A species specific satellite DNA family in the genome of the(3)

Meloidogyne exigua satellite DNA

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Quanti cation and copy number in the genome The relative abundance of the Bgl II repetitive sequence was assessed from dot blot experiments. Increasing amounts of a cloned monomer (pMeL8) and M. exigua (isolate ex1) genomic DNA were blotted on to nylon membranes and hybridized with the DNA insert released from the same clone. pBs was used as a background control and Caenorhabditis elegans, Drosophila melanogaster and calf thymus DNAs were used as negative controls (data not shown). The satellite DNA family appeared to make up to 9.7% of the nematode genomic DNA, as calculated from values obtained by scintillation measurements. Assuming that the genome size of Meloidogyne is about 51 Mb (Pableo and Triantaphyllou, 1989), and based on a monomer size of 277 bp (see below), this fraction corresponds to approximately 17 900 copies per haploid genome. Monomer sequence analysis The≈ 270 bp Bgl II restriction fragments were isolated from the gel and subcloned into the pBS vector. Twenty positive clones, named pMeL(n), were selected at random and sequenced (Fig. 3). Most of these sequences were 277 bp long, except for ve clones which exhibited lengths of 273 bp (pMeL13 and 15), 280 bp (pMeL3), and 281 bp (pMeL9 and 18), respectively. This length closely agreed with the estimate based on the

electrophoretic mobility of the restriction fragments.

An unambiguous 277 bp long consensus sequence was derived from the complete data set, and is listed at the top of Fig. 3. It was deposited in the GENBANK Nucleotide Sequence Database under accession no. AY078994. The consensus has an A+ T content of 54.2%. Analysis of the primary structure of the 20 monomers in relation to the consensus sequence revealed a high homogeneity of this reiterated DNA family, with an overall average pair-wise sequence variability of only 2.4%. Compared to the consensus sequence, the individual divergence of the cloned monomers to the consensus ranged from 0.7% to 5.4%, with a total of 32 variable positions (11.6%). The variable positions were not distributed at random, but were exclusively concentrated between positions 49 and 144, and 217 and 242. Most of the mutations to the consensus sequence were single-point substitutions, except for three short deletions (1 or 4 bp) in clones pMeL3, 13 and 15, and three short insertions (4 bp) in clones pMeL3, 9 and 18. Interestingly, the same 4 bp motif (TCTT) was involved in both the insertions and deletions. In general, the observed nucleotide substitutions appeared to be shared between two or more monomers (e.g. a C-to-T substitution in positions 52 for 9 clones, or an A-to-G substitution in position 123 for 7 clones; see Fig. 3). In addition, since dimers were still present after 6 h of digestion with Bgl II (Fig. 2), mutations should also have occurred at the enzyme restriction site.

Fig. 3 Nucleotide sequences of 20 randomly cloned Meloidogyne exigua satellite DNA monomers, and the derived consensus sequence. In the monomer sequences, positions showing no variation from the consensus are shown with a dot and deletions are indicated with a dash. The black arrow indicates the position of a 4 bp insertion (TCTT) in clones pMeL3, 9 and 18.

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

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