A species specific satellite DNA family in the genome of the

MOLECULAR PLANT PATHOLOGY (2002) 3(6), 431–437

A species-speci c satellite DNA family in the genome of the coffee root-knot nematode Meloidogyne exigua: application to molecular diagnostics of the parasiteO N I VA L D O R A N D I G 1, M I C H E L B O N G I O VA N N I 1, R E G I N A M . D. G. C A R N E I R O 2, J E A N - L O U I S S A RA H 3 A N D P H I L I P P E C A S T A G N O N E - S E R E N O 1, *1 2

Blackwell Science, Ltd

INRA, Unité Interactions Plantes–Microorganismes et Santé Végétale, BP2078, 06606 Antibes Cedex, France, EMBRAPA, Recursos Genéticos e Biotecnologia, CP 02372, 70849-970 Brazilia, DF, Brazil, 3 CIRAD-AMIS, Laboratoire Phytrop, TA 40/02, Avenue Agropolis, 34398 Montpellier Cedex 5, France

SUMMARYA new Bgl II satellite DNA has been isolated, cloned and sequenced from the coffee root-knot nematode, Meloidogyne exigua (Nematoda: Tylenchida). It is represented as tandemly repeated sequences with a monomeric unit of 277 bp. The monomers are present at approximately 17 900 copies per haploid genome, and represent about 9.7% of the total genomic DNA. Twenty randomly chosen monomers have been sequenced. The deduced unambiguous consensus sequence is 277 bp long, and displays an A+ T content of 54.2%. The monomers are very homogenous in sequence, showing on average 2.4% divergence from their consensus. Therefore, it is hypothesized that this repeated family may have recently appeared in the genome of the nematode, through some extensive ampli cation burst. Using a cloned monomer as a probe, dot-blot experiments demonstrated the species-speci c distribution of the Bgl II satellite DNA. Moreover, squash-blot assays allowed us to detect single M. exigua individuals, at any developmental stage, and even within root tissues, without the need for preliminary DNA puri cation. From these results, it is concluded that the procedure described, using the satellite DNA as a sensitive species-speci c probe, should constitute an improved and accurate diagnosis method for the detection and identi cation of the nematode, which would contribute to the implementation of targeted pest management strategies in all coffee growing countries of South and Central America.

I N T RO D U C T I O NNematodes belonging to the genus Meloidogyne, commonly known as root-knot nematodes (RKN), constitute the most widely distributed*Correspondence: E-mail: pca@antibes.inra.fr

and damaging group of plant-parasitic nematodes, being responsible for at least 5% of crop losses on a worldwide basis (Sasser and Carter, 1985). These sedentary biotrophic endoparasites have evolved very complex relationships with the root tissues of their hosts, in which they induce the formation of specialized feeding structures known as giant cells (Williamson and Hussey, 1996). The disease is characterized by galls or root-knots on infected plants. Symptoms include poor fruit yield, stunted growth, wilting, and increased susceptibility to other pathogens. O

n coffee (Coffea arabica), RKN are the most damaging nematode species, causing great losses to farmers and to the economy of developing countries. Among them, M. exigua is certainly the most widely distributed and serious species, present in nearly all the major coffee growing countries of South and Central America, either as separate species or mixed with other species (Campos et al., 1990). Its economic importance is so great that coffee has been replaced by other crops in heavily infested areas, as has been well documented for Brazil (Campos et al., 1990). Considering the agricultural impact of M. exigua, and the diversity of nematode populations in the eld, it would be highly useful to develop speci c DNA probes for diagnostic purposes. However, little is known about the basic molecular biology of these organisms. Thus, characterization of the highly repetitive fraction of the genome of this nematode has been initiated. Tandemly arranged repetitive DNA sequences, referred to as satellite DNA (satDNA), are ubiquitous constituents of all eukaryotic genomes and are widely represented in animal and plant species (Beridze, 1986). They consist of very high repetitions of a basic monomeric unit 70–2000 bp long and appear to be clustered in heterochromatin centromeric and telomeric regions (Charlesworth et al., 1994). Although no de ned function has yet been established, satDNA has been suggested to be involved in evolutionary processes and the stability of genome structure (Csink and Henikoff, 1998). Because it diverged rapidly during evolution, and is constantly homogenized, it often gives rise to sequences that are species- or genome-speci c (Bachmann et al.,

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