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Key message In Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency. Abstract Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA–DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA–DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p

We studied a unique case of prolonged viral shedding in an immunocompromised patient that generated a series of SARS-CoV-2 immune escape mutations over a period of seven months. During the persisting SARS-CoV-2 infection seventeen non-synonymous mutations were observed, thirteen (13/17; 76.5%) of which occurred in the genomic region coding for spike. Fifteen (15/17; 88.2%) of these mutations have already been described in the context of variants of concern and include the prominent immune escape mutations S:E484K, S:D950N, S:P681H, S:N501Y, S:del(9), N:S235F and S:H655Y. Fifty percent of all mutations acquired by the investigated strain (11/22) are found in similar form in the Omicron variant of concern. The study shows the chronology of the evolution of intra-host mutations, which can be seen as the straight mutational response of the virus to specific antibodies and should therefore be given special attention in the rating of immune escape mutations of SARS-CoV-2.

Different scenarios explaining the emergence of novel variants of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including their evolution in scarcely monitored populations, in animals as alternative hosts, or in immunocompromised individuals. Here we report SARS-CoV-2 immune escape mutations over a period of seven months in an immunocompromised patient with prolonged viral shedding. Signs of infection, viral shedding and mutation events are periodically analyzed using RT-PCR and next-generation sequencing based on naso-pharyngeal swabs, with the results complemented by immunological diagnostics to determine humoral and T cell immune responses. Throughout the infection course, 17 non-synonymous intra-host mutations are noted, with 15 (88.2%) having been previously described as prominent immune escape mutations (S:E484K, S:D950N, S:P681H, S:N501Y, S:del(9), N:S235F and S:H655Y) in VOCs. The high frequency of these non-synonymous mutations is consistent with multiple events of convergent evolution. Thus, our results suggest that specific mutations in the SARS-CoV-2 genome may represent positions with a fitness advantage, and may serve as targets in future vaccine and therapeutics development for COVID-19.

Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome, might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant bryophytes. Whereas angiosperms repair DNA double strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in bryophytes homologous recombination (HR) is the main DNA-DSB repair pathway facilitating the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in the moss Physcomitrella using haploid plants and autodiploid plants, generated via an artificial duplication of the whole genome. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the whole genome duplication event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p

Coordinated by ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR), two highly conserved kinases, DNA damage repair ensures genome integrity and survival in all organisms. The Arabidopsis thaliana (A. thaliana) orthologues are well characterized and exhibit typical mammalian characteristics. We mutated the Physcomitrella patens (P. patens) PpATM and PpATR genes by deleting functionally important domains using gene targeting. Both mutants showed growth abnormalities, indicating that these genes, particularly PpATR, are important for normal vegetative development. ATR was also required for repair of both direct and replication-coupled double-strand breaks (DSBs) and dominated the transcriptional response to direct DSBs, whereas ATM was far less important, as shown by assays assessing resistance to DSB induction and SuperSAGE-based transcriptomics focused on DNA damage repair genes. These characteristics differed significantly from the A. thaliana genes but resembled those in yeast (Saccharomyces cerevisiae). PpATR was not important for gene targeting, pointing to differences in the regulation of gene targeting and direct DSB repair. Our analysis suggests that ATM and ATR functions can be substantially diverged between plants. The differences in ATM and ATR reflect the differences in DSB repair pathway choices between A. thaliana and P. patens, suggesting that they represent adaptations to different demands for the maintenance of genome stability.

SARS-CoV-2 is assumed to use angiotensin-converting enzyme 2 (ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory-confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and there has been speculation that SARS-CoV-2 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctivas, 12 melanomas, seven squamous cell carcinomas, and seven papilloma samples, were analyzed using high-throughput RNA sequencing to assess messenger RNA (mRNA) expression of the SARS-CoV-2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS-CoV-2 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA (median: 0.0 transcripts per million [TPM], min: 0.0 TPM, max: 1.7 TPM) and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS-CoV-2 via these mediators unlikely.

The Lathyrus cicera transcriptome was analysed in response to rust (Uromyces pisi) infection to develop novel molecular breeding tools with potential for genetic mapping of resistance in this robust orphan legume species. One RNA-seq library each was generated from control and rust-inoculated leaves from two L. cicera genotypes with contrasting quantitative resistance, de novo assembled into contigs and sequence polymorphisms were identified. In toto, 19,224 SNPs differentiate the susceptible from the partially resistant genotype’s transcriptome. In addition, we developed and tested 341 expressed E-SSR markers from the contigs, of which 60.7% varied between the two L. cicera genotypes. A first L. cicera linkage map was created using part of the developed markers in a RIL population from the cross of the two genotypes. This map contains 307 markers, covered 724.2 cM and is organised in 7 major and 2 minor linkage groups, with an average mapping interval of 2.4 cM. The genic markers also enabled us to compare their position in L. cicera map with the physical position of the same markers mapped on Medicago truncatula genome, highlighting a high macrosyntenic conservation between both species. This study provides a large new set of genic polymorphic molecular markers with potential for mapping rust resistances. It represents the first step towards genomics-assisted precision breeding in L. cicera. Genetic analysis of Lathyrus cicera, or ‘chickling pea’, a plant most commonly cultivated for animal feed, identified DNA polymorphisms that may be associated to resistance to the fungal disease known as rust. Researchers in Europe led by Maria Carlota Vaz Patto at the NOVA University of Lisbon, Portugal, discovered thousands of genetic differences between plants that were either susceptible to or at least partially resistant to the rust fungus Uromyces pisi. They created a ‘genetic linkage map’, which reveals the likelihood that different genetic characteristics will be inherited together through the generations. This map can now serve as a tool to help geneticists and plant breeders understand and promote resistance to rust and potentially other fungal diseases in L cicera. The results may also be applied to other legumes, including peas and beans more commonly cultivated for human consumption.

Preserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation. To investigate the impact of Xellulin on the endothelial cell phenotype in detail, we applied quantitative transcriptomics and proteomics and compared the molecular makeup of native HUVEC, HUVEC on collagen-coated Xellulin and collagen-coated cell culture plastic (polystyrene).Statistical analysis of 12,475 transcripts and 7831 proteins unveiled massive quantitative differences of the compared transcriptomes and proteomes. K-means clustering followed by network analysis showed that HUVEC on plastic upregulate transcripts and proteins controlling proliferation, cell cycle and protein biosynthesis. In contrast, HUVEC on Xellulin maintained, by and large, the expression levels of genes supporting their native biological functions and signaling networks such as integrin, receptor tyrosine kinase MAP/ERK and PI3K signaling pathways, while decreasing the expression of proliferation associated proteins. Moreover, CD34-an endothelial cell differentiation marker usually lost early during cell culture - was re-expressed within 2 weeks on Xellulin but not on plastic. And HUVEC on Xellulin showed a significantly stronger functional responsiveness to a prototypic pro-inflammatory stimulus than HUVEC on plastic.Taken together, this is one of the most comprehensive transcriptomic and proteomic studies of native and propagated HUVEC, which underscores the importance of the morphology of the cellular microenvironment to regulate cellular differentiation, and demonstrates, for the first time, the potential of Xellulin as versatile tool promoting an in vivo-like phenotype in primary and propagated cell culture.

Aim of study: The purpose was to identify some general and genotypic-specific defense responses, in order to obtain a set of candidate genes presumably involved in the resistance.Area of study: The experiment was carried out in León, Spain.Material and methods: We have analyzed the response of three lentil genotypes to Ascochyta lentis (isolate AL 84) at transcriptomic level using the Massive Analysis of cDNA Ends (MACE) technique: the susceptible cultivar 'Lupa', the moderately resistant 'ILL5588' and the resistant wild accession 'BG 16880' (L. culinaris subsp. orientalis).Main results: MACE results yielded a total of 50,935 contigs. The average number of detected contigs in each of the six samples was approximately of 40,000. In response to Ascochyta infection, the jasmonic acid pathway and the lignin biosynthesis were up-regulated in resistant genotypes, while they were down-regulated in the susceptible one. The response to chitin, the salicylic pathway and the auxin response were activated only in the resistant L. c. culinaris genotype, while the giberellin synthesis was only induced in the susceptible L. c. culinaris cv. 'Lupa'. A set of 18 lentil gene sequences putatively involved in the response to the pathogen were validated by RT-qPCR.Research highlights: It can be concluded that in response to the infection by Ascochyta, the lignin biosynthesis and the JA pathway were critical for the resistance, while the giberellin synthesis seems to be related with susceptibility to the pathogen.

Recent studies deciphering the transcriptional profile of choroidal neovascularization (CNV) in body donor eyes with neovascular age-related macular degeneration are limited by the time span from death to preservation and the associated 5′-RNA degradation. This study therefore used CNV and control specimens that were formalin-fixed and paraffin-embedded immediately after surgical extraction and analyzed them by a 3′-RNA sequencing approach. Transcriptome profiles were analyzed to estimate content of immune and stromal cells and to define disease-associated gene signatures by using statistical and bioinformatics methods. This study identified 158 differentially expressed genes (DEGs) that were significantly increased in CNV compared with control tissue. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of endothelial cells, macrophages, T cells, and natural killer T cells in the CNV. Gene ontology enrichment analysis found that DEGs contributed to blood vessel development, extracellular structure organization, response to wounding, and several immune-related terms. The S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9) emerged among the top DEGs, as confirmed by immunohistochemistry on CNV tissue and protein analysis of vitreous samples. This study provides a high-resolution RNA-sequencing–based transcriptional signature of human CNV, characterizes its compositional pattern of immune and stromal cells, and reveals S100A8/A9 to be a novel biomarker and promising target for therapeutics and diagnostics directed at age-related macular degeneration.

Coordinated by ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR), two highly conserved kinases, DNA damage repair ensures genome integrity and survival in all organisms. The Arabidopsis thaliana (A. thaliana) orthologues are well characterized and exhibit typical mammalian characteristics. We mutated the Physcomitrella patens (P. patens) PpATM and PpATR genes by deleting functionally important domains using gene targeting. Both mutants showed growth abnormalities, indicating that these genes, particularly PpATR, are important for normal vegetative development. ATR was also required for repair of both direct and replication-coupled double-strand breaks (DSBs) and dominated the transcriptional response to direct DSBs, whereas ATM was far less important, as shown by assays assessing resistance to DSB induction and SuperSAGE-based transcriptomics focused on DNA damage repair genes. These characteristics differed significantly from the A. thaliana genes but resembled those in yeast (Saccharomyces cerevisiae). PpATR was not important for gene targeting, pointing to differences in the regulation of gene targeting and direct DSB repair. Our analysis suggests that ATM and ATR functions can be substantially diverged between plants. The differences in ATM and ATR reflect the differences in DSB repair pathway choices between A. thaliana and P. patens, suggesting that they represent adaptations to different demands for the maintenance of genome stability.

To investigate the expression profile of the hypoxia-inducible transcription factor-1α (HIF-1α) and its downstream targets in malignancies of the ocular adnexa and to determine its relevance as a prognostic factor for clinical outcome. We included 49 subjects with malignant tumours (25 squamous cell carcinomas (SCC), 15 non-Hodgkin lymphomas, 9 melanomas) and 30 patients with benign tumours of the ocular adnexa (13 papillomas, 7 reactive lymphoid hyperplasias (RLHs) and 10 nevi) as controls. We quantified HIF-1α protein expression by immunohistochemistry and assessed the association between HIF-1α and clinical outcome via Kaplan–Meier analysis. Furthermore, we assessed the expression of HIF-1α downstream factors by transcriptional sequencing using the MACE (massive analysis of cDNA ends) technology. SCCs revealed a strong HIF-1α expression in 61% of tumour cells in comparison with only 22% in papillomas (p

In the present paper we show a survey of the Asterias rubens sea star genome for genes associated with NF-kappa-B proteins implied in the immune response. The NF-kappa B gene, into 2 subunits, was found in this invertebrate.

We present an integrative model predicting associations among epiphytism, the tank habit, entangling seeds, C3 vs. CAM photosynthesis, avian pollinators, life in fertile, moist montane habitats, and net rates of species diversification in the monocot family Bromeliaceae. We test these predictions by relating evolutionary shifts in form, physiology, and ecology to time and ancestral distributions, quantifying patterns of correlated and contingent evolution among pairs of traits and analyzing the apparent impact of individual traits on rates of net species diversification and geographic expansion beyond the ancestral Guayana Shield. All predicted patterns of correlated evolution were significant, and the temporal and spatial associations of phenotypic shifts with orogenies generally accorded with predictions. Net rates of species diversification were most closely coupled to life in fertile, moist, geographically extensive cordilleras, with additional significant ties to epiphytism, avian pollination, and the tank habit. The highest rates of net diversification were seen in the bromelioid tank-epiphytic clade (Dcrown=1.05My-1), associated primarily with the Serra do Mar and nearby ranges of coastal Brazil, and in the core tillandsioids (Dcrown=0.67My-1), associated primarily with the Andes and Central America. Six large-scale adaptive radiations and accompanying pulses of speciation account for 86% of total species richness in the family. This study is among the first to test a priori hypotheses about the relationships among phylogeny, phenotypic evolution, geographic spread, and net species diversification, and to argue for causality to flow from functional diversity to spatial expansion to species diversity. © 2013 Elsevier Inc., The authors gratefully acknowledge financial support for this investigation by Grants from the National Science Foundation to PEB, KJS, and TJG (DEB-9981587), TJG (DEB-0830036 and DEB- 0830036), KJS (DEB-0431258), and GKB and TME (DEB-0129446 and DEB-0129414), and from the Hertel Gift Fund to TJG; from the Commission for Interdisciplinary Ecological Studies (KIÖS) at the Austrian Academy of Sciences (ÖAW; 2007-02 to WT and MHJB; and from the Deutsche Forschungsgemeinshaft (ZI 557/7- 1, SCHU 2426/1-1) and the Hessian initiative for the development of scientific and economic excellence (LOEWE) at the Biodiversity and Climate Research Centre, Frankfurt am Mein, to GZ and KS.

In chicken, the left and right female gonads undergo a completely different program during development. To learn more about the molecular factors underlying side-specific development and to identify potential sex- and side-specific genes in developing gonads, we separately performed next-generation sequencing-based deepSuperSAGE transcription profiling from left and right, female and male gonads of 19-day-old chicken embryos. A total of 836 transcript variants were significantly differentially expressed (p < 10-5) between combined male and female gonads. Left-right comparison revealed 1,056 and 822 differentially (p < 10-5) expressed transcript variants for male and female gonads, respectively, of which 72 are side-specific in both sexes. At least some of these may represent key players for lateral development in birds. Additionally, several genes with laterally differential expression in the ovaries seem to determine female gonads for growth or regression, whereas right-left differences in testes are mostly limited to the differentially expressed genes present in both sexes. With a few exceptions, side-specific genes are not located on the sex chromosomes. The large differences in lateral gene expression in the ovaries in almost all metabolic pathways suggest that the regressing right gonad might have undergone a change of function during evolution.

 Premise : Bromeliaceae form a large, ecologically diverse family of angiosperms native to the New World. We use a bromeliad phylogeny based on eight plastid regions to analyze relationships within the family, test a new, eight-subfamily classifi cation, infer the chronology of bromeliad evolution and invasion of different regions, and provide the basis for future analyses of trait evolution and rates of diversifi cation.  Methods : We employed maximum-parsimony, maximum-likelihood, and Bayesian approaches to analyze 9341 aligned bases for four outgroups and 90 bromeliad species representing 46 of 58 described genera. We calibrate the resulting phylogeny against time using penalized likelihood applied to a monocot-wide tree based on plastid ndhF sequences and use it to analyze patterns of geographic spread using parsimony, Bayesian inference, and the program S-DIVA.  Results : Bromeliad subfamilies are related to each other as follows: (Brocchinioideae, (Lindmanioideae, (Tillandsioideae, (Hechtioideae, (Navioideae, (Pitcairnioideae, (Puyoideae, Bromelioideae))))))). Bromeliads arose in the Guayana Shield ca. 100 million years ago (Ma), spread centrifugally in the New World beginning ca. 16 – 13 Ma, and dispersed to West Africa ca. 9.3 Ma. Modern lineages began to diverge from each other roughly 19 Ma.  Conclusions : Nearly two-thirds of extant bromeliads belong to two large radiations: the core tillandsioids, originating in the Andes ca. 14.2 Ma, and the Brazilian Shield bromelioids, originating in the Serro do Mar and adjacent regions ca. 9.1 Ma.

A reconstruction of the phylogeny of Bromeliaceae based on sequence data from three noncoding chloroplast DNA markers (trnL intron, trnT-trnL, and trnT-trnF intergenic spacer [IGS]) is presented, including 26 genera and 33 species. Relationships of Bromelioideae and phylogeny within this subfamily were analyzed in more detail on the basis of two of these markers (trnL intron and trnL-trnF IGS) using a set of 37 genera/74 species of Bromeliaceae, including 28 genera/60 species of Bromelioideae. Sister group relationships of Bromelioideae were not resolved with sufficient reliability, but the most likely candidates are the genera Fosterella and Puya. The basal phylogeny of Bromelioideae also was not resolved. Greigia, Ochagavia/Fascicularia/Fernseea, Deinacanthon, Bromelia, and a "core group" of the remaining Bromelioideae formed a basal polytomy. Within Bromelioideae, the AFLP technique was applied to assess relationships among selected groups of genera. In the Ochagavia/Fascicularia group (5 species and subspecies/16 accessions), AFLP data fully confirmed the systematic relationships based on morphological and anatomical characters. Investigation of 30 Aechmea species (33 accessions), including all subgenera and one species each from the related genera Ursulaea, Portea, Chevaliera, and Streptocalyx produced no resolution for several of the species. Clades that received good bootstrap support generally did not correspond with the delimitation of subgenera of Aechmea. Additionally, leaf blade anatomy of these species was investigated. The results corresponded partly with those of the AFLP analysis. Generic rank for Ursulaea and Portea was not supported.

New cytological information and chromosome counts are presented for 19 taxa of 15 genera of the Bromeliaceae, among them, data for 15 taxa and five genera are reported for the first time. The basic number x = 25 is confirmed and polyploidy seems to be the main evolutionary mechanism in Bromeliaceae. Most of the analyzed species presented 2n = 50. Polyploids have been detected in Deinacanthon urbanianum with 2n = ca.160 and Bromelia laciniosa with 2n = ca.150. In Deuterocohnia lorentziana we observed individuals with two different ploidy levels (2n = 50 and 2n = 100) growing together in the same pot. Ayensua uaipanensis showed the uncommon number 2n = 46. After triple staining with CMA3/Actinomycin/DAPI one or two CMA+/DAPI− bands could be observed in the studied species (Aechmea bromeliifolia, Greigia sphacelata and Ochagavia litoralis). The role of these features in the evolution of the family is discussed, revealing new aspects of the evolution of the Bromeliaceae.

Four cultivars of chickpea (C. arietinum) were used in this study, two Desi (ICC 4958 and ICC 6098) cultivars and two Kabuli (Beja-1 and Amdoun-1) each one with a water stress tolerant and sensitive variety, inoculated with a Mesorhizobium strain. The effects of drought stress on plant growth and the response of genes related to metabolism were analysed by means of qRT-PCR at various time points of treatments. Nodules and root tissue was used for RNA extraction. We measured the expression of key genes involved by qRT-PCR on samples from stressed and control roots and nodules of a different cultivar using available Taqman assays. In addition phenotype characterisation was performed, such as Relative water content (RWC), quantification of biomass, evaluation ''in situ' of' acetylene reduction activity at the end of the vegetative stage, and nitrogenase activity. The expression analysis was done after 7 days of drought stress and after 10 days of drought stress. We studied genes implicated in the metabolism (glutamine synthase and AAT) or transport of proteins (peptide amino acid transporter like protein PAAT and proline rich protein PPP), sucrose synthase, malate dehydrogenase, sucrose phosphate synthase, sugar transporter) and Sucrose transporter, the antioxidant genes ascorbate peroxidase, Glutathione-S-transferase, Leghemoglobin, catalase, and two lipoxygenase genes.

Phylogenetic relationships within Bromeliaceae were assessed by comparative sequencing of chloroplast trnL(UAA) intronic DNA. Sixty-seven taxa were analyzed, encompassing 62 species, two subspecies and two varieties from 32 bromeliad genera. One representative of Rapateaceae and two of Velloziaceae were used as outgroups. Introns were amplified by the polymerase chain reaction and conserved flanking primers. Amplification products were sequenced directly. Of 700 base pairs total trnL (UAA) intron size, 551 bp could be unambiguously aligned. Sequence divergence among ingroup taxa ranged from zero to 4.6 %. Phylogenetic reconstruction by parsimony analysis of 73 informative base substitutions yielded 1092 most parsimonious trees. The topology of the resulting strict consensus tree was only partially consistent with current classification schemes. Brocchinia and Ayensua grouped close together and formed a well-supported basal clade, suggesting a sister group relationship of both to the remainder of the family. The remaining ingroup species were divided into an unresolved polytomy of three lineages, comprising (1) all members of subfamily Tillandsioideae, (2) the three investigated Hechtia species, and (3) all Bromelioideae and the remaining Pitcairnioideae (except Brocchinia, Ayensua and Hechtia). The latter clade was split into another polytomy of five sublineages, one of which comprised all Bromelioideae. Whereas monophyly of Tillandsioideae and Bromelioideae was supported by trnL intron data, Pitcairnioideae as traditionally circumscribed are clearly polyphyletic. There is evidence that the genera Tillandsia, Vriesea, Guzmania, Abromeitiella, Deuterocohnia, Dyckia, Pitcairnia and Aechmea in the present circumscription are not monophyletic.

The genus Fascicularia Mez is revised as part of a study of the Bromeliaceae for the Flora de Chile. Morphological and anatomical investigation of herbarium and living material from cultivation as well as DNA-studies (RAPDs) in cultivated material has led us to conclude that Fascicularia bicolor (Ruiz & Pav.) Mez, the only one species in the genus, comprises two subspecies which are distinguished by their leaf anatomy and morphology.

BACKGROUND: Pollen of common ragweed (Ambrosia artemisiifolia) is a main cause of allergic diseases in Northern America. The weed has recently become spreading as a neophyte in Europe, while climate change may also affect the growth of the plant and additionally may also influence pollen allergenicity. To gain better insight in the molecular mechanisms in the development of ragweed pollen and its allergenic proteins under global change scenarios, we generated SuperSAGE libraries to identify differentially expressed transcripts. RESULTS: Ragweed plants were grown in a greenhouse under 380ppm CO2 and under elevated level of CO2 (700ppm). In addition, drought experiments under both CO2 concentrations were performed. The pollen viability was not altered under elevated CO2, whereas drought stress decreased its viability. Increased levels of individual flavonoid metabolites were found under elevated CO2 and/or drought. Total RNA was isolated from ragweed pollen, exposed to the four mentioned scenarios and four SuperSAGE libraries were constructed. The library dataset included 236,942 unique sequences, showing overlapping as well as clear differently expressed sequence tags (ESTs). The analysis targeted ESTs known in Ambrosia, as well as in pollen of other plants. Among the identified ESTs, those encoding allergenic ragweed proteins (Amb a) increased under elevated CO2 and drought stress. In addition, ESTs encoding allergenic proteins in other plants were also identified. CONCLUSIONS: The analysis of changes in the transcriptome of ragweed pollen upon CO2 and drought stress using SuperSAGE indicates that under global change scenarios the pollen transcriptome was altered, and impacts the allergenic potential of ragweed pollen.

Background The rationale for gathering information from plants procuring nitrogen through symbiotic interactions controlled by a common genetic program for a sustainable biofuel production is the high energy demanding application of synthetic nitrogen fertilizers. We curated sequence information publicly available for the biofuel plant sugarcane, performed an analysis of the common SYM pathway known to control symbiosis in other plants, and provide results, sequences and literature links as an online database. Methods Sugarcane sequences and informations were downloaded from the nucEST database, cleaned and trimmed with seqclean, assembled with TGICL plus translating mapping method, and annotated. The annotation is based on BLAST searches against a local formatted plant Uniprot90 generated with CD-HIT for functional assignment, rpsBLAST to CDD database for conserved domain analysis, and BLAST search to sorghum's for Gene Ontology (GO) assignment. Gene expression was normalized according the Unigene standard, presented as ESTs/100 kb. Protein sequences known in the SYM pathway were used as queries to search the SymGRASS sequence database. Additionally, antimicrobial peptides described in the PhytAMP database served as queries to retrieve and generate expression profiles of these defense genes in the libraries compared to the libraries obtained under symbiotic interactions. Results We describe the SymGRASS, a database of sugarcane orthologous genes involved in arbuscular mycorrhiza (AM) and root nodule (RN) symbiosis. The database aggregates knowledge about sequences, tissues, organ, developmental stages and experimental conditions, and provides annotation and level of gene expression for sugarcane transcripts and SYM orthologous genes in sugarcane through a web interface. Several candidate genes were found for all nodes in the pathway, and interestingly a set of symbiosis specific genes was found. Conclusions The knowledge integrated in SymGRASS may guide studies on molecular, cellular and physiological mechanisms by which sugarcane controls the establishment and efficiency of endophytic associations. We believe that the candidate sequences for the SYM pathway together with the pool of exclusively expressed tentative consensus (TC) sequences are crucial for the design of molecular studies to unravel the mechanisms controlling the establishment of symbioses in sugarcane, ultimately serving as a basis for the improvement of grass crops.

Purpose Extracellular microRNAs (miRNAs) in aqueous humor were suggested to have a role in transcellular signaling and may serve as disease biomarkers. The authors adopted next-generation sequencing (NGS) techniques to further characterize the miRNA profile in single samples of 60 to 80 μL human aqueous humor. Methods Samples were obtained at the outset of cataract surgery in nine independent, otherwise healthy eyes. Four samples were used to extract RNA and generate sequencing libraries, followed by an adapter-driven amplification step, electrophoretic size selection, sequencing, and data analysis. Five samples were used for quantitative PCR (qPCR) validation of NGS results. Published NGS data on circulating miRNAs in blood were analyzed in comparison. Results One hundred fifty-eight miRNAs were consistently detected by NGS in all four samples; an additional 59 miRNAs were present in at least three samples. The aqueous humor miRNA profile shows some overlap with published NGS-derived inventories of circulating miRNAs in blood plasma with high prevalence of human miR-451a, -21, and -16. In contrast to blood, miR-184, -4448, -30a, -29a, -29c, -19a, -30d, -205, -24, -22, and -3074 were detected among the 20 most prevalent miRNAs in aqueous humor. Relative expression patterns of miR-451a, -202, and -144 suggested by NGS were confirmed by qPCR. Conclusions Our data illustrate the feasibility of miRNA analysis by NGS in small individual aqueous humor samples. Intraocular cells as well as blood plasma contribute to the extracellular aqueous humor miRNome. The data suggest possible roles of miRNA in intraocular cell adhesion and signaling by TGF-β and Wnt, which are important in intraocular pressure regulation and glaucoma.

Drought is one of the major constraints in subtropical agriculture. Therefore improving water stress tolerance is of great importance to breed for drought tolerance in future. The first plant organ sensing dehydration is the root. Aim of the present work was to clarify the potential impact of the phyto-oxylipins pathway on drought tolerance of chickpea (Cicer arietinum), the third important legume crop worldwide. Therefore, we measured the expression of key genes involved in oxylipins metabolism by qPCR on samples from stressed and non-stressed roots of a drought-tolerant and a drought-sensitive chickpea variety using commercially available TaqMan assays. We demonstrate that the drought tolerant variety reacts to drought with sustained and earlier activation of a specific lipoxygenase (Mt-LOX 1) gene, two hydroperoxide lyases (Mt-HPL 1 and Mt-HPL 2), an allene oxide synthase (Mt-AOS), and an oxo-phytodienoate reductase (Mt-OPR). We further show that gene over-expression positively correlates with the levels of major oxylipin metabolites from the AOS branch of the pathway, which finally leads to the synthesis of jasmonates. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the active form JA-isoleucine (JA-Ile) were especially detected in the root tissues of the tolerant variety, prompting us to assume a role of jasmonates in the early signalling of drought stress in chickpea and its involvement in the tolerance mechanism of the drought-tolerant variety.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Common bean (Phaseolus vulgaris L.) is often subject to various environmental constraints including soil salinity and phosphorus deficiency as major limitations for the yield of most grain legumes, especially when the plant growth depends upon N-2 fixation. In order to assess the genetic variation for tolerance to moderate salinity and phosphorus deficiency and identify the related morphological, physiological and genetic traits, 37 common bean recombinant inbred lines (RILs) were inoculated with Rhizobium tropici CIAT899, and grown in a glasshouse with 25 mM NaCl or 75 mu mol P plant(-1) week(-1), compared to optimal nutrient solution in hydroaeroponic culture system. Large genotypic variation in tolerance to P deficiency and salt was found with some RILs being tolerant to both constraints. By contrast some of the RILs showed tolerance to only one constraint while the most sensitive to salinity were also sensitive to P-deficiency. By using 18 microsatellite primer-pairs with six most contrasting RILs, 4 alleles were found to discriminate among the RILs. It is concluded that these genotypes and the microsatellites primers can be used to identify genes involved in salinity and P deficiency tolerance of N-2-dependent legume.

Molluscs are raising attention as ecotoxicological test organisms due to their high diversity and ecological importance. The ovoviviparous prosobranch gastropod Potamopyrgus antipodarum (freshwater mudsnail) responds very sensitively to xenobiotics and has therefore been proposed as OECD standard test organism. Endocrine disrupting chemicals influence the reproduction of P. antipodarum, which can be assessed by embryo numbers in the brood pouch. However, the knowledge about the endocrine system of P. antipodarum is rather limited. The aim of this study was to identify an estrogen receptor in the endocrine system of P. antipodarum and to investigate if this receptor is differentially expressed under exposure to (xeno-)hormones (17α-ethinylestradiol, bisphenol A and 17α-methyltestosterone). The DNA-binding domain of the identified ER-like transcript has an amino acid identity of 92 percent compared to the ER of the gastropod Nucella lapillus (84 percent to human ERα) and 83 percent in the ligand binding domain (38 percent to human ERα). Furthermore, the P. antipodarum ER is transcriptionally regulated as shown by quantitative real-time PCRs of (xeno-)hormone exposed snails. 17α-ethinylestradiol and bisphenol A exposure resulted in a transitory ER-mRNA increase while17α-methyltestosterone caused a transitory reduction of ER-mRNA. In addition the solvent dimethyl sulfoxide had also a modulating effect on the receptor.

26 páginas, 6 figuras, 7 tablas., [Background]: The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche’s 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level. [Results]: We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress. Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available. [Conclusions]: This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25 mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE, Work of the authors was supported by a grant from the Aquarhiz project (INCOCT- 2004-509115) to GK, grants from the Grain Legumes project (Food-CT- 2004-506223) to GK and GenXPro GmbH, and from DFG to GK (Ka 332/22-1).

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago. Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1265-1) contains supplementary material, which is available to authorized users.

� Background and Aims Germination and establishment of seeds are complex traits affected by a wide range of internal and external influences. The effects of parental temperature preconditioning and temperature during germination on germination and establishment of Arabidopsis thaliana were examined. � Methods Seeds from parental plants grown at 14 and at 22 � C were screened for germination (protrusion of radicle) and establishment (greening of cotyledons) at three different temperatures (10, 18 and 26 � C). Seventy-three accessions from across the entire distribution range of A. thaliana were included. � Key Results Multifactorial analyses of variances revealed significant differences in the effects of genotypes, preconditioning, temperature treatment, and their interactions on duration of germination and establishment. Reaction norms showed an enormous range of plasticity among the preconditioning and different germination temperatures. Correlations of percentage total germination and establishment after 38 d with the geographical origin of accessions were only significant for 14 � C preconditioning but not for 22 � C preconditioning. Correlations with temperature and precipitation on the origin of the accessions were mainly found at the lower germination temperatures (10 and 18 � C) and were absent at higher germination temperatures (26 � C). � Conclusions Overall, the data show huge variation of germination and establishment among natural accessions of A. thaliana and might serve as a valuable source for further germination and plasticity studies.

d Background and Aims Estimates of the amount of nuclear DNA of Arabidopsis thaliana, known to be among the lowest within angiosperms, vary considerably. This study aimed to determine genome size of a range of accessions from throughout the entire Eurasian range of the species. d Methods Twenty accessions from all over Europe and one from Japan were examined using flow cytometry. d Key Results Significant differences in mean C-values were detected over a 1·1-fold range. Mean haploid (1C) genome size was 0·215 pg (211 Mbp) for all analysed accessions. Two accessions were tetraploid. d Conclusions A closer investigation of the DNA fractions involved in intraspecific genome size differences in this experimentally accessible species may provide information on the factors involved in stability and evolution of genome sizes. a 2004 Annals of Botany Company

Background Drought is the major constraint to increase yield in chickpea (Cicer arietinum). Improving drought tolerance is therefore of outmost importance for breeding. However, the complexity of the trait allowed only marginal progress. A solution to the current stagnation is expected from innovative molecular tools such as transcriptome analyses providing insight into stress-related gene activity, which combined with molecular markers and expression (e)QTL mapping, may accelerate knowledge-based breeding. SuperSAGE, an improved version of the serial analysis of gene expression (SAGE) technique, generating genome-wide, high-quality transcription profiles from any eukaryote, has been employed in the present study. The method produces 26 bp long fragments (26 bp tags) from defined positions in cDNAs, providing sufficient sequence information to unambiguously characterize the mRNAs. Further, SuperSAGE tags may be immediately used to produce microarrays and probes for real-time-PCR, thereby overcoming the lack of genomic tools in non-model organisms. Results We applied SuperSAGE to the analysis of gene expression in chickpea roots in response to drought. To this end, we sequenced 80,238 26 bp tags representing 17,493 unique transcripts (UniTags) from drought-stressed and non-stressed control roots. A total of 7,532 (43%) UniTags were more than 2.7-fold differentially expressed, and 880 (5.0%) were regulated more than 8-fold upon stress. Their large size enabled the unambiguous annotation of 3,858 (22%) UniTags to genes or proteins in public data bases and thus to stress-response processes. We designed a microarray carrying 3,000 of these 26 bp tags. The chip data confirmed 79% of the tag-based results, whereas RT-PCR confirmed the SuperSAGE data in all cases. Conclusion This study represents the most comprehensive analysis of the drought-response transcriptome of chickpea available to date. It demonstrates that – inter alias – signal transduction, transcription regulation, osmolyte accumulation, and ROS scavenging undergo strong transcriptional remodelling in chickpea roots already 6 h after drought stress. Certain transcript isoforms characterizing these processes are potential targets for breeding for drought tolerance. We demonstrate that these can be easily accessed by micro-arrays and RT-PCR assays readily produced downstream of SuperSAGE. Our study proves that SuperSAGE owns potential for molecular breeding also in non-model crops.