1 microl CE but it is possible that using lower amountsof input CE could extend this estimate even further Thepresence of non-DNA contaminants within crude nema-tode extracts prevented accurate measurements of theirDNA concentrations so it was not possible to directlyequate concentrations of CE and MDA genomic DNANevertheless these experiments provide a practical com-parison of PCR results using MDA-produced genomicDNA and the nematode CE volumes typically employed

The ribosomal internal transcribed spacer ITS1 and theLSU D2-D3 expansion regions are two readily amplifiedribosomal genes that have been previously examinedfrom root-knot nematodes (Powers amp Harris 1993 Chenet al 2003) thus providing an established experimentalcontext in which to evaluate template produced by MDAThe sizes of ITS1 PCR products amplified from MDADNA were consistent with those reported previously(Powers et al 1997) For most root-knot species bothCE and MDA templates produced strong bands at allDNA concentrations tested (Fig 2) As low as 001 microlMDA DNA produced strong ITS1 products that werecomparable to or greater than the signals resulting from05 microl nematode CE The exception was M hapla whichproduced a relatively strong ITS1 signal from 5 microl CE

DNA that gradually disappeared at lower levels of CEWhile high levels of MDA DNA from M hapla producedstrong bands at the expected size (ca 450 bp) loweramounts of template produced additional bands at ca 600and ca 800 bp The identity of these products is unclearthey could be an artefact of MDA amplification resultfrom mispriming in the rDNA or may indicate that thecrude DNA preparation used for MDA of M hapla was oflesser quality than for the other nematodes

The results from the D2-D3 comparison generally gavesimilar results to the ITS1 although the signal strengths ofthe D2-D3 products were lower overall (Fig 3) Some ofthe CE PCR reactions were higher or lower than expectedfor serial dilutions of the DNA It is likely these datapoints resulted from pipetting errors (Fig 3 M incognitaand M javanica 1 microl CE) Again the M hapla CE andMDA results were not as robust as those from the othernematodes CE reactions gave no products and MDAtemplates produced a smaller band in addition to the ca600 bp D2-D3 band These reactions were performed withthe same CE and MDA DNA as for ITS1 indicatingthat both genes in M hapla were affected similarly Aswith any method involving DNA polymerase activity thatreplicates input DNA without bias MDA results can beaffected by contamination Therefore nematodes used forMDA should be prepared with care to ensure they are freefrom microbial contamination The precise reason for thelower quality PCR results with M hapla is not clear butas a cautionary measure fresh nematode CE and MDADNA were prepared for the next set of experiments

The M3FM2R Hsp90 reactions were successful for allconcentrations of CE and MDA for all nematodes (Fig 5)with the exception of the lowest CE levels for M haplaand the 05 microl CE point for M javanica M3F (two-fold degenerate) and M2R (four-fold) were designed toamplify Hsp90 within the genus Meloidogyne with theinitial aim of developing this fragment as a diagnosticmarker While the PCR procedure was straightforwardwe discovered no apparent size variation in this fragmentamong the root-knot nematodes (Fig 5) and a low level ofsequence variation among the mitotic parthenogens (datanot shown) thus limiting its diagnostic potential for thegenus However increasing the degeneracy of primersM3F and M2R may yet prove useful for examiningvariation across a broader range of nematode taxa

The U831BL1110B Hsp90 fragment was of interestbecause significant sequence variation in this region hadbeen previously detected among several root-knot nema-tode species (Skantar amp Carta 2005) This variability is

290 Nematology

MDA in Meloidogyne PCR

apparent in the slightly different sizes of PCR products(Fig 6) such as the slower-migrating band for M incog-nita and the slightly faster migrating bands for M are-naria and M hapla U831B and L1110B are less degen-erate modifications (24-fold and four-fold respectively)of a previous primer set (Skantar amp Carta 2000) How-ever even with increased specificity these primers some-times required two cycles of amplification to producestrong PCR products from the extracts of single nema-todes (Skantar amp Carta 2005) The benefit of MDA foramplification of Hsp90 with these degenerate primers wasreflected in the strong products obtained from a singleround of PCR It is likely that adding more genomic DNAincreased the number of productive interactions betweenthe degenerate primers and the target gene leading tohigher product yield Raising the level of crude nema-tode extracts in Hsp90 PCR reactions generally does notachieve the same advantage (data not shown) as high lev-els of impure extract may contribute an excess of PCR-inhibiting contaminants While genomic DNA prepara-tions from bulk quantities of nematodes also give strongHsp90 PCR products (data not shown) preparing themrequires significantly more nematodes time and labourthan MDA

The technical improvements shown here should signifi-cantly broaden the appeal of Hsp90 and open the door foranalysis of other single-copy genes for nematode identifi-cation and phylogenetics The combination of MDA anddegenerate primers will facilitate future surveys of Hsp90across the phylum Nematoda On a finer scale with MDAit will be possible to address questions about Hsp90 al-lelic variation within and among individuals in a popula-tion Whether Hsp90 sequences can be used to developreliable diagnostics for the major root-knot nematodes orfor species of regulatory importance remains to be vali-dated through the examination of multiple populations ofthese species

The potential applications of MDA to nematode iden-tification and systematics are far reaching For instancewith MDA it should be possible to archive the geneticmaterial from individual nematodes thereby eliminatingthe need for more labour-intensive culture methods suchas propagating single egg-mass cultures (Hugall et al1999) MDA could facilitate the development and produc-tion of DNA lsquotype specimensrsquo that may be shared amongscientists Thus it would be possible to generate a set ofDNA standards that could be widely distributed provid-ing a practical tool for greater uniformity in nematodemolecular diagnostics Another possible advance result-

ing from MDA is the ability to archive large quantitiesof genetic material from rare difficult-to-maintain or his-torically valuable specimens It will now be feasible toperform morphological analysis on a specimen catalogueits unique features in a digital archive (De Ley amp Bert2002) and with MDA produce a corresponding geneticarchive Also MDA provides an attractive alternative tothe international or intrastate exchange of live specimensa practice that presents a number of regulatory challengesand restrictions

Acknowledgements

We thank Maria Hult Sharon Ochs and Donna Elling-ton for technical assistance We thank the following peo-ple for providing nematode isolates Saad Hafez M chit-woodi Janete Brito M mayaguensis Andy NyzcepirM floridensis Don Schmitt M hapla

Mention of trade names or commercial products inthis publication is solely for the purpose of providingspecific information and does not imply recommendationor endorsement by the US Department of Agriculture

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmental evolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

BAUM TJ GRESSHOFF PM LEWIS SA amp DEAN RA(1994) Characterization and phylogenetic analysis of fourroot-knot nematode species using DNA amplification finger-printing and automated polyacrylamide gel electrophoresisMolecular Plant-Microbe Interactions 7 39-47

BECKENBACH K SMITH MJ amp WEBSTER JM (1992)Taxonomic affinities and intra- and interspecific variation inBursaphelenchus spp as determined by polymerase chainreaction Journal of Nematology 24 140-147

BIRNBY DA LINK EM VOWELS JJ TIAN H CO-LACURCIO PL amp THOMAS JH (2000) A transmembraneguanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate acommon set of chemosensory behaviors in Caenorhabditiselegans Genetics 155 85-104

BLOK VC PHILLIPS MS MCNICOL JW amp FARGETTEM (1997a) Genetic variation in tropical Meloidogyne spp

Vol 7(2) 2005 291

MDA in Meloidogyne PCR

Fig 4 Schematic diagram of Hsp90 with positions of PCR primers M3F M2R U831B and L1110B marked by arrows The conservedamino acid domains corresponding to the primer sequences are indicated in parentheses

Fig 5 Comparison of Hsp90 amplified from four different root-knot nematodes PCR reactions of 25 microl included primers M3F and M2Rand DNA template from either crude nematode extracts (CE) or multiple displacement amplification (MDA) Agarose gel electrophoresisof PCR products produced from Meloidogyne arenaria M incognita M javanica and M hapla Numbers below represent the amountof template DNA added to 25 microl PCR reactions Arrows denote position of expected PCR products M = 100 bp ladder (New EnglandBiolabs)

PCR products However the M hapla CE reactions wererelatively weaker than the other species reactions at lowlevels of DNA (Fig 5) MDA DNA from all four speciesproduced strong PCR bands over the entire 200-fold rangeof template DNA added

The next experiment involved an examination of Hsp90fragments produced with MDA DNA and the U831B andL1110B primers MDA DNA was generated from CE pre-pared from one two or nine J2 of several root-knot nema-tode species The number of nematodes in a given speciespool was purely incidental reflecting the sometimes lim-ited material obtained as interceptions Meloidogyne are-naria M incognita M javanica and M hapla were allincluded because of their worldwide distribution and im-portance to agriculture Three other species of regulatoryimportance M mayaguensis M floridensis and M chit-woodi were also tested Strong Hsp90 amplification wasobserved in all of the MDA reactions (Fig 6) Two Hsp90PCR products of slightly different size were detected for

M incognita but single bands were visible for all otherspecies with small differences in migration distance ap-parent for some of them

Discussion

We have demonstrated that MDA is an effective methodof WGA for several species of root-knot nematode whichfurther suggests that this method will have broad applica-bility in nematode molecular systematics and identifica-tion MDA produced robust amounts of genomic DNA re-gardless of the number of nematodes in the crude extractand worked equally well with mechanically disrupted ju-veniles and females MDA from single M chitwoodi fe-males produced micrograms of genomic DNA from as lit-tle as 5 of the crude extracts and similar yields were ob-tained from pools of two to nine juveniles Because suc-cessful PCR was achieved with as low as 001 microl MDADNA we estimate that crude extracts subjected to MDA

Vol 7(2) 2005 289

AM Skantar amp LK Carta

Fig 6 Agarose gel electrophoresis of Hsp90 PCR productsproduced with degenerate primers U831B and L1110B andMDA DNA generated from 1 microl of lsquonematode smashrsquo crudeextracts Extracts from Ma Mi Mj and Mh were prepared frompools of nine J2 Mm and Mh were from pools of two J2Mc was from a single female Ma = Meloidogyne arenariaMi = M incognita Mj = M javanica Mh = M hapla Mm= M mayaguensis Mf = M floridensis Mc = M chitwoodiMW = 100 bp ladder (New England Biolabs)

can produce sufficient template for as many as 10 000 to40 000 PCR reactions depending upon the number sizeand stage of nematodes in the starting material and thegene being amplified We did not attempt MDA with lessthan 1 microl CE but it is possible that using lower amountsof input CE could extend this estimate even further Thepresence of non-DNA contaminants within crude nema-tode extracts prevented accurate measurements of theirDNA concentrations so it was not possible to directlyequate concentrations of CE and MDA genomic DNANevertheless these experiments provide a practical com-parison of PCR results using MDA-produced genomicDNA and the nematode CE volumes typically employed

The ribosomal internal transcribed spacer ITS1 and theLSU D2-D3 expansion regions are two readily amplifiedribosomal genes that have been previously examinedfrom root-knot nematodes (Powers amp Harris 1993 Chenet al 2003) thus providing an established experimentalcontext in which to evaluate template produced by MDAThe sizes of ITS1 PCR products amplified from MDADNA were consistent with those reported previously(Powers et al 1997) For most root-knot species bothCE and MDA templates produced strong bands at allDNA concentrations tested (Fig 2) As low as 001 microlMDA DNA produced strong ITS1 products that werecomparable to or greater than the signals resulting from05 microl nematode CE The exception was M hapla whichproduced a relatively strong ITS1 signal from 5 microl CE

DNA that gradually disappeared at lower levels of CEWhile high levels of MDA DNA from M hapla producedstrong bands at the expected size (ca 450 bp) loweramounts of template produced additional bands at ca 600and ca 800 bp The identity of these products is unclearthey could be an artefact of MDA amplification resultfrom mispriming in the rDNA or may indicate that thecrude DNA preparation used for MDA of M hapla was oflesser quality than for the other nematodes

The results from the D2-D3 comparison generally gavesimilar results to the ITS1 although the signal strengths ofthe D2-D3 products were lower overall (Fig 3) Some ofthe CE PCR reactions were higher or lower than expectedfor serial dilutions of the DNA It is likely these datapoints resulted from pipetting errors (Fig 3 M incognitaand M javanica 1 microl CE) Again the M hapla CE andMDA results were not as robust as those from the othernematodes CE reactions gave no products and MDAtemplates produced a smaller band in addition to the ca600 bp D2-D3 band These reactions were performed withthe same CE and MDA DNA as for ITS1 indicatingthat both genes in M hapla were affected similarly Aswith any method involving DNA polymerase activity thatreplicates input DNA without bias MDA results can beaffected by contamination Therefore nematodes used forMDA should be prepared with care to ensure they are freefrom microbial contamination The precise reason for thelower quality PCR results with M hapla is not clear butas a cautionary measure fresh nematode CE and MDADNA were prepared for the next set of experiments

The M3FM2R Hsp90 reactions were successful for allconcentrations of CE and MDA for all nematodes (Fig 5)with the exception of the lowest CE levels for M haplaand the 05 microl CE point for M javanica M3F (two-fold degenerate) and M2R (four-fold) were designed toamplify Hsp90 within the genus Meloidogyne with theinitial aim of developing this fragment as a diagnosticmarker While the PCR procedure was straightforwardwe discovered no apparent size variation in this fragmentamong the root-knot nematodes (Fig 5) and a low level ofsequence variation among the mitotic parthenogens (datanot shown) thus limiting its diagnostic potential for thegenus However increasing the degeneracy of primersM3F and M2R may yet prove useful for examiningvariation across a broader range of nematode taxa

The U831BL1110B Hsp90 fragment was of interestbecause significant sequence variation in this region hadbeen previously detected among several root-knot nema-tode species (Skantar amp Carta 2005) This variability is

290 Nematology

MDA in Meloidogyne PCR

apparent in the slightly different sizes of PCR products(Fig 6) such as the slower-migrating band for M incog-nita and the slightly faster migrating bands for M are-naria and M hapla U831B and L1110B are less degen-erate modifications (24-fold and four-fold respectively)of a previous primer set (Skantar amp Carta 2000) How-ever even with increased specificity these primers some-times required two cycles of amplification to producestrong PCR products from the extracts of single nema-todes (Skantar amp Carta 2005) The benefit of MDA foramplification of Hsp90 with these degenerate primers wasreflected in the strong products obtained from a singleround of PCR It is likely that adding more genomic DNAincreased the number of productive interactions betweenthe degenerate primers and the target gene leading tohigher product yield Raising the level of crude nema-tode extracts in Hsp90 PCR reactions generally does notachieve the same advantage (data not shown) as high lev-els of impure extract may contribute an excess of PCR-inhibiting contaminants While genomic DNA prepara-tions from bulk quantities of nematodes also give strongHsp90 PCR products (data not shown) preparing themrequires significantly more nematodes time and labourthan MDA

The technical improvements shown here should signifi-cantly broaden the appeal of Hsp90 and open the door foranalysis of other single-copy genes for nematode identifi-cation and phylogenetics The combination of MDA anddegenerate primers will facilitate future surveys of Hsp90across the phylum Nematoda On a finer scale with MDAit will be possible to address questions about Hsp90 al-lelic variation within and among individuals in a popula-tion Whether Hsp90 sequences can be used to developreliable diagnostics for the major root-knot nematodes orfor species of regulatory importance remains to be vali-dated through the examination of multiple populations ofthese species

The potential applications of MDA to nematode iden-tification and systematics are far reaching For instancewith MDA it should be possible to archive the geneticmaterial from individual nematodes thereby eliminatingthe need for more labour-intensive culture methods suchas propagating single egg-mass cultures (Hugall et al1999) MDA could facilitate the development and produc-tion of DNA lsquotype specimensrsquo that may be shared amongscientists Thus it would be possible to generate a set ofDNA standards that could be widely distributed provid-ing a practical tool for greater uniformity in nematodemolecular diagnostics Another possible advance result-

ing from MDA is the ability to archive large quantitiesof genetic material from rare difficult-to-maintain or his-torically valuable specimens It will now be feasible toperform morphological analysis on a specimen catalogueits unique features in a digital archive (De Ley amp Bert2002) and with MDA produce a corresponding geneticarchive Also MDA provides an attractive alternative tothe international or intrastate exchange of live specimensa practice that presents a number of regulatory challengesand restrictions

Acknowledgements

We thank Maria Hult Sharon Ochs and Donna Elling-ton for technical assistance We thank the following peo-ple for providing nematode isolates Saad Hafez M chit-woodi Janete Brito M mayaguensis Andy NyzcepirM floridensis Don Schmitt M hapla

Mention of trade names or commercial products inthis publication is solely for the purpose of providingspecific information and does not imply recommendationor endorsement by the US Department of Agriculture

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmental evolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

BAUM TJ GRESSHOFF PM LEWIS SA amp DEAN RA(1994) Characterization and phylogenetic analysis of fourroot-knot nematode species using DNA amplification finger-printing and automated polyacrylamide gel electrophoresisMolecular Plant-Microbe Interactions 7 39-47

BECKENBACH K SMITH MJ amp WEBSTER JM (1992)Taxonomic affinities and intra- and interspecific variation inBursaphelenchus spp as determined by polymerase chainreaction Journal of Nematology 24 140-147

BIRNBY DA LINK EM VOWELS JJ TIAN H CO-LACURCIO PL amp THOMAS JH (2000) A transmembraneguanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate acommon set of chemosensory behaviors in Caenorhabditiselegans Genetics 155 85-104

BLOK VC PHILLIPS MS MCNICOL JW amp FARGETTEM (1997a) Genetic variation in tropical Meloidogyne spp

Vol 7(2) 2005 291

AM Skantar amp LK Carta

Fig 6 Agarose gel electrophoresis of Hsp90 PCR productsproduced with degenerate primers U831B and L1110B andMDA DNA generated from 1 microl of lsquonematode smashrsquo crudeextracts Extracts from Ma Mi Mj and Mh were prepared frompools of nine J2 Mm and Mh were from pools of two J2Mc was from a single female Ma = Meloidogyne arenariaMi = M incognita Mj = M javanica Mh = M hapla Mm= M mayaguensis Mf = M floridensis Mc = M chitwoodiMW = 100 bp ladder (New England Biolabs)

can produce sufficient template for as many as 10 000 to40 000 PCR reactions depending upon the number sizeand stage of nematodes in the starting material and thegene being amplified We did not attempt MDA with lessthan 1 microl CE but it is possible that using lower amountsof input CE could extend this estimate even further Thepresence of non-DNA contaminants within crude nema-tode extracts prevented accurate measurements of theirDNA concentrations so it was not possible to directlyequate concentrations of CE and MDA genomic DNANevertheless these experiments provide a practical com-parison of PCR results using MDA-produced genomicDNA and the nematode CE volumes typically employed

The ribosomal internal transcribed spacer ITS1 and theLSU D2-D3 expansion regions are two readily amplifiedribosomal genes that have been previously examinedfrom root-knot nematodes (Powers amp Harris 1993 Chenet al 2003) thus providing an established experimentalcontext in which to evaluate template produced by MDAThe sizes of ITS1 PCR products amplified from MDADNA were consistent with those reported previously(Powers et al 1997) For most root-knot species bothCE and MDA templates produced strong bands at allDNA concentrations tested (Fig 2) As low as 001 microlMDA DNA produced strong ITS1 products that werecomparable to or greater than the signals resulting from05 microl nematode CE The exception was M hapla whichproduced a relatively strong ITS1 signal from 5 microl CE

DNA that gradually disappeared at lower levels of CEWhile high levels of MDA DNA from M hapla producedstrong bands at the expected size (ca 450 bp) loweramounts of template produced additional bands at ca 600and ca 800 bp The identity of these products is unclearthey could be an artefact of MDA amplification resultfrom mispriming in the rDNA or may indicate that thecrude DNA preparation used for MDA of M hapla was oflesser quality than for the other nematodes

The results from the D2-D3 comparison generally gavesimilar results to the ITS1 although the signal strengths ofthe D2-D3 products were lower overall (Fig 3) Some ofthe CE PCR reactions were higher or lower than expectedfor serial dilutions of the DNA It is likely these datapoints resulted from pipetting errors (Fig 3 M incognitaand M javanica 1 microl CE) Again the M hapla CE andMDA results were not as robust as those from the othernematodes CE reactions gave no products and MDAtemplates produced a smaller band in addition to the ca600 bp D2-D3 band These reactions were performed withthe same CE and MDA DNA as for ITS1 indicatingthat both genes in M hapla were affected similarly Aswith any method involving DNA polymerase activity thatreplicates input DNA without bias MDA results can beaffected by contamination Therefore nematodes used forMDA should be prepared with care to ensure they are freefrom microbial contamination The precise reason for thelower quality PCR results with M hapla is not clear butas a cautionary measure fresh nematode CE and MDADNA were prepared for the next set of experiments

The M3FM2R Hsp90 reactions were successful for allconcentrations of CE and MDA for all nematodes (Fig 5)with the exception of the lowest CE levels for M haplaand the 05 microl CE point for M javanica M3F (two-fold degenerate) and M2R (four-fold) were designed toamplify Hsp90 within the genus Meloidogyne with theinitial aim of developing this fragment as a diagnosticmarker While the PCR procedure was straightforwardwe discovered no apparent size variation in this fragmentamong the root-knot nematodes (Fig 5) and a low level ofsequence variation among the mitotic parthenogens (datanot shown) thus limiting its diagnostic potential for thegenus However increasing the degeneracy of primersM3F and M2R may yet prove useful for examiningvariation across a broader range of nematode taxa

The U831BL1110B Hsp90 fragment was of interestbecause significant sequence variation in this region hadbeen previously detected among several root-knot nema-tode species (Skantar amp Carta 2005) This variability is

290 Nematology

MDA in Meloidogyne PCR

apparent in the slightly different sizes of PCR products(Fig 6) such as the slower-migrating band for M incog-nita and the slightly faster migrating bands for M are-naria and M hapla U831B and L1110B are less degen-erate modifications (24-fold and four-fold respectively)of a previous primer set (Skantar amp Carta 2000) How-ever even with increased specificity these primers some-times required two cycles of amplification to producestrong PCR products from the extracts of single nema-todes (Skantar amp Carta 2005) The benefit of MDA foramplification of Hsp90 with these degenerate primers wasreflected in the strong products obtained from a singleround of PCR It is likely that adding more genomic DNAincreased the number of productive interactions betweenthe degenerate primers and the target gene leading tohigher product yield Raising the level of crude nema-tode extracts in Hsp90 PCR reactions generally does notachieve the same advantage (data not shown) as high lev-els of impure extract may contribute an excess of PCR-inhibiting contaminants While genomic DNA prepara-tions from bulk quantities of nematodes also give strongHsp90 PCR products (data not shown) preparing themrequires significantly more nematodes time and labourthan MDA

The technical improvements shown here should signifi-cantly broaden the appeal of Hsp90 and open the door foranalysis of other single-copy genes for nematode identifi-cation and phylogenetics The combination of MDA anddegenerate primers will facilitate future surveys of Hsp90across the phylum Nematoda On a finer scale with MDAit will be possible to address questions about Hsp90 al-lelic variation within and among individuals in a popula-tion Whether Hsp90 sequences can be used to developreliable diagnostics for the major root-knot nematodes orfor species of regulatory importance remains to be vali-dated through the examination of multiple populations ofthese species

The potential applications of MDA to nematode iden-tification and systematics are far reaching For instancewith MDA it should be possible to archive the geneticmaterial from individual nematodes thereby eliminatingthe need for more labour-intensive culture methods suchas propagating single egg-mass cultures (Hugall et al1999) MDA could facilitate the development and produc-tion of DNA lsquotype specimensrsquo that may be shared amongscientists Thus it would be possible to generate a set ofDNA standards that could be widely distributed provid-ing a practical tool for greater uniformity in nematodemolecular diagnostics Another possible advance result-

ing from MDA is the ability to archive large quantitiesof genetic material from rare difficult-to-maintain or his-torically valuable specimens It will now be feasible toperform morphological analysis on a specimen catalogueits unique features in a digital archive (De Ley amp Bert2002) and with MDA produce a corresponding geneticarchive Also MDA provides an attractive alternative tothe international or intrastate exchange of live specimensa practice that presents a number of regulatory challengesand restrictions

Acknowledgements

We thank Maria Hult Sharon Ochs and Donna Elling-ton for technical assistance We thank the following peo-ple for providing nematode isolates Saad Hafez M chit-woodi Janete Brito M mayaguensis Andy NyzcepirM floridensis Don Schmitt M hapla

Mention of trade names or commercial products inthis publication is solely for the purpose of providingspecific information and does not imply recommendationor endorsement by the US Department of Agriculture

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmental evolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

BAUM TJ GRESSHOFF PM LEWIS SA amp DEAN RA(1994) Characterization and phylogenetic analysis of fourroot-knot nematode species using DNA amplification finger-printing and automated polyacrylamide gel electrophoresisMolecular Plant-Microbe Interactions 7 39-47

BECKENBACH K SMITH MJ amp WEBSTER JM (1992)Taxonomic affinities and intra- and interspecific variation inBursaphelenchus spp as determined by polymerase chainreaction Journal of Nematology 24 140-147

BIRNBY DA LINK EM VOWELS JJ TIAN H CO-LACURCIO PL amp THOMAS JH (2000) A transmembraneguanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate acommon set of chemosensory behaviors in Caenorhabditiselegans Genetics 155 85-104

BLOK VC PHILLIPS MS MCNICOL JW amp FARGETTEM (1997a) Genetic variation in tropical Meloidogyne spp

Vol 7(2) 2005 291

MDA in Meloidogyne PCR

apparent in the slightly different sizes of PCR products(Fig 6) such as the slower-migrating band for M incog-nita and the slightly faster migrating bands for M are-naria and M hapla U831B and L1110B are less degen-erate modifications (24-fold and four-fold respectively)of a previous primer set (Skantar amp Carta 2000) How-ever even with increased specificity these primers some-times required two cycles of amplification to producestrong PCR products from the extracts of single nema-todes (Skantar amp Carta 2005) The benefit of MDA foramplification of Hsp90 with these degenerate primers wasreflected in the strong products obtained from a singleround of PCR It is likely that adding more genomic DNAincreased the number of productive interactions betweenthe degenerate primers and the target gene leading tohigher product yield Raising the level of crude nema-tode extracts in Hsp90 PCR reactions generally does notachieve the same advantage (data not shown) as high lev-els of impure extract may contribute an excess of PCR-inhibiting contaminants While genomic DNA prepara-tions from bulk quantities of nematodes also give strongHsp90 PCR products (data not shown) preparing themrequires significantly more nematodes time and labourthan MDA

The technical improvements shown here should signifi-cantly broaden the appeal of Hsp90 and open the door foranalysis of other single-copy genes for nematode identifi-cation and phylogenetics The combination of MDA anddegenerate primers will facilitate future surveys of Hsp90across the phylum Nematoda On a finer scale with MDAit will be possible to address questions about Hsp90 al-lelic variation within and among individuals in a popula-tion Whether Hsp90 sequences can be used to developreliable diagnostics for the major root-knot nematodes orfor species of regulatory importance remains to be vali-dated through the examination of multiple populations ofthese species

The potential applications of MDA to nematode iden-tification and systematics are far reaching For instancewith MDA it should be possible to archive the geneticmaterial from individual nematodes thereby eliminatingthe need for more labour-intensive culture methods suchas propagating single egg-mass cultures (Hugall et al1999) MDA could facilitate the development and produc-tion of DNA lsquotype specimensrsquo that may be shared amongscientists Thus it would be possible to generate a set ofDNA standards that could be widely distributed provid-ing a practical tool for greater uniformity in nematodemolecular diagnostics Another possible advance result-

ing from MDA is the ability to archive large quantitiesof genetic material from rare difficult-to-maintain or his-torically valuable specimens It will now be feasible toperform morphological analysis on a specimen catalogueits unique features in a digital archive (De Ley amp Bert2002) and with MDA produce a corresponding geneticarchive Also MDA provides an attractive alternative tothe international or intrastate exchange of live specimensa practice that presents a number of regulatory challengesand restrictions

Acknowledgements

We thank Maria Hult Sharon Ochs and Donna Elling-ton for technical assistance We thank the following peo-ple for providing nematode isolates Saad Hafez M chit-woodi Janete Brito M mayaguensis Andy NyzcepirM floridensis Don Schmitt M hapla

Mention of trade names or commercial products inthis publication is solely for the purpose of providingspecific information and does not imply recommendationor endorsement by the US Department of Agriculture

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmental evolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

BAUM TJ GRESSHOFF PM LEWIS SA amp DEAN RA(1994) Characterization and phylogenetic analysis of fourroot-knot nematode species using DNA amplification finger-printing and automated polyacrylamide gel electrophoresisMolecular Plant-Microbe Interactions 7 39-47

BECKENBACH K SMITH MJ amp WEBSTER JM (1992)Taxonomic affinities and intra- and interspecific variation inBursaphelenchus spp as determined by polymerase chainreaction Journal of Nematology 24 140-147

BIRNBY DA LINK EM VOWELS JJ TIAN H CO-LACURCIO PL amp THOMAS JH (2000) A transmembraneguanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate acommon set of chemosensory behaviors in Caenorhabditiselegans Genetics 155 85-104

BLOK VC PHILLIPS MS MCNICOL JW amp FARGETTEM (1997a) Genetic variation in tropical Meloidogyne spp

Vol 7(2) 2005 291

AM Skantar amp LK Carta

as shown by RAPD-PCR Fundamental and Applied Nema-tology 20 127-133

BLOK VC PHILLIPS MS amp FARGETTE M (1997b)Comparison of sequences from the ribosomal DNA intergenicregion of Meloidogyne mayaguensis and other major root-knot nematodes Journal of Nematology 29 16-22

BLOK VC WISHART J FARGETTE M BERTHIER Kamp PHILLIPS MS (2002) Mitochondrial DNA differencesdistinguishing Meloidogyne mayaguensis from the majorspecies of tropical root-knot nematodes Nematology 4 773-781

CENIS JL (1993) Identification of four major Meloidogynespp as shown by random amplified polymorphic DNA(RAPD-PCR) Phytopathology 83 76-78

CHEN P ROBERTS PA METCALF AE amp HYMANBC (2003) Nucleotide substitution patterning within theMeloidogyne rDNA D3 region and its evolutionary implica-tions Journal of Nematology 35 404-410

CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribed space region of Belono-laimus (Nemata Belonolaimidae) Journal of Nematology 2923-29

COURTRIGHT EM WALL DH VIRGINIA RA FRISSELM VIDA JT amp THOMAS WK (2000) Nuclear andmitochondrial DNA sequence diversity in the Antarctic ne-matode Scottnema lindsayae Journal of Nematology 32 143-153

DEAN FB NELSON JR GIESLER TL amp LASKEN RS(2001) Rapid amplification of plasmid and phage DNA usingphi29 DNA polymerase and multiply-primed rolling circleamplification Genome Research 11 1095-1099

DEAN FB HOSONO S FANG L WU X FARUQI AFBRAY-WARD P SUN Z ZONG Q DU Y DU JDRISCOLL M SONG W KINGSMORE SF amp EGHOLMM (2002) Comprehensive human genome amplificationusing multiple displacement amplification Proceedings of theNational Academy of Sciences USA 99 5261-5266

DE LEY P amp BERT W (2002) Video capture and editing as atool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

EISENBACK JD amp TRIANTAPHYLLOU HH (1991) Root-knot nematodes Meloidogyne species and races In NickleWR (Ed) Manual of agricultural nematology New YorkUSA Marcel Dekker pp 191-274

ESBENSHADE PR amp TRIANTAPHYLLOU AC (1985) Useof enzyme phenotypes for identification of Meloidogynespecies (Nematoda Tylenchida) Journal of Nematology 176-20

GORROCHOTEGUI-ESCALANTE N amp BLACK IV WC(2003) Amplifying whole insect genomes with multiple dis-placement amplification Insect Molecular Biology 12 195-200

HARRIS TS SANDALL LJ amp POWERS TO (1990)Identification of single Meloidogyne juveniles by polymerasechain reaction amplification of mitochondrial DNA Journalof Nematology 22 518-524

HASHMI G HASHMI S SELVAN S GREWAL PS ampGAUGLER R (1997) Polymorphism in heat shock proteingene (hsp70) in entomopathogenic nematodes (Rhabditida)Journal of Thermal Biology 22 143-149

HUGALL A MORITZ C STANTON J amp WOLSTEN-HOLME DR (1994) Low but strongly structured mitochon-drial DNA diversity in root-knot nematodes (Meloidogyne)Genetics 136 903-912

HUGALL A STANTON J amp MORITZ C (1999) Reticulateevolution and the origins of ribosomal internal transcribedspacer diversity in apomictic Meloidogyne Molecular Biol-ogy and Evolution 16 157-164

JEYAPRAKASH A amp HOY MA (2004) Multiple displace-ment amplification in combination with high-fidelity PCRimproves detection of bacteria from single females or eggsof Metaseiulus occidentalis (Nesbitt) (Acari Phytoseiidae)Journal of Invertebrate Pathology 86 111-116

LASKEN RS amp EGHOLM M (2003) Whole genome ampli-fication abundant supplies of DNA from precious samples orclinical specimens Trends in Biotechnology 21 531-535

PAEZ JG LIN M BEROUKHIM R LEE JC ZHAO XRICHTER DJ GABRIEL S HERMAN P SASAKI HALTSCHULER D LI C MEYERSON M amp SELLERSWR (2004) Genome coverage and sequence fidelity ofphi29 polymerase-based multiple strand displacement wholegenome amplification Nucleic Acids Research 32 e71

POWERS TO amp HARRIS TS (1993) A polymerase chainreaction method for identification of five major Meloidogynespecies Journal of Nematology 25 1-6

POWERS TO TODD TC BURNELL AM MURRAYPCB FLEMING CC SZALANSKI AL ADAMS BAamp HARRIS TS (1997) The rDNA internal transcribedspacer region as a taxonomic marker for nematodes Journalof Nematology 29 441-450

SETTERQUIST RA SMITH GK JONES R amp FOX GE(1996) Diagnostic probes targeting the major sperm proteingene that may be useful in the molecular identification ofnematodes Journal of Nematology 28 414-421

SKANTAR AM amp CARTA LK (2000) Amplification ofHSP90 homologs from plant-parasitic nematodes using de-generate primers and ramped annealing PCR BioTechniques29 1182-1185

SKANTAR AM amp CARTA LK (2005) Molecular character-ization and phylogenetic evaluation of the Hsp90 gene fromselected nematodes Journal of Nematology in press

THOMAS WK VIDA JT FRISSE LM MUNDO Mamp BALDWIN JG (1997) DNA sequences from formalin-fixed nematodes Integrating molecular and morphologicalapproaches to taxonomy Journal of Nematology 29 250-254

292 Nematology

MDA in Meloidogyne PCR

VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecific rDNA restriction frag-ment length polymorphism in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-573

WISHART J PHILLIPS MS amp BLOK VC (2002) Riboso-mal intergenic spacer A polymerase chain reaction diagnos-tic Phytopathology 92 884-892

YAN J FENG J HOSONO S amp SOMMER SS (2004) As-sessment of multiple displacement amplification in molecularepidemiology BioTechniques 37 136-138

ZIJLSTRA C LEVER AEM UENK BJ amp VAN SIL-FHOUT CH (1995) Differences between ITS regions of iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237

ZIJLSTRA C UENK BJ amp VAN SILFHOUT CH (1997)A reliable precise method to differentiate species of root-knot nematode in mixtures on the basis of ITS-RFLPsFundamental and Applied Nematology 20 59-63

Vol 7(2) 2005 293