Development of Bursaphelenchus xylophilus-specific microsatellite markers to assess the genetic diversity of populations from European forests.

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer, 1934), Nickle (Nematoda: Aphelenchoididae) is the causal agent of the pine wilt disease and is currently considered as one of the most important pests and pathogens in the world. Its introduction and spread in new forest ecosystems have considerable consequences both economically and environmentally. Therefore, it is of crucial importance to identify its invasion routes, to determine the origin of new outbreaks and to understand the invasion process of this species to prevent further dissemination of the disease in Europe. In order to address these questions using population genetic approaches, we have been developing a set of PWN-specific microsatellite markers, usable in routine conditions at the individual level, thanks to multiplex PCR coupled with a fast DNA extraction method. Microsatellites were isolated from a genomic library using a procedure combining DNA enrichment and high throughput pyrosequencing as recently described by Malausa et al. (2011). Primers were designed for 71 and 23 perfect and compound microsatellites, respectively, 26 of which were experimentally validated so far. Among them, 18 markers exhibited polymorphism after several rounds of amplification tests. Preliminary results on a set of 190 nematodes from 13 populations indicate a very low level of polymorphism in PWN populations from Portugal and Madeira Island, compared to populations from the native area in North America. The genotyping of a wide collection of samples from Europe, Asia and North America is currently underway in the laboratory. Assessing the genetic diversity of populations indeed constitutes the cornerstone to determine whether the European invasive PWN populations are the result of a single or several independent events of introduction

Variability among four populations of Meloidogyne javanica from Brazil

La morphologie, les profils isoenzymatiques, la caryolgie, la gamme d'hôtes et l'analyse de l'ADN (PCR-RAPD) de quatre populations atypiques de #Meloidogyne javanica originaires du Brésil (P1-P4) ont été comparés à ceux de deux populations typiques appartenant à la même espèce (S1 et S2). Ces approches ont permis la séparation de ces populations en deux groupes : P1, S1 et S2, d'une part et P3 et P4, d'autre part. En se basant sur l'analyse de l'ADN, la population P2 présente une position intermédiaire, mais semble très proche de P1 d'après ses caractères morphologiques et le nombre de ses chromosomes. Elle en diffère cependant par son phénotype estérasique et par sa gamme d'hôtes. La technique de PCR-RAPD a permis la détection d'une variabilité intraspécifique notable. Cela représente un avantage certain par rapport à l'utilisation des isoenzymes et des gammes d'hôtes, dans la mesure où le nombre d'hôtes différentiels et de systèmes enzymatiques disponsibles est limité. Par comparaison avec d'autres études, nos résultats mettent en évidence une variabilité intraspécifique élevée chez #M. javanica, espèce considérée jusque là comme relativement homogène. (Résumé d'auteur

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation

Sequence variability of the MspI satellite DNA family of the pinewood nematode Bursaphelenchus xylophilus at different geographic scales

Tandemly repeated sequences known as satellite DNA (satDNA) generally exhibit complex evolutionary patterns of concerted evolution in which mutations are homogenized and fixed in a stochastic process of molecular drive. Here, the nucleotidic variability of the MspI satDNA family of the pinewood nematode Bursaphelenchus xylophilus is analyzed in order to understand the evolutionary dynamics of satDNA at the intraspecific level. A total of 425 MspI monomer units, either PCR-amplified from isolates of local (Peninsula of Setúbal, Portugal) or worldwide origin, or retrieved from the B. xylophilus genome sequence, were characterized and compared. Whatever their origin, sliding window analysis of sequence variability patterns among monomers revealed low, moderate and highly variant domains, indicating that variable levels of evolutionary constraint may act upon the entire monomers. The phylogenetic inference based on the different sets of MspI satDNA family for this species shows a broad polymorphism of the individual monomers, which were distributed into four main clusters. However, such clustering appeared indepen- dent from the geographic origin of the nematodes, and could not discriminate isolates or groups of geo- graphically close isolates. Rather, the formation of different phylogenetic groups within this satDNA family suggests an a priori embodying of a set of diverging repeats from a common ancestor satDNA library, which have been differently amplified along the evolutionary pathway of this species. The present work improves knowledge on the evolutionary dynamics of satDNA at the intraspecific level, and pro- vides new information on satDNA sequence variability among natural populations sampled at a local geo- graphic scale

First insights into the genetic diversity of the pinewood nematode in its native area and around the world.

The pinewood nematode (PWN), Bursaphelenchus xylophilus is the causal agent of the pine wilt disease and is currently considered as one of the most important pests and pathogens in forest ecosystems. Native to North America, it has been introduced and it has spread in pine forests in Asia and more recently in Europe where it has now considerable economic and environmental impacts (annual loss of millions of pine trees worldwide). Anticipating the possibility of expansion of the PWN in European forests is essential. It is therefore important to decipher the invasion routes and better understand the invasion process of this species. To do this, 16 microsatellite markers have been developed and the study of genetic variability of the PWN was undertaken

Conserved DNA motifs, including the CENP-B box-like, are involved in satellite DNA array rearrangements

Satellite DNAs (satDNAs), despite rapid evolution that continuously remodel the genomic landscape, occupy functionally essential centromeric regions. Difficult to be explored due to their repetitive nature and divergence, satDNAs are still hardly accessible frontiers of eukaryotic genomes and knowledge concerning functional significance of satellite DNAs is rather limited. In this work, we provide a comprehensive analysis of six satDNAs in the library of recently separated root-knot nematodes Meloidogyne chitwoodi and M. fallax. We disclosed two different conserved regions common for analyzed satDNAs. One appeared to be highly similar to the CENP-B box of human alpha satDNA, which emerged, in sequence alignment, as a conserved segment common for six divergent satDNAs shared by closely related genomes. Observed results emphasize it as the most prominent example of the CENP-B box-like motif out of mammals. The proposed feature of the CENP-B box-like motif is to act as a promoter in the hypothesized cut-and-paste transposition-related mechanism. This observation could represent a novel role of the CENP-B box, in addition to the known function in centromere protein binding. We propose that the second conserved sequence motif detected in explored satDNAs is involved in illegitimate recombination. In parallel to alpha satDNAs, we found organization of satDNA arrays in nematodes comparable to that found in human and primates, in the form of simple and complex higher order repeats (HORs). In contrast to human satDNA organization, characterized by phylogenetically distinct HOR and monomeric forms, organizational patterns observed in nematodes are consistent with frequent and continuous shuffling of sequences between HORs and monomeric arrays. Our results suggest the role of conserved domains in mechanisms that cause rapid shuffling of sequences among divergent satDNAs, on the level of short-segment tracts. In context of satDNA evolution, our finding provides, for the first time, an experimentally verified link between conserved domains and satDNA rearrangement events