A species specific satellite DNA family in the genome of the(4)

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Fig. 4 Nucleotide consensus sequence of the Bgl II monomer of the pMeL satellite DNA family from Meloidogyne exigua. The 7 bp (7a, 7b) and 8 bp (8a, 8b) direct repeats are indicated with grey arrows. Restriction sites are shown with black dashes.

A computer search for evidence of internal repeated subunits in the pMeL consensus sequence allowed the detection of several direct subrepeats: two 8 bp motives repeated two times each, and two 7 bp motives repeated two and three times, respectively (Fig. 4). Analysis of the restriction sites in the consensus sequence showed a good correlation with the patterns observed on the Southern blot (Fig. 1): one Bgl II, two RsaI and two Sau3A I restriction sites, respectively. No Alu I restriction site was detected in the consensus sequence, but four monomers (pMeL1, 5, 11 and 14) exhibited such a restriction site in positions 65– 68, due to a C-to-A substitution in position 66 (Fig. 3). A search in the EMBL and GENBANK nucleic acid databases revealed no signi cant similarity with any recorded seq

uence, thus suggesting that the pMeL family represents a novel satellite DNA family in RKN. Speci c distribution of the satellite DNA and detection of single nematodes To test the speci city of the distribution of the Bgl II satellite DNA in RKN, the genomic DNA of 12 isolates belonging to eight Meloidogyne species was dotted on to a nylon membrane and hybridized with the probe consisting of a cloned monomer (pMeL8). The autoradiography shown in Fig. 5 indicates that hybridization only occurred with the three M. exigua isolates tested, thus demonstrating the species-speci c distribution of the pMeL satellite DNA family. The pMeL8 probe was also tested directly on the biological material itself, using a squash-blot procedure that avoided any time-consuming DNA extraction step. As shown in Fig. 6A, either one second-stage juvenile, one female, one egg-mass or one female in galled root tissues could be unambiguously identi ed after 3–5 h exposure of the membrane. At the same time, the

Fig. 5 Dot-blot experiment using a cloned satellite DNA monomer as a probe. Dotted genomic DNAs are as follows: A1, A2, A3= M. exigua ex1, ex2, ex3, respectively; B1= M. arenaria are1; B2= M. hapla ha1; B3, C1, C2= M. incognita inc2, inc3, inc5, respectively; C3= M. javanica jav1; D1= M. paranaenesis par5; D2= M. chitwoodi chi1; D3= M. fallax fa1. Population codes are given in Table 1.

absence of cross-hybridization of the probe with non-infested roots was checked (data not shown). Moreover, the same speci city of hybridization as displayed in the previous dot-blot experiment was observed, with strong signals detected with M. exigua isolates only (Fig. 6A,B). In conclusion, the results of the squash-blot experiments con rmed the species-speci c distribution of the Bgl II satellite DNA, and demonstrated the extreme sensitivity of the detection where it was used as a probe.

DISCUSSIONIn the present study, we have isolated and characterized a new Bgl II satDNA family from the coffee RKN M. exigua, which consists of tandemly arranged monomeric units of 277 bp. There are approximately 17 900 copies of this element, which comprise about 9.7% of the nematode genome. This value is in good agreement with the genomic content of satDNA sequences previously found in other Meloidogyne species, which ranges from 2.5% in M. incognita (Piotte et al ., 1994) up to 20% in M. fallax (Castagnone-Sereno et al., 1998). Twenty independent monomers of the Bgl II satDNA family were randomly selected, sequenced and compared, assuming

MOLECULAR PLANT PATHOLOGY (2002) 3(6), 431–437© 2002 BLACKWELL SCIENCE LTD

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