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

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

Satellitome analyses in nematodes illuminate complex species history and show conserved features in satellite DNAs

Background: Satellite DNAs (satDNAs) are tandemly repeated non-coding DNA sequences that belong to the most abundant and the fastest evolving parts of the eukaryotic genome. A satellitome represents the collection of different satDNAs in a genome. Due to extreme diversity and methodological difficulties to characterize and compare satDNA collection in complex genomes, knowledge on their putative functional constraints and capacity to participate in genome evolution remains rather elusive. SatDNA transcripts have been detected in many species, however comparative studies of satDNA transcriptome between species are extremely rare. Results: We conducted a genome-wide survey and comparative analyses of satellitomes among different closely related Meloidogyne spp. nematodes. The evolutionary trends of satDNAs suggest that each round of proposed polyploidization in the evolutionary history is concomitant with the addition of a new set of satDNAs in the satellitome of any particular Meloidogyne species. Successive incorporation of new sets of satDNAs in the genome along the process of polyploidization supports multiple hybridization events as the main factor responsible for the formation of these species. Through comparative analyses of 83 distinct satDNAs, we found a CENP-B box-like sequence motif conserved among 11 divergent satDNAs (similarity ranges from 36 to 74%). We also found satDNAs that harbor a splice leader (SL) sequence which, in spite of overall divergence, shows conservation across species in two putative functional regions, the 25-nt SL exon and the Sm binding site. Intra- and interspecific comparative expression analyses of the complete satDNA set in the analyzed Meloidogyne species revealed transcription profiles including a subset of 14 actively transcribed satDNAs. Among those, 9 show active transcription in every species where they are found in the genome and throughout developmental stages. Conclusions: Our results demonstrate the feasibility and power of comparative analysis of the non-coding repetitive genome for elucidation of the origin of species with a complex history. Although satDNAs generally evolve extremely quickly, the comparative analyses of 83 satDNAs detected in the analyzed Meloidogyne species revealed conserved sequence features in some satDNAs suggesting sequence evolution under selective pressure. SatDNAs that are actively transcribed in related genomes and throughout nematode development support the view that their expression is not stochastic

As traduções de Le Fanatisme ou Mahomet le Prophète na cena e na página: um caso de voltairomania nas últimas décadas do século XVIII português

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