identification of mariner-like elements from the root-knot nematode meloidogyne spp

Molecular and Biochemical Parasitology 107 (2000) 181–190

Identification of mariner-like elements from the root-knotnematode Meloidogyne spp.

Helene Leroy a, Frederic Leroy a, Corinne Auge-Gouillou b,Philippe Castagnone-Sereno a, Flavie Vanlerberghe-Masutti a, Yves Bigot b,

Pierre Abad a,*a Unite Sante des Plantes et En6ironnement, INRA, 123, Bd Francis Meilland, BP 2078, 06606 Antibes cedex, France

b Institut de Recherche sur la Biologie de l’Insecte, UPRESA CNRS 6035, Faculte des Sciences, Parc Grandmont,37200 Tours, France

Received 15 July 1999; received in revised form 22 December 1999; accepted 4 January 2000

Abstract

The Meloidogyne species are agriculturally important pests widespread in the world. These polyphagous endopara-sitic nematodes possess an astonishing ability to bypass the plant resistance genes in few generations. However, thegenes and mechanisms involved in this molecular determinism are not yet known. Except cytogenetic and cytotaxo-nomic studies, few data are available concerning their genome. There is therefore an important need of moleculartools for genetic investigation of their virulence character and other aspects of host–pathogen interactions. In thatrespect, the presence of mariner-like-elements (MLEs) was assessed in these endoparasitic nematodes by a polymerasechain reaction (PCR) assay using degenerate primers designed from two conserved regions of the mariner transposaseopen reading frame (ORF). Four Meloidogyne species of the five tested revealed the presence of MLEs in theirgenome. Southern blot analysis indicated that sequences hybridizing to the mariner transposase-like PCR clones occurat a moderate to low copy number in the different Meloidogyne spp. genomes. The phylogenetic analysis show thatthe Meloidogyne MLEs may form new subfamilies of mariner. Moreover, five PCR clones were shown to possess acontinuous ORF suggesting the presence of putative transposase-like coding regions. © 2000 Elsevier Science B.V. Allrights reserved.

Keywords: Transposable element; Mariner ; Plant-parasitic nematode; Meloidogyne

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1. Introduction

Transposable elements are widespread in allmajor phylogenetic groups. Because of their abil-ity to move from one location to another, they arevaluable tools for genetic analyses as mutagenicagents for the mapping and isolation of genes. In

Abbre6iations: MLE, mariner-like element; ORF, open read-ing frame; PCR, polymerase chain reaction.

* Corresponding author. Tel.: +33-4-9367-8943; fax: +33-4-9367-8955.

E-mail address: [email protected] (P. Abad)

0166-6851/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

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H. Leroy et al. / Molecular and Biochemical Parasitology 107 (2000) 181–190182

the nematode Caenorhabditis elegans, the Tc1 ele-ment has proved a valuable tool for reverse genet-ics [1]. Due to the intense interest in C. elegans asan experimental organism for developmental ge-netic studies and the availability of sophisticatedgenetics, most information about transposons isknown in this species. However, except thismodel, few elements are known in other nematodespecies. Some of them are present in nematodes ofmedical interest, such as TAS in Ascaris lumbri-coides [2] or mle-1 in Trichostrongylus colubri-formis [3]. But so far, there is no data concerningplant parasitic nematodes.

Endoparasitic nematodes, such as Meloidogyne,Globodera or Heterodera species, are pests of greatagronomic importance, infecting thousands ofplant species [4]. Some of them includingMeloidogyne ja6anica, Meloidogyne incognita,Meloidogyne arenaria, reproduce exclusively bymitotic parthenogenesis. Nevertheless, in theirclonal progeny, virulent individuals can appear onplants carrying genetic resistance. However, dueto lack of sexual reproduction, the inheritance ofavirulence and virulence cannot be directly testedbut must await development of new strategies tostudy genes involved in this mechanism. In thisconnection, transposable elements could be usedin reverse genetics as molecular tools for genetagging, since such a strategy has been used toisolate a range of genes in a large number ofspecies [5–7].

The mariner element was first found inDrosophila mauritiana [8]. Afterwards, it has beenidentified in an exceedingly wide variety ofgenomes ranging from fungi to plants or humans[9–14]. Mariner-like-elements (MLEs) are mem-bers of the mariner/Tc1 transposon superfamily.Members of this superfamily contain a singleopen reading frame (ORF) encoding a trans-posase, possess inverted terminal repeats, andtranspose via a DNA intermediate expressed inboth the germline and the soma [for review see[15]]. MLEs are a family of about 1250 bp trans-posons with 20–40 bp ITRs, encoding a trans-posase of about 345 amino acids. However,functional elements constitute only a small subsetof the mariner elements that have been sequenced.To date, the only known MLEs naturally active

are represented by Mos1 and closely relatedmauritiana MLEs [16]. Mos1 is an autonomouscopy of mariner isolated from D. mauritiana [17].All other MLEs that have been examined arenonfunctional either because of missense muta-tions in the transposase ORF or because of smalldeletions or point mutations that frameshift theORF. Most of the MLEs have been detected viaPCR experiments by using primers designed onconserved regions of the mariner transposasegene, representing a third of the element [10,18].Several MLEs have been isolated from nematodesand cestodes, two types have been found in thegenome of the nematode C. elegans and a relatedsequence has been isolated from the planarianworm Dugesia tigrina [11,19,20].

In this study, data on the presence of MLEs inthe plant parasitic nematode Meloidogyne is re-ported. These mariner elements, identified for thefirst time in phytoparasitic nematodes, may formnew distinct groups as compared with the marinersubfamilies known until now. Among them, fivePCR fragments show a continuous ORF, withoutstop codons, frameshifts or deletions. These datastrongly suggest that some Meloidogyne speciesmay possess mariner transposase-like codingregions.

2. Materials and methods

2.1. Nematode species

Each Meloidogyne isolate used in this studyoriginated from the progeny of a single female.The nematodes have been maintained in theINRA Antibes collection for several years andwere identified, at the species level, according totheir isoesterase electrophoretic pattern [21]. Thisbiochemical characterization was performed onseveral individual females to ensure that no mix-ture had occurred during nematode multiplica-tion. The Meloidogyne species tested were thefollowing: M. arenaria, M. incognita, M. ja6anica,Meloidogyne hapla and Meloidogyne chitwoodi.Except for the M. hapla and M. chitwoodi isolates,which reproduce by both facultative amphimixisand meiotic parthenogenesis, all the nematode

H. Leroy et al. / Molecular and Biochemical Parasitology 107 (2000) 181–190 183

isolates used in this study belong to mitoticparthenogenetic species. Two other phytoparasiticnematode species were analysed, Globodera pallidaand Heterodera schachtii that reproduce by sexualmating.

2.2. DNA extraction and Southern blotting

For each nematode isolate, total genomic DNAwas purified from 100 to 200 ml of second-stagejuveniles pooled together. Frozen nematodes wereground with a pestle and mortar in liquid nitrogenand DNA was extracted from the resulting pow-der by a phenol:chloroform procedure followedby ethanol precipitation [22]. Genomic DNA sam-ples from G. pallida and H. schachtii were kindlysupplied by Eric Grenier from INRA Rennes.

For Southern blot, the purified DNAs weredigested with restriction enzymes (BamHI andHindIII) and run on a 1% agarose gel. TheseDNAs were transferred onto Nylon membranes(Hybond-N, Amersham) according to standardprocedures [23]. Probes were labelled with [a-32P]dCTP (Amersham) by the random primingmethod using the Ready-to-Go kit (Pharmacia).Hybridizations were conducted overnight underhigh stringency conditions at 65°C [22]. Afterhybridization, filters were washed at 65°C for 30min in 2×SSPE–0.1% SDS and then for a fur-ther 30 min in 1×SSPE–0.1% SDS (1×SSPE:EDTA 1 mM, NaCl 150 mM, NaH2PO4 8.5 mM)and exposed to X-ray film with intensifying screenat −80°C.

2.3. PCR reactions

Amplifications of a conserved region of themariner transposase were performed on 50 ng ofgenomic DNA from each isolate using degenerateprimers previously described by Robertson [18]:MAR124F 5%-TGGGTNCCNCAYGARYT-3%(WVPHEL) and MAR276R 5%-GGNGCNAR-RTCNGGNSWRTA-3% (YSPDLAP), (Y=C orT; R=A or G; S=C or G; W=A or T; N=A,C, G or T). The reactions were performed in atotal volume of 50 ml and conducted in AppligeneTaq buffer adding 2 mM of each primer, 150 mMof each dNTP and 1 U of Taq polymerase. Each

reaction was overlaid with 100 ml of mineral oil toprevent evaporation. Amplifications were per-formed in a Biometra TRIO-Thermo-block ther-mal cycler. The cycling conditions used were asfollows: denaturation at 94°C for 1 min, anneal-ing at 52°C for 1 min and extension at 72°C for 1min, repeated for 40 cycles. A 5-min incubationperiod at 72°C followed the last cycle in order tocomplete any partially synthesized second strands.

2.4. Cloning, sequencing and sequence analysis

The amplified fragments were subcloned into apGEM®-T vector. Samples were sequenced usingT7 DNA polymerase (Pharmacia). Nucleotide se-quences were aligned using Clustal V [24] whileprotein sequences were manually adjusted withjudicious use of gaps and frameshifts to improvethe alignment.

Infobiogen facilities were used for phylogeneticanalysis [25]. The phylogenetic studies were per-formed using Phylip version 3.5c [26]. The analy-ses were done using parsimony programs with theprotpars option.

3. Results

3.1. PCR amplification, cloning and sequenceanalysis

The presence of MLEs in seven species of plantparasitic nematodes was examined by PCR usingdegenerate primers designed from two conservedamino acid motifs of the known MLE trans-posases. Fragments of weak intensity but with theexpected size (0.45–0.5 kb) were obtained fromMeloidogyne species and G. pallida, although sev-eral additional intense bands of higher molecularmass were detected. No PCR products were visu-alized on agarose gel for Heterodera species (datanot shown).

PCR products were purified and cloned into theplasmid pGEM®-T easy. At least three cloneswere sequenced for each species. Clones from fourof the five species of Meloidogyne (M. incognita,M. ja6anica, M. hapla and M. chitwoodi ) showedclear sequence similarities to putative mariner


transposase sequences. No similarities could befound for the clones from M. arenaria and G.pallida (data not shown). These mariners arenamed following the convention of Robertsonand Asplund [27].

Among the 13 PCR clones showing similaritiesto the known MLE transposases, the nucleotidesimilarities ranged from 54 to 63%. Within eachof the following species, M. incognita, M. haplaand M. chitwoodii, the sequenced clones showed agreat diversity ranging from 45 to 90% suggestingthe occurrence of element originating from differ-ent subfamilies of mariner in these species. On theother hand, the sequences from M. ja6anica cloneswere 98% homologous among themselves at thenucleic acid sequence level (data not shown).

In addition, the translation of five clones re-vealed a coding phase with no stop codons,frameshifts nor deletions. These uninterrupted se-quences were: Mimar1 and Mimar5 from M.incognita and Mcmar1, Mcmar2 and Mcmar4from M. chitwoodi. All the other sequences con-tain stop codons, frameshifts, and deletions (Fig.1).

An alignment of the amino acid sequencestranslated from the PCR products was made withthe homologous region of several known MLEsequences representative of the major marinersubfamilies previously defined. These sequencesare the active D. mauritiana mariner 1 [17], the D.tigrina mariner 1 [20], the Hyalophora cecropiamariner 1 [9], the Chrysolerpa plorabunda mariner1 [28], the Apis mellifera mariner 1 consensustransposase [27], the C. elegans mariner 1 consen-sus sequence (consensus transposase of three ele-ments according to Robertson [29]) and mariner 2elements [11], the Bombyx mori mariner 1 [27](Fig. 1).

The alignment showed several interestingpoints. First, all the elements clearly share highsimilarity and stretches of identity for this am-plified region which is thought to constitute theenzymatic core of the transposase. The consensussequence deduced from this alignment points outsome signature sequences already referenced asmariner signature sequences [29]. Except for Mh-mar3, all the PCR fragments possess the residuesAsp(D), Asp(D) (Fig. 1) which represent the two

first residues of the triad D,D34D known to beinvolved directly in the catalysis reaction [30].Second, mariner sequences from M. ja6anica havea deletion in their C terminus (positions 141–155and 159–160 in the alignment). Lastly, marinersequences from M. ja6anica and the clone Mc-mar1 possess a FQQD domain which constitutes apart of the Tc-like motif FQQDND (Fig. 1) in-stead of the mariner motif HDNARPH at their Cterminus.

3.2. Phylogenetic analysis

The 13 PCR clones were added to a previousalignment (deposited in EMBL under accessionnumber DS36877) of 25 mariner elements fromdifferent genomes (Bigot, unpublished data), suchas insects and related arthropods (18 species),human (two clones), nematodes (two species) andfungus (1 clone).

The phylogenetic analysis indicates that theMLEs from Meloidogyne spp. are distributedwithin two clusters (Fig. 2). The clones from M.ja6anica species are all clustered together while theother MLE clones are dispersed in the tree. Twonodes are supported by strong bootstrap values(99.7 and 89.1%). The first node branches to-gether Mcmar4, Mimar1, Mcmar2, Mimar5 andMcmar3 clones whereas the second assembles theMhmar2, Mhmar1 clones with all the M. ja6anicaclones. However, the bootstrap values designatedin Fig. 2 with arrows are not consistent enough(B50%) to determine the relative positions be-tween Meloidogyne clones, elegans, mauritiana,mellifera and capitata subfamilies. Nevertheless,mariner clones from Meloidogyne spp. appearedto be more closely related to elegans, mauritiana,mellifera, capitata and cecropia subfamilies thanthe irritans subfamily.

3.3. Distribution and copy number

To estimate the copy number and the distribu-tion of the mariner elements in the genome ofMeloidogyne, a Southern blotting analysis wasperformed using a mix of the PCR amplificationproducts as a probe to allow the detection of allthe copies originating from the different sub-


families present in the different Meloidogyne spe-cies. Fig. 3 shows many bands of different sizes andallows estimation of the copy number of MLEs atabout 20 copies per genome in M. ja6anica, M.incognita and M. arenaria. These results are consis-tent with those from C. elegans [11] and H. bacte-riophora [31]. However, M. hapla and M. chitwoodipossess only two or three copies per genome.

In addition, it can be seen that the probe cross-hybridizes with genomic DNA of M. arenaria(Fig. 3), a species for which the PCR amplifica-tion products had no homology with a marinerelement. However, when the PCR clones fromeach Meloidogyne species have been used as inde-pendent probes on the genomic DNA of M. are-naria, signals have been only obtained using

Fig. 1. Alignment of the translations of mariner transposase polymerase chain reaction (PCR) fragments from M. incognita, M.ja6anica, M. hapla and M. chitwoodi with those of insects, other invertebrates and the Caenorhabditis elegans Tc1 element. Thesequences aligned correspond to the mariner transposase region comprised between the N-terminal WVPHEL and the C-terminalYSPDLAP motifs. The Meloidogyne sequences are separated from the other sequences by a consensus sequence derived from themajority of available sequences at each position. Conserved residues with the mariner consensus sequence are highlighted in bold.Residues D, are indicated below the mariner consensus sequence by a plus sign (+ ), and the FQQD motif is boxed in grey.Alignment gaps are indicated by dashes (–), stop codons by asterisks (*), and frameshifts required to maintain an aligned readingframe by hash marks (c ).


Fig. 2. Phylogenetic relationships of the Meloidogyne mariner-like elements and other members of the mariner family. The tree wasobtained using the protein parsimony procedure in the Phylip package [26]. The values given at each node correspond to thebootstrap values (1000 repetitions). Some of the values, less than 50% and discussed in the text, are arrowed. The mariner-likesequences used and their accession number: Dmmar1 for Drosophila mauritiana (X78906), Mdmar1 for Mayetiola destructor(U24436), Btmar1 for Bombus terrestris (Bigot, unpublished data), Momar1 for Metaseuilius occidentalis (U12279), Cemar1 for thefirst type of MLE in Caenorhabditis elegans (M98552), Cemar2 for the second type of MLE in C. elegans (X77804), Cpmar1 forChrysoperla plorabunda (U11641), Dtmar1 for Dugesia tigrina (X79719), Himar1 for Haematobia irritans (U11646), Damar1 forDrosophila ananassae (U11656), Mpmar1 for Mantispa pulchella (U11649), Hcmar1 for Hyalophora cecropia (M63844), Demar1 forDrosophila erecta (U08094), Gpmar1 for Glossina palpalis (U18308), Ammar1 for Apis mellifera (U19902), Aamar1 for Attacus atlas(AB006464), Ccmar1 for Ceratitis capitata (U40493), Hsmar1 for the cecropia element from Homo sapiens (U52077), Hsmar2 for theirritans element from Homo sapiens (U49974), Hbmar1 for Heterorhabditis bacteriophora (U04455), Bmmar1 for Bombyx mori(AB006196), the Impala element from Fusarium oxysporum (S75103), the Bari element from Drosophila melanogaster (X67681) andthe Tc1 element from C. elegans (X01005).


Fig. 3. Autoradiogram of a Southern blot of Meloidogynegenomic DNAs digested with two restriction enzymes(BamHI/HindIII). Two micrograms of the digested DNAswere loaded in each lane for agarose gel electrophoresis. Theprobe was a mix consisted of all mariner cloned fragments, andwas directly made by mixing each DNA at the same concen-tration. The specific activity of this probe was 30. A total of108 dpm/microgram of DNA. Lane (1) M. incognita ; (2) M.arenaria ; (3) M. ja6anica ; (4) M. hapla ; (5) M. chitwoodi.

(data not shown).

4. Discussion

In this work, the presence of MLEs in theendoparasitic nematode Meloidogyne, are shownusing degenerate PCR primers from conservedamino acid regions of the mariner putative trans-posase. However, the two other tested species G.pallida and H. glycines did not yield any marinerelement . This result does not mean that they donot have any mariner in their genomes, as it mightbe due to a lack of sequence similarity. Robertsonfound MLEs in only 15% of 400 insect speciesusing different pairs of degenerate PCR primers[10]. Furthermore, this observation is supportedby the fact that the PCR clones from M. arenariadid not yield any similarity with a mariner ele-ment, but in the genomic DNA of M. arenaria,these elements were revealed by hybridizationwith a mariner probe from elements of M.incognita.

Inside the Nematoda phylum, only two kinds ofMLE subfamilies have been found: the eleganssubfamily from the bacteriophagous species,Caenorhabditis spp. [11,19], and a cecropia sub-family element from the entomopathogenous spe-cies, H. bacteriophora [31]. Therefore, the presenceof mariner elements in Meloidogyne species ex-tends the host range and the diversity of marinerfamily transposons. The exact relationships be-tween the subfamilies are difficult to determinewithout full-length sequences from genomic li-braries. Nevertheless, it has been shown that PCRfragments from the conserved region, localizedbetween the sequence coding for the WVPHEL-YSPDLAP motifs, usually represent mariner thatcan be recovered from these genomes [32].

The estimation of the copy number of MLEs inMeloidogyne spp. was done by Southern blotanalysis, and was about 20 copies per genome.However, two species possess a lower number ofcopies: two copies per genome were estimated tobe present in M. hapla and three copies pergenome for M. chitwoodi. This situation could beexplained by the fact that these two species arediploid, while the other species studied in this

clones from M. incognita. In that case, the patternobtained was the same as the one shown in Fig. 3


work are polyploid [33]. Nevertheless, these copynumbers are much less than the 1000 copies ofMLEs in H. cecropia or the 8000 copies in aplanarian (Plathelminthe) [34], but comparable tothe 20 copies found in D. mauritiana [8] or in C.elegans N2 strain [11].

The data obtained from the phylogenetic analy-sis revealed three interesting points. First, thebootstrap analysis gave 99.7 and 89.1% support totwo Meloidogyne mariner branches. TheseMeloidogyne clones seem then to form new MLEsubfamilies. Second, the presence of mariner sub-families may be evident in some Meloidogynespecies. According to the definition of a subfamilyas a distinct grouping in the phylogenetic treebased on sequence comparisons, it is notable thatM. incognita, M. hapla and M. chitwoodi possesssequences from at least two types of MLEs. Thishas already been described in other species whichhave multiple types of mariner elements, e.g. sev-eral insects have up to six kinds of MLEs [35].Third, from this study, it appears that the Mc-mar4 and Mimar1 clones are grouped in the tree,indicating that they belong to the same subfamilyof MLEs, but they have originated from twodifferent species, respectively M. chitwoodi and M.incognita. In addition, M. hapla possesses twokinds of MLE since two clones, Mhmar1 andMhmar2, are related to those of M. ja6anicaclones. Because the bootstrap analysis gave 51.1%support to the node grouping cecropia subfamilyelements (elements known to belong to thisgrouping [10]), Mhmar3 can be considered relatedto the elegans subfamily since the bootstrap sup-port gave 52.2%. Such peculiar distribution ofMLE in different species of the Meloidogynegenus could be explained by two alternative, butnot exclusive hypotheses: (i) transposable ele-ments have evolved from a common ancestor.Based on cytogenetic information, it can be con-sidered that the ancestral root-knot nematodeswere amphimictic animals, from which theparthenogenetic forms have evolved [33]. More-over, M. hapla and M. chitwoodi, which are mei-otic parthenogenetic nematodes with facultativeamphimixis, may hold a position closer to theancestral forms of root-knot nematodes and mayhave played a significant role in the evolution of

all other forms such as M. ja6anica, M. incognitaand M. arenaria, which are mitotic partheno-genetic animals. In that respect, an ancestralmariner sequence may have evolved from an am-phimictic Meloidogyne species by vertical trans-mission towards the parthenogenetic species,resulting in the occurrence of different kinds ofMLEs in the divergent species. (ii) The secondassumption is that mariner transposable elementphylogeny is dominated by horizontal transferevents. The occurrence of recent horizontal trans-fers of mariner elements has been documented.Horizontal transmission was first suggested by theisolation of an MLE from Zaprionus tuberculatus97% identical in sequence with the Mos1 elementfrom D. mauritiana, whereas a mauritiana sub-family element from Drosophila tsacasi belongingto the same subgenus was only 92% identical withMos1 [36]. Other cases of probable horizontaltransmission were discovered by Robertson [18]and Robertson and MacLeod [10] in a study ofthe presence of MLEs in insects. The distributionof MLEs is often nonuniform and inconsistentwith the phylogeny of the host species [37]. Todate, the presence of MLEs in nematodes havebeen detected in only three genera: Caenorhabditis(bacteriophagous nematodes), Heterorhabditis(entomoparasitic nematodes) and Trichostrongylus(mammalian parasitic nematodes). Therefore, thepresence of MLEs in a phytoparasitic nematoderaises the question of their origin. Even if trans-posable elements are usually transmitted fromgeneration to generation by vertical transmission,they can exceptionally be transmitted laterallyacross the barriers of species with the help ofvectors and/or mechanisms yet unknown. Thereare many possibilities: parasites, symbionts, infec-tious agents, DNA or RNA viruses, retroviruses.An example can illustrate this hypothesis as theMcmar4 clone found in M. chitwoodi is closer tothe Mimar1 clone from M. incognita than anyother M. chitwoodi clones (Fig. 2). Consideringthese two nonexclusive hypotheses, both verticaland horizontal transmissions may have con-tributed to the dispersion of MLEs in these endo-parasitic nematodes.

Some results obtained in this study show aparadoxical situation concerning the M. ja6anica


species, as on the one hand very homologousmariner sequences have been identified, and onthe other hand these elements have accumulatedmutations, deletions and stop codons resulting inan interrupted ORF. A reduced number of fullyactive transposons could be responsible for themaintenance and spread of the element, while themariner sequences as an average could be evolvingneutrally, rapidly acquiring mutations [27]. Ac-cording to recent understanding of the naturalhistory of MLEs [35], vertical inactivation andstochastic loss are the long term fate of MLEs inthe genome. Thus, MLEs from M. ja6anica wouldbe in the inactivated state. In eukaryotes, mosttransposons are expected to be inactive because oflack of selection pressure [38], a prediction that isconfirmed for most mariner elements found todate [rewieved in [39]]. Therefore, an evolutionaryquestion still remains for M. ja6anica clones con-cerning the paradox between the fact that they aredegenerate mariner elements and they are so con-served in this genome.

Most of the PCR clones analysed in this workproved to be from highly defective elements, hav-ing accumulated many mutations including stopcodons and frameshifts, as usually observed inmariner elements from other organisms. However,five PCR clones, Mimar1, Mimar5, Mcmar1, Mc-mar2 and Mcmar4 were recovered with a continu-ous ORF. Although this PCR experiment did notallow recovery of the whole sequence of the cata-lytic domain of the putative transposase (the Cterminal part of this domain is missing), these fiveclones all possess a D,D motif which is part of thesequence signature of MLEs (the triad D,D34D).Therefore, these data raise the possibility thatfully functional mariner-like elements might existin M. chitwoodi and M. incognita, which couldprovide valuable tools for gene tagging in thesetwo species.

Acknowledgements

Nucleotide sequence data reported in this paperare available in the EMBL, Genbank™ andDDJB databases under the accession numbersAJ251404 to AJ251416.

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identification of mariner-like elements from the root-knot nematode meloidogyne spp

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