Postembryonic establishment of megabase-scale gene silencing in nucleolar dominance

Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor’s rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, theA. thaliana –derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generatio

Interplay of ribosomal DNA Loci in nucleolar dominance: dominant NORs are up-regulated by chromatin dynamics in the wheat-rye system

Background: Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions. Methodology/Principal Findings: Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs. Conclusions/Significance: We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are alsomodified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to ‘mutual amphiplasty’ in the nucleolar dominance process.This work was supported by the Fundação para a Ciência e Tecnologia (projects POCI/BIA-BDE/57575/2004 to M.S. and POCI/BIA-BCM/59389/2004 to N.N.

Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences

The tobamovirus resistance gene L3 of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinenseL3 gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L3-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L3 gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L3 locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus

Differing Requirements for RAD51 and DMC1 in Meiotic Pairing of Centromeres and Chromosome Arms in Arabidopsis thaliana

During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequence

Tracking seasonal changes in North Sea zooplankton trophic dynamics using stable isotopes

Trophodynamics of meso-zooplankton in the North Sea (NS) were assessed at a site in the southern NS, and at a shallow and a deep site in the central NS. Offshore and neritic species from different ecological niches, including Calanus spp., Temora spp. and Sagitta spp., were collected during seven cruises over 14 months from 2007 to 2008. Bulk stable isotope (SI) analysis, phospholipid-derived fatty acid (PLFA) compositions, and δ 13CPLFA data of meso-zooplankton and particulate organic matter (POM) were used to describe changes in zooplankton relative trophic positions (RTPs) and trophodynamics. The aim of the study was to test the hypothesis that the RTPs of zooplankton in the North Sea vary spatially and seasonally, in response to hydrographic variability, with the microbial food web playing an important role at times. Zooplankton RTPs tended to be higher during winter and lower during the phytoplankton bloom in spring. RTPs were highest for predators such as Sagitta sp. and Calanus helgolandicus and lowest for small copepods such as Pseudocalanus elongatus and zoea larvae (Brachyura). δ 15NPOM-based RTPs were only moderate surrogates for animals’ ecological niches, because of the plasticity in source materials from the herbivorous and the microbial loop food web. Common (16:0) and essential (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) structural lipids showed relatively constant abundances. This could be explained by incorporation of PLFAs with δ 13C signatures which followed seasonal changes in bulk δ 13CPOM and PLFA δ 13CPOM signatures. This study highlighted the complementarity of three biogeochemical approaches for trophodynamic studies and substantiated conceptual views of size-based food web analysis, in which small individuals of large species may be functionally equivalent to large individuals of small species. Seasonal and spatial variability was also important in altering the relative importance of the herbivorous and microbial food webs