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Background and AimsEndogenous pararetroviruses (EPRVs) are widespread components of plant genomes that originated from episomal DNA viruses of the Caulimoviridae family. Due to fragmentation and rearrangements, most EPRVs have lost their ability to replicate through reverse transcription and to initiate viral infection. Similar to the closely related retrotransposons, extant EPRVs were retained and often amplified in plant genomes for several million years. Here, we characterize the complete genomic EPRV fraction of the crop sugar beet (Beta vulgaris, Amaranthaceae) to understand how they shaped the beet genome and to suggest explanations for their absent virulence.MethodsUsing next- and third-generation sequencing data and genome assembly, we reconstructed full-length in silico representatives for the three host-specific EPRVs (beetEPRVs) in the B. vulgaris genome. Focusing on the endogenous caulimovirid beetEPRV3, we investigated its chromosomal localization, abundance and distribution by fluorescent in situ and Southern hybridization.Key ResultsFull-length beetEPRVs range between 7.5 and 10.7 kb in size, are heterogeneous in structure and sequence, and occupy about 0.3 % of the beet genome. Although all three beetEPRVs were assigned to the florendoviruses, they showed variably arranged protein-coding domains, different fragmentation, and preferences for diverse sequence contexts. We observed small RNAs that specifically target the individual beetEPRVs, indicating stringent epigenetic suppression. BeetEPRV3 sequences occur along all sugar beet chromosomes, preferentially in the vicinity of each other and are associated with heterochromatic, centromeric and intercalary satellite DNAs. BeetEPRV3 members also exist in genomes of related wild species, indicating an initial beetEPRV3 integration 13.4–7.2 million years ago.ConclusionsOur study in beet illustrates the variability of EPRV structure and sequence in a single host genome. Evidence of sequence fragmentation and epigenetic silencing implies possible plant strategies to cope with long-term persistence of EPRVs, including amplification, fixation in the heterochromatin, and containment of EPRV virulence.

[BACKGROUND] Long terminal repeat (LTR) retrotransposons are widespread in plant genomes and play a large role in the generation of genomic variation. Despite this, their identification and characterization remains challenging, especially for non-model genomes. Hence, LTR retrotransposons remain undercharacterized in Vaccinium genomes, although they may be beneficial for current berry breeding efforts. [OBJECTIVE] Exemplarily focusing on the genome of American cranberry (Vaccinium macrocarpon Aiton), we aim to generate an overview of the LTR retrotransposon landscape, highlighting the abundance, transcriptional activity, sequence, and structure of the major retrotransposon lineages. [METHODS] Graph-based clustering of whole genome shotgun Illumina reads was performed to identify the most abundant LTR retrotransposons and to reconstruct representative in silico full-length elements. To generate insights into the LTR retrotransposon diversity in V. macrocarpon, we also queried the genome assembly for presence of reverse transcriptases (RTs), the key domain of LTR retrotransposons. Using transcriptomic data, transcriptional activity of retrotransposons corresponding to the consensuses was analyzed. [RESULTS] We provide an in-depth characterization of the LTR retrotransposon landscape in the V. macrocarpon genome. Based on 475 RTs harvested from the genome assembly, we detect a high retrotransposon variety, with all major lineages present. To better understand their structural hallmarks, we reconstructed 26 Ty1-copia and 28 Ty3-gypsy in silico consensuses that capture the detected diversity. Accordingly, we frequently identify association with tandemly repeated motifs, extra open reading frames, and specialized, lineage-typical domains. Based on the overall high genomic abundance and transcriptional activity, we suggest that retrotransposons of the Ale and Athila lineages are most promising to monitor retrotransposon-derived polymorphisms across accessions. [CONCLUSIONS]We conclude that LTR retrotransposons are major components of the V. macrocarpon genome. The representative consensuses provide an entry point for further Vaccinium genome analyses and may be applied to derive molecular markers for enhancing cranberry selection and breeding.

BACKGROUNDLong terminal repeat (LTR) retrotransposons are widespread in plant genomes and play a large role in the generation of genomic variation. Despite this, their identification and characterization remains challenging, especially for non-model genomes. Hence, LTR retrotransposons remain undercharacterized in Vaccinium genomes, although they may be beneficial for current berry breeding efforts.OBJECTIVEExemplarily focusing on the genome of American cranberry (Vaccinium macrocarpon Aiton), we aim to generate an overview of the LTR retrotransposon landscape, highlighting the abundance, transcriptional activity, sequence, and structure of the major retrotransposon lineages.METHODSGraph-based clustering of whole genome shotgun Illumina reads was performed to identify the most abundant LTR retrotransposons and to reconstruct representative in silico full-length elements. To generate insights into the LTR retrotransposon diversity in V. macrocarpon, we also queried the genome assembly for presence of reverse transcriptases (RTs), the key domain of LTR retrotransposons. Using transcriptomic data, transcriptional activity of retrotransposons corresponding to the consensuses was analyzed.RESULTSWe provide an in-depth characterization of the LTR retrotransposon landscape in the V. macrocarpon genome. Based on 475 RTs harvested from the genome assembly, we detect a high retrotransposon variety, with all major lineages present. To better understand their structural hallmarks, we reconstructed 26 Ty1-copia and 28 Ty3-gypsy in silico consensuses that capture the detected diversity. Accordingly, we frequently identify association with tandemly repeated motifs, extra open reading frames, and specialized, lineage-typical domains. Based on the overall high genomic abundance and transcriptional activity, we suggest that retrotransposons of the Ale and Athila lineages are most promising to monitor retrotransposon-derived polymorphisms across accessions.CONCLUSIONSWe conclude that LTR retrotransposons are major components of the V. macrocarpon genome. The representative consensuses provide an entry point for further Vaccinium genome analyses and may be applied to derive molecular markers for enhancing cranberry selection and breeding.

In eukaryotic genomes, cycles of repeat expansion and removal lead to large-scale genomic changes and propel organisms forward in evolution. However, in conifers, active repeat removal is thought to be limited, leading to expansions of their genomes, mostly exceeding 10 gigabasepairs. As a result, conifer genomes are largely littered with fragmented and decayed repeats. Here, we aim to investigate how the repeat landscapes of two related conifers have diverged, given the conifers’ accumulative genome evolution mode. For this, we applied low coverage sequencing and read clustering to the genomes of European and Japanese larch, Larix decidua (Lamb.) Carrière and Larix kaempferi (Mill.), that arose from a common ancestor, but are now geographically isolated. We found that both Larix species harbored largely similar repeat landscapes, especially regarding the transposable element content. To pin down possible genomic changes, we focused on the repeat class with the fastest sequence turnover: satellite DNAs (satDNAs). Using comparative bioinformatics, Southern, and fluorescent in situ hybridization, we reveal the satDNAs’ organizational patterns, their abundances, and chromosomal locations. Four out of the five identified satDNAs are widespread in the Larix genus, with two even present in the more distantly related Pseudotsuga and Abies genera. Unexpectedly, the EulaSat3 family was restricted to L. decidua and absent from L. kaempferi, indicating its evolutionarily young age. Taken together, our results exemplify how the accumulative genome evolution of conifers may limit the overall divergence of repeats after speciation, producing only few repeat-induced genomic novelties.

Background and AimsEndogenous pararetroviruses (EPRVs) are widespread components of plant genomes that originated from episomal DNA viruses of the Caulimoviridae family. Due to fragmentation and rearrangements, most EPRVs have lost their ability to replicate through reverse transcription and to initiate viral infection. Similar to the closely related retrotransposons, extant EPRVs were retained and often amplified in plant genomes for several million years. Here, we characterize the complete genomic EPRV fraction of the crop sugar beet (Beta vulgaris, Amaranthaceae) to understand how they shaped the beet genome and to suggest explanations for their absent virulence.MethodsUsing next- and third-generation sequencing data and the genome assembly, we reconstructed full-length in silico representatives for the three host-specific EPRV families (beetEPRVs) in the B. vulgaris genome. Focusing on the canonical family beetEPRV3, we investigated its chromosomal localization, abundance, and distribution by fluorescent in situ and Southern hybridization.Key ResultsBeetEPRVs range between 7.5 and 10.7 kb (0.3 % of the B. vulgaris genome) and are heterogeneous in structure and sequence. Although all three beetEPRV families were assigned to the florendoviruses, they showed variably arranged protein-coding domains, different degrees of fragmentation, and preferences for diverse sequence contexts. We observed small RNAs that target beetEPRVs in a family-specific manner, indicating stringent epigenetic suppression. We localized beetEPRV3 on all 18 sugar beet chromosomes, occurring preferentially in clusters and associated with heterochromatic, centromeric and intercalary satellite DNAs. BeetEPRV3 variants also exist in the genomes of related wild species, indicating an initial beetEPRV3 integration 13.4 to 7.2 million years ago.ConclusionsOur study in beet illustrates the variability of EPRV structure and sequence in a single host genome. Evidence of sequence fragmentation and epigenetic silencing imply possible plant strategies to cope with long-term persistence of EPRVs, including amplification, fixation in the heterochromatin, and containment of EPRV virulence.

Background and Aims Plant genomes contain many retrotransposons and their derivatives, which are subject to rapid sequence turnover. As non-autonomous retrotransposons do not encode any proteins, they experience reduced selective constraints leading to their diversification into multiple families, usually limited to a few closely related species. In contrast, the non-coding Cassandra terminal repeat retrotransposons in miniature (TRIMs) are widespread in many plants. Their hallmark is a conserved 5S rDNA-derived promoter in their long terminal repeats (LTRs). As sugar beet (Beta vulgaris) has a well-described LTR retrotransposon landscape, we aim to characterize TRIMs in beet and related genomes. Methods We identified Cassandra retrotransposons in the sugar beet reference genome and characterized their structural relationships. Genomic organization, chromosomal localization, and distribution of Cassandra-TRIMs across the Amaranthaceae were verified by Southern and fluorescent in situ hybridization. Key results All 638 Cassandra sequences in the sugar beet genome contain conserved LTRs and thus constitute a single family. Nevertheless, variable internal regions required a subdivision into two Cassandra subfamilies within B. vulgaris. The related Chenopodium quinoa harbours a third subfamily. These subfamilies vary in their distribution within Amaranthaceae genomes, their insertion times and the degree of silencing by small RNAs. Cassandra retrotransposons gave rise to many structural variants, such as solo LTRs or tandemly arranged Cassandra retrotransposons. These Cassandra derivatives point to an interplay of template switch and recombination processes – mechanisms that likely caused Cassandra’s subfamily formation and diversification. Conclusions We traced the evolution of Cassandra in the Amaranthaceae and detected a considerable variability within the short internal regions, whereas the LTRs are strongly conserved in sequence and length. Presumably these hallmarks make Cassandra a prime target for unequal recombination, resulting in the observed structural diversity, an example of the impact of LTR-mediated evolutionary mechanisms on the host genome.

To reach customers by dialog marketing campaigns is more and more difficult. This is a common problem of companies and marketing agencies worldwide: information overload, multi-channel-communication and a confusing variety of offers make it hard to gain the attention of the target group. The contribution of this paper is four-fold: we provide an overview of the current state of print dialog marketing activities and trends (I). Based on this corpus we identify the main key performance indicators of dialog marketing customer interaction (II). A qualitative user experience study identifies the customer wishes and needs, focusing on lottery offers for senior citizens (III). Finally, we evaluate the success of two different dialog marketing campaigns with 20,000 clients and compare the key performance indicators of the original hands-on experience-based print mailings with user experience tested and optimized mailings (IV).

SUMMARYIf two related plant species hybridise, their genomes are combined within a single nucleus, thereby forming an allotetraploid. How the emerging plant balances two co-evolved genomes is still a matter of ongoing research. Here, we focus on satellite DNA (satDNA), the fastest turn-over sequence class in eukaryotes, aiming to trace its emergence, amplification and loss during plant speciation and allopolyploidisation. As a model, we used Chenopodium quinoa Willd. (quinoa), an allopolyploid crop with 2n=4x=36 chromosomes. Quinoa originated by hybridisation of an unknown female American Chenopodium diploid (AA genome) with an unknown male Old World diploid species (BB genome), dating back 3.3 to 6.3 million years. Applying short read clustering to quinoa (AABB), C. pallidicaule (AA), and C. suecicum (BB) whole genome shotgun sequences, we classified their repetitive fractions, and identified and characterised seven satDNA families, together with the 5S rDNA model repeat. We show unequal satDNA amplification (two families) and exclusive occurrence (four families) in the AA and BB diploids by read mapping as well as Southern, genomic and fluorescent in situ hybridisation. As C. pallidicaule harbours a unique satDNA profile, we are able to exclude it as quinoa’s parental species. Using quinoa long reads and scaffolds, we detected only limited evidence of interlocus homogenisation of satDNA after allopolyploidisation, but were able to exclude dispersal of 5S rRNA genes between subgenomes. Our results exemplify the complex route of tandem repeat evolution through Chenopodium speciation and allopolyploidisation, and may provide sequence targets for the identification of quinoa’s progenitors.

† Background and Aims The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. † Methods A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100‐500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. † Key Results Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S‐5.8S ‐25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. † Conclusions The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

BACKGROUND: Carboplatin plus paclitaxel administered every 3 weeks is standard first-line chemotherapy for patients with advanced ovarian cancer. A weekly paclitaxel schedule combined with carboplatin every 3 weeks prolonged progression-free survival and overall survival in a Japanese phase 3 trial. The aim of our study was to assess whether a weekly schedule of carboplatin plus paclitaxel is more effective than the same drugs given every 3 weeks. METHODS: We did a multicentre, randomised, phase 3 study at 67 institutions in Italy and France. Women with FIGO stage IC-IV ovarian cancer, an ECOG performance status of 2 or lower, and who had never received chemotherapy were randomly allocated in a 1:1 ratio to receive either carboplatin (AUC 6 mg/mL per min) plus paclitaxel (175 mg/m(2)) every 3 weeks for six cycles or carboplatin (AUC 2 mg/mL per min) plus paclitaxel (60 mg/m(2)) every week for 18 weeks. Randomisation was done by computer-based minimisation, stratified by centre, residual disease after surgery, and ECOG performance status. The study was not blinded. Coprimary endpoints were progression-free survival and quality of life (assessed by the Functional Assessment of Cancer Therapy Ovarian Trial Outcome Index [FACT-O/TOI] score), and analysis was by modified intention to treat. This report presents the final analysis. The study is registered with ClinicalTrials.gov, number NCT00660842. FINDINGS: 822 patients were enrolled into the study between Nov 20, 2008, and March 1, 2012; 12 withdrew their consent immediately after randomisation and were excluded, and 810 were eligible for analysis. 404 women were allocated treatment every 3 weeks and 406 were assigned to the weekly schedule. After median follow-up of 22·3 months (IQR 16·2-30·9), 449 progression-free survival events were recorded. Median progression-free survival was 17·3 months (95% CI 15·2-20·2) in patients assigned to treatment every 3 weeks, versus 18·3 months (16·8-20·9) in women allocated to the weekly schedule (hazard ratio 0·96, 95% CI 0·80-1·16; p=0·66). FACT-O/TOI scores differed significantly between the two schedules (treatment-by-time interaction p

Long terminal repeat (LTR) retrotransposons are major components of plant genomes influencing genome size and evolution. Using two separate approaches, we identified the Ty1-copia retrotransposon families Cotzilla and SALIRE in the Beta vulgaris (sugar beet) genome. While SALIRE elements are similar to typical Ty1-copia retrotransposons, Cotzilla elements belong to a lineage called Sireviruses. Hallmarks of Cotzilla retrotransposons are the existence of an additional putative env-like open reading frame upstream of the 3'LTR, an extended gag region, and a frameshift separating the gag and pol genes. Detected in a c ( 0 ) t-1 DNA library, Cotzilla elements belong to the most abundant retrotransposon families in B. vulgaris and are relatively homogenous and evolutionarily young. In contrast, the SALIRE family has relatively few copies, is diverged, and most likely ancient. As revealed by fluorescent in situ hybridization, SALIRE elements target predominantly gene-rich euchromatic regions, while Cotzilla retrotransposons are abundant in the intercalary and pericentromeric heterochromatin. The analysis of two retrotransposons from the same subclass contrasting in abundance, age, sequence diversity, and localization gives insight in the heterogeneity of LTR retrotransposons populating a plant genome.

LTR retrotransposons belong to a major group of DNA sequences that are often localized in plant centromeres. Using BAC inserts originating from the centromere of a monosomic wild beet (Beta procumbens) chromosome fragment in Beta vulgaris, two complete LTR retrotransposons were identified. Both elements, designated Beetle1 and Beetle2, possess a coding region with genes in the order characteristic for Ty3-gypsy retrotransposons. Beetle1 and Beetle2 have a chromodomain in the C-terminus of the integrase gene and are highly similar to the centromeric retrotransposons (CRs) of rice, maize, and barley. Both retroelements were localized in the centromeric region of B. procumbens chromosomes by fluorescence in-situ hybridization. They can therefore be classified as centromere-specific chromoviruses. PCR analysis using RNA as template indicated that Beetle1 and Beetle2 are transcriptionally active. On the basis of the sequence diversity between the LTR sequences, it was estimated that Beetle1 and Beetle2 transposed within the last 60,000 years and 130,000 years, respectively. The centromeric localization of Beetle1 and Beetle2 and their transcriptional activity combined with high sequence conservation within each family play an important structural role in the centromeres of B. procumbens chromosomes.

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.

Background Chromoviruses are one of the three genera of Ty3-gypsy long terminal repeat (LTR) retrotransposons, and are present in high copy numbers in plant genomes. They are widely distributed within the plant kingdom, with representatives even in lower plants such as green and red algae. Their hallmark is the presence of a chromodomain at the C-terminus of the integrase. The chromodomain exhibits structural characteristics similar to proteins of the heterochromatin protein 1 (HP1) family, which mediate the binding of each chromovirus type to specific histone variants. A specific integration via the chromodomain has been shown for only a few chromoviruses. However, a detailed study of different chromoviral clades populating a single plant genome has not yet been carried out. Results We conducted a comprehensive survey of chromoviruses within the Beta vulgaris (sugar beet) genome, and found a highly diverse chromovirus population, with significant differences in element size, primarily caused by their flanking LTRs. In total, we identified and annotated full-length members of 16 families belonging to the four plant chromoviral clades: CRM, Tekay, Reina, and Galadriel. The families within each clade are structurally highly conserved; in particular, the position of the chromodomain coding region relative to the polypurine tract is clade-specific. Two distinct groups of chromodomains were identified. The group II chromodomain was present in three chromoviral clades, whereas families of the CRM clade contained a more divergent motif. Physical mapping using representatives of all four clades identified a clade-specific integration pattern. For some chromoviral families, we detected the presence of expressed sequence tags, indicating transcriptional activity. Conclusions We present a detailed study of chromoviruses, belonging to the four major clades, which populate a single plant genome. Our results illustrate the diversity and family structure of B. vulgaris chromoviruses, and emphasize the role of chromodomains in the targeted integration of these viruses. We suggest that the diverse sets of plant chromoviruses with their different localization patterns might help to facilitate plant-genome organization in a structural and functional manner.

Exposure is considered to be an essential ingredient of cognitive-behavioral therapy treatment of social phobia and of most anxiety disorders. To assess the impact of the amount of exposure on outcome, 30 social phobic patients were randomly allocated to 1 of 2 group treatments of 8 weekly sessions: Self-Focused Exposure Therapy which is based essentially on prolonged exposure to public speaking combined with positive feedback or a more standard cognitive and behavioral method encompassing psychoeducation, cognitive work, working through exposure hierarchies of feared situations for exposure within and outside the group. The results show that the 2 methods led to significant and equivalent symptomatic improvements which were maintained at 1-year follow-up. There was a more rapid and initially more pronounced decrease in negative cognitions with the Self-Focused Exposure Therapy, which included no formal cognitive work, than with the more standard approach in which approximately a third of the content was cognitive. In contrast, decrease in social avoidance was more persistent with standard cognitive-behavior therapy which involved less exposure. The results indicate that positive cognitive change can be achieved more rapidly with non cognitive methods while avoidance decreases more reliably with a standard approach rather than an approach with an exclusive focus on exposure.