Nonadditive gene expression is correlated with nonadditive phenotypic expression in interspecific triploid hybrids of willow (Salix spp.).

Many studies have highlighted the complex and diverse basis for heterosis in inbred crops. Despite the lack of a consensus model, it is vital that we turn our attention to understanding heterosis in undomesticated, heterozygous, and polyploid species, such as willow (Salix spp.). Shrub willow is a dedicated energy crop bred to be fast-growing and high yielding on marginal land without competing with food crops. A trend in willow breeding is the consistent pattern of heterosis in triploids produced from crosses between diploid and tetraploid species. Here, we test whether differentially expressed genes are associated with heterosis in triploid families derived from diploid Salix purpurea, diploid Salix viminalis, and tetraploid Salix miyabeana parents. Three biological replicates of shoot tips from all family progeny and parents were collected after 12 weeks in the greenhouse and RNA extracted for RNA-Seq analysis. This study provides evidence that nonadditive patterns of gene expression are correlated with nonadditive phenotypic expression in interspecific triploid hybrids of willow. Expression-level dominance was most correlated with heterosis for biomass yield traits and was highly enriched for processes involved in starch and sucrose metabolism. In addition, there was a global dosage effect of parent alleles in triploid hybrids, with expression proportional to copy number variation. Importantly, differentially expressed genes between family parents were most predictive of heterosis for both field and greenhouse collected traits. Altogether, these data will be used to progress models of heterosis to complement the growing genomic resources available for the improvement of heterozygous perennial bioenergy crops.

Sequencing and analysis of globally obtained human parainfluenza viruses 1 and 3 genomes.

Human Parainfluenza viruses (HPIV) type 1 and 3 are important causes of respiratory tract infections in young children globally. HPIV infections do not confer complete protective immunity so reinfections occur throughout life. Since no effective vaccine is available for the two virus subtypes, comprehensive understanding of HPIV-1 and HPIV-3 genetic and epidemic features is important for diagnosis, prevention, and treatment of HPIV-1 and HPIV-3 infections. Relatively few whole genome sequences are available for both HPIV-1 and HPIV-3 viruses, so our study sought to provide whole genome sequences from multiple countries to further the understanding of the global diversity of HPIV at a whole-genome level. We collected HPIV-1 and HPIV-3 samples and isolates from Argentina, Australia, France, Mexico, South Africa, Switzerland, and USA from the years 2003-2011 and sequenced the genomes of 40 HPIV-1 and 75 HPIV-3 viruses with Sanger and next-generation sequencing with the Ion Torrent, Illumina, and 454 platforms. Phylogenetic analysis showed that the HPIV-1 genome is evolving at an estimated rate of 4.97 × 10-4 mutations/site/year (95% highest posterior density 4.55 × 10-4 to 5.38 × 10-4) and the HPIV-3 genome is evolving at a similar rate (3.59 × 10-4 mutations/site/year, 95% highest posterior density 3.26 × 10-4 to 3.94 × 10-4). There were multiple genetically distinct lineages of both HPIV-1 and 3 circulating on a global scale. Further surveillance and whole-genome sequencing are greatly needed to better understand the spatial dynamics of these important respiratory viruses in humans.

Population Gene Introgression and High Genome Plasticity for the Zoonotic Pathogen Streptococcus agalactiae.

The influence that bacterial adaptation (or niche partitioning) within species has on gene spillover and transmission among bacterial populations occupying different niches is not well understood. Streptococcus agalactiae is an important bacterial pathogen that has a taxonomically diverse host range making it an excellent model system to study these processes. Here, we analyze a global set of 901 genome sequences from nine diverse host species to advance our understanding of these processes. Bayesian clustering analysis delineated 12 major populations that closely aligned with niches. Comparative genomics revealed extensive gene gain/loss among populations and a large pan genome of 9,527 genes, which remained open and was strongly partitioned among niches. As a result, the biochemical characteristics of 11 populations were highly distinctive (significantly enriched). Positive selection was detected and biochemical characteristics of the dispensable genes under selection were enriched in ten populations. Despite the strong gene partitioning, phylogenomics detected gene spillover. In particular, tetracycline resistance (which likely evolved in the human-associated population) from humans to bovine, canines, seals, and fish, demonstrating how a gene selected in one host can ultimately be transmitted into another, and biased transmission from humans to bovines was confirmed with a Bayesian migration analysis. Our findings show high bacterial genome plasticity acting in balance with selection pressure from distinct functional requirements of niches that is associated with an extensive and highly partitioned dispensable genome, likely facilitating continued and expansive adaptation.

Dominance and Sexual Dimorphism Pervade the Salix purpurea L. Transcriptome.

The heritability of gene expression is critical in understanding heterosis and is dependent on allele-specific regulation by local and remote factors in the genome. We used RNA-Seq to test whether variation in gene expression among F1 and F2 intraspecific Salix purpurea progeny is attributable to cis- and trans-regulatory divergence. We assessed the mode of inheritance based on gene expression levels and allele-specific expression for F1 and F2 intraspecific progeny in two distinct tissue types: shoot tip and stem internode. In addition, we explored sexually dimorphic patterns of inheritance and regulatory divergence among F1 progeny individuals. We show that in S. purpurea intraspecific crosses, gene expression inheritance largely exhibits a maternal dominant pattern, regardless of tissue type or pedigree. A significantly greater number of cis- and trans-regulated genes coincided with upregulation of the maternal parent allele in the progeny, irrespective of the magnitude, whereas the paternal allele was higher expressed for genes showing cis × trans or compensatory regulation. Importantly, consistent with previous genetic mapping results for sex in shrub willow, we have delimited sex-biased gene expression to a 2 Mb pericentromeric region on S. purpurea chr15 and further refined the sex determination region. Altogether, our results offer insight into the inheritance of gene expression in S. purpurea as well as evidence of sexually dimorphic expression which may have contributed to the evolution of dioecy in Salix.

Araport11: a complete reannotation of the Arabidopsis thaliana reference genome.

The flowering plant Arabidopsis thaliana is a dicot model organism for research in many aspects of plant biology. A comprehensive annotation of its genome paves the way for understanding the functions and activities of all types of transcripts, including mRNA, the various classes of non-coding RNA, and small RNA. The TAIR10 annotation update had a profound impact on Arabidopsis research but was released more than 5 years ago. Maintaining the accuracy of the annotation continues to be a prerequisite for future progress. Using an integrative annotation pipeline, we assembled tissue-specific RNA-Seq libraries from 113 datasets and constructed 48 359 transcript models of protein-coding genes in eleven tissues. In addition, we annotated various classes of non-coding RNA including microRNA, long intergenic RNA, small nucleolar RNA, natural antisense transcript, small nuclear RNA, and small RNA using published datasets and in-house analytic results. Altogether, we identified 635 novel protein-coding genes, 508 novel transcribed regions, 5178 non-coding RNAs, and 35 846 small RNA loci that were formerly unannotated. Analysis of the splicing events and RNA-Seq based expression profiles revealed the landscapes of gene structures, untranslated regions, and splicing activities to be more intricate than previously appreciated. Furthermore, we present 692 uniformly expressed housekeeping genes, 43% of whose human orthologs are also housekeeping genes. This updated Arabidopsis genome annotation with a substantially increased resolution of gene models will not only further our understanding of the biological processes of this plant model but also of other species.

ThaleMine: A Warehouse for Arabidopsis Data Integration and Discovery.

ThaleMine (https://apps.araport.org/thalemine/) is a comprehensive data warehouse that integrates a wide array of genomic information of the model plant Arabidopsis thaliana. The data collection currently includes the latest structural and functional annotation from the Araport11 update, the Col-0 genome sequence, RNA-seq and array expression, co-expression, protein interactions, homologs, pathways, publications, alleles, germplasm and phenotypes. The data are collected from a wide variety of public resources. Users can browse gene-specific data through Gene Report pages, identify and create gene lists based on experiments or indexed keywords, and run GO enrichment analysis to investigate the biological significance of selected gene sets. Developed by the Arabidopsis Information Portal project (Araport, https://www.araport.org/), ThaleMine uses the InterMine software framework, which builds well-structured data, and provides powerful data query and analysis functionality. The warehoused data can be accessed by users via graphical interfaces, as well as programmatically via web-services. Here we describe recent developments in ThaleMine including new features and extensions, and discuss future improvements. InterMine has been broadly adopted by the model organism research community including nematode, rat, mouse, zebrafish, budding yeast, the modENCODE project, as well as being used for human data. ThaleMine is the first InterMine developed for a plant model. As additional new plant InterMines are developed by the legume and other plant research communities, the potential of cross-organism integrative data analysis will be further enabled.

The pangenome of an agronomically important crop plant Brassica oleracea.

There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis.

Sequencing and analysis of globally obtained human respiratory syncytial virus A and B genomes.

BACKGROUND: Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level. METHODS: We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Phylogenetic analyses were performed using the Bayesian and maximum likelihood methods of phylogenetic inference. RESULTS: We sequenced the genomes of 34 RSVA and 23 RSVB viruses. Phylogenetic analysis showed that the RSVA genome evolves at an estimated rate of 6.72 × 10(-4) substitutions/site/year (95% HPD 5.61 × 10(-4) to 7.6 × 10(-4)) and for RSVB the evolutionary rate was 7.69 × 10(-4) substitutions/site/year (95% HPD 6.81 × 10(-4) to 8.62 × 10(-4)). We found multiple clades co-circulating globally for both RSV A and B. The predominant clades were GA2 and GA5 for RSVA and BA for RSVB. CONCLUSIONS: Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized.

Optical mapping in plant comparative genomics.

Optical mapping has been widely used to improve de novo plant genome assemblies, including rice, maize, Medicago, Amborella, tomato and wheat, with more genomes in the pipeline. Optical mapping provides long-range information of the genome and can more easily identify large structural variations. The ability of optical mapping to assay long single DNA molecules nicely complements short-read sequencing which is more suitable for the identification of small and short-range variants. Direct use of optical mapping to study population-level genetic diversity is currently limited to microbial strain typing and human diversity studies. Nonetheless, optical mapping shows great promise in the study of plant trait development, domestication and polyploid evolution. Here we review the current applications and future prospects of optical mapping in the field of plant comparative genomics.

Polyribosomal RNA-Seq reveals the decreased complexity and diversity of the Arabidopsis translatome.

Recent RNA-seq studies reveal that the transcriptomes in animals and plants are more complex than previously thought, leading to the inclusion of many more splice isoforms in annotated genomes. However, it is possible that a significant proportion of the transcripts are spurious isoforms that do not contribute to functional proteins. One of the current hypotheses is that commonly used mRNA extraction methods isolate both pre-mature (nuclear) mRNA and mature (cytoplasmic) mRNA, and these incompletely spliced pre-mature mRNAs may contribute to a large proportion of these spurious transcripts. To investigate this, we compared a traditional RNA-seq dataset (total RNA-seq) and a ribosome-bound RNA-seq dataset (polyribosomal RNA-seq) from Arabidopsis thaliana. An integrative framework that combined de novo assembly and genome-guided assembly was applied to reconstruct transcriptomes for the two datasets. Up to 44.8% of the de novo assembled transcripts in total RNA-seq sample were of low abundance, whereas only 0.09% in polyribosomal RNA-seq de novo assembly were of low abundance. The final round of assembly using PASA (Program to Assemble Spliced Alignments) resulted in more transcript assemblies in the total RNA-seq than those in polyribosomal sample. Comparison of alternative splicing (AS) patterns between total and polyribosomal RNA-seq showed a significant difference (G-test, p-value<0.01) in intron retention events: 46.4% of AS events in the total sample were intron retention, whereas only 23.5% showed evidence of intron retention in the polyribosomal sample. It is likely that a large proportion of retained introns in total RNA-seq result from incompletely spliced pre-mature mRNA. Overall, this study demonstrated that polyribosomal RNA-seq technology decreased the complexity and diversity of the coding transcriptome by eliminating pre-mature mRNAs, especially those of low abundance.

Araport: the Arabidopsis information portal.

The Arabidopsis Information Portal (https://www.araport.org) is a new online resource for plant biology research. It houses the Arabidopsis thaliana genome sequence and associated annotation. It was conceived as a framework that allows the research community to develop and release 'modules' that integrate, analyze and visualize Arabidopsis data that may reside at remote sites. The current implementation provides an indexed database of core genomic information. These data are made available through feature-rich web applications that provide search, data mining, and genome browser functionality, and also by bulk download and web services. Araport uses software from the InterMine and JBrowse projects to expose curated data from TAIR, GO, BAR, EBI, UniProt, PubMed and EPIC CoGe. The site also hosts 'science apps,' developed as prototypes for community modules that use dynamic web pages to present data obtained on-demand from third-party servers via RESTful web services. Designed for sustainability, the Arabidopsis Information Portal strategy exploits existing scientific computing infrastructure, adopts a practical mixture of data integration technologies and encourages collaborative enhancement of the resource by its user community.

MTGD: The Medicago truncatula genome database.

Medicago truncatula, a close relative of alfalfa (Medicago sativa), is a model legume used for studying symbiotic nitrogen fixation, mycorrhizal interactions and legume genomics. J. Craig Venter Institute (JCVI; formerly TIGR) has been involved in M. truncatula genome sequencing and annotation since 2002 and has maintained a web-based resource providing data to the community for this entire period. The website (http://www.MedicagoGenome.org) has seen major updates in the past year, where it currently hosts the latest version of the genome (Mt4.0), associated data and legacy project information, presented to users via a rich set of open-source tools. A JBrowse-based genome browser interface exposes tracks for visualization. Mutant gene symbols originally assembled and curated by the Frugoli lab are now hosted at JCVI and tie into our community annotation interface, Medicago EuCAP (to be integrated soon with our implementation of WebApollo). Literature pertinent to M. truncatula is indexed and made searchable via the Textpresso search engine. The site also implements MedicMine, an instance of InterMine that offers interconnectivity with other plant 'mines' such as ThaleMine and PhytoMine, and other model organism databases (MODs). In addition to these new features, we continue to provide keyword- and locus identifier-based searches served via a Chado-backed Tripal Instance, a BLAST search interface and bulk downloads of data sets from the iPlant Data Store (iDS). Finally, we maintain an E-mail helpdesk, facilitated by a JIRA issue tracking system, where we receive and respond to questions about the website and requests for specific data sets from the community.

Consequences of Whole-Genome Triplication as Revealed by Comparative Genomic Analyses of the Wild Radish Raphanus raphanistrum and Three Other Brassicaceae Species.

Polyploidization events are frequent among flowering plants, and the duplicate genes produced via such events contribute significantly to plant evolution. We sequenced the genome of wild radish (Raphanus raphanistrum), a Brassicaceae species that experienced a whole-genome triplication event prior to diverging from Brassica rapa. Despite substantial gene gains in these two species compared with Arabidopsis thaliana and Arabidopsis lyrata, ∼70% of the orthologous groups experienced gene losses in R. raphanistrum and B. rapa, with most of the losses occurring prior to their divergence. The retained duplicates show substantial divergence in sequence and expression. Based on comparison of A. thaliana and R. raphanistrum ortholog floral expression levels, retained radish duplicates diverged primarily via maintenance of ancestral expression level in one copy and reduction of expression level in others. In addition, retained duplicates differed significantly from genes that reverted to singleton state in function, sequence composition, expression patterns, network connectivity, and rates of evolution. Using these properties, we established a statistical learning model for predicting whether a duplicate would be retained postpolyploidization. Overall, our study provides new insights into the processes of plant duplicate loss, retention, and functional divergence and highlights the need for further understanding factors controlling duplicate gene fate.

An improved genome release (version Mt4.0) for the model legume Medicago truncatula.

BACKGROUND: Medicago truncatula, a close relative of alfalfa, is a preeminent model for studying nitrogen fixation, symbiosis, and legume genomics. The Medicago sequencing project began in 2003 with the goal to decipher sequences originated from the euchromatic portion of the genome. The initial sequencing approach was based on a BAC tiling path, culminating in a BAC-based assembly (Mt3.5) as well as an in-depth analysis of the genome published in 2011. RESULTS: Here we describe a further improved and refined version of the M. truncatula genome (Mt4.0) based on de novo whole genome shotgun assembly of a majority of Illumina and 454 reads using ALLPATHS-LG. The ALLPATHS-LG scaffolds were anchored onto the pseudomolecules on the basis of alignments to both the optical map and the genotyping-by-sequencing (GBS) map. The Mt4.0 pseudomolecules encompass ~360 Mb of actual sequences spanning 390 Mb of which ~330 Mb align perfectly with the optical map, presenting a drastic improvement over the BAC-based Mt3.5 which only contained 70% sequences (~250 Mb) of the current version. Most of the sequences and genes that previously resided on the unanchored portion of Mt3.5 have now been incorporated into the Mt4.0 pseudomolecules, with the exception of ~28 Mb of unplaced sequences. With regard to gene annotation, the genome has been re-annotated through our gene prediction pipeline, which integrates EST, RNA-seq, protein and gene prediction evidences. A total of 50,894 genes (31,661 high confidence and 19,233 low confidence) are included in Mt4.0 which overlapped with ~82% of the gene loci annotated in Mt3.5. Of the remaining genes, 14% of the Mt3.5 genes have been deprecated to an "unsupported" status and 4% are absent from the Mt4.0 predictions. CONCLUSIONS: Mt4.0 and its associated resources, such as genome browsers, BLAST-able datasets and gene information pages, can be found on the JCVI Medicago web site (http://www.jcvi.org/medicago). The assembly and annotation has been deposited in GenBank (BioProject: PRJNA10791). The heavily curated chromosomal sequences and associated gene models of Medicago will serve as a better reference for legume biology and comparative genomics.

Phylogenomics and the dynamic genome evolution of the genus Streptococcus.

The genus Streptococcus comprises important pathogens that have a severe impact on human health and are responsible for substantial economic losses to agriculture. Here, we utilize 46 Streptococcus genome sequences (44 species), including eight species sequenced here, to provide the first genomic level insight into the evolutionary history and genetic basis underlying the functional diversity of all major groups of this genus. Gene gain/loss analysis revealed a dynamic pattern of genome evolution characterized by an initial period of gene gain followed by a period of loss, as the major groups within the genus diversified. This was followed by a period of genome expansion associated with the origins of the present extant species. The pattern is concordant with an emerging view that genomes evolve through a dynamic process of expansion and streamlining. A large proportion of the pan-genome has experienced lateral gene transfer (LGT) with causative factors, such as relatedness and shared environment, operating over different evolutionary scales. Multiple gene ontology terms were significantly enriched for each group, and mapping terms onto the phylogeny showed that those corresponding to genes born on branches leading to the major groups represented approximately one-fifth of those enriched. Furthermore, despite the extensive LGT, several biochemical characteristics have been retained since group formation, suggesting genomic cohesiveness through time, and that these characteristics may be fundamental to each group. For example, proteolysis: mitis group; urea metabolism: salivarius group; carbohydrate metabolism: pyogenic group; and transcription regulation: bovis group.

Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses.

From 1 January 2009 to 31 May 2013, 15 287 respiratory specimens submitted to the Clinical Virology Laboratory at the Children's Hospital Colorado were tested for human coronavirus RNA by reverse transcription-PCR. Human coronaviruses HKU1, OC43, 229E and NL63 co-circulated during each of the respiratory seasons but with significant year-to-year variability, and cumulatively accounted for 7.4-15.6 % of all samples tested during the months of peak activity. A total of 79 (0.5 % prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human tracheal-bronchial epithelial cells cultured at the air-liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of the deduced amino acid sequences of seven full-length genomes of Colorado HKU1 viruses and the spike glycoproteins from four additional HKU1 viruses from Colorado and three from Brazil demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by three amino acid substitutions (W197F, F613Y and S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N-terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of these varying tandem repeats will increase our understanding of the replication process and pathogenicity of HKU1 and potentially of other coronaviruses.

An atlas of over 90,000 conserved noncoding sequences provides insight into crucifer regulatory regions.

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species.

The receptor like kinase at Rhg1-a/Rfs2 caused pleiotropic resistance to sudden death syndrome and soybean cyst nematode as a transgene by altering signaling responses.

BACKGROUND: Soybean (Glycine max (L. Merr.)) resistance to any population of Heterodera glycines (I.), or Fusarium virguliforme (Akoi, O'Donnell, Homma & Lattanzi) required a functional allele at Rhg1/Rfs2. H. glycines, the soybean cyst nematode (SCN) was an ancient, endemic, pest of soybean whereas F. virguliforme causal agent of sudden death syndrome (SDS), was a recent, regional, pest. This study examined the role of a receptor like kinase (RLK) GmRLK18-1 (gene model Glyma_18_02680 at 1,071 kbp on chromosome 18 of the genome sequence) within the Rhg1/Rfs2 locus in causing resistance to SCN and SDS. RESULTS: A BAC (B73p06) encompassing the Rhg1/Rfs2 locus was sequenced from a resistant cultivar and compared to the sequences of two susceptible cultivars from which 800 SNPs were found. Sequence alignments inferred that the resistance allele was an introgressed region of about 59 kbp at the center of which the GmRLK18-1 was the most polymorphic gene and encoded protein. Analyses were made of plants that were either heterozygous at, or transgenic (and so hemizygous at a new location) with, the resistance allele of GmRLK18-1. Those plants infested with either H. glycines or F. virguliforme showed that the allele for resistance was dominant. In the absence of Rhg4 the GmRLK18-1 was sufficient to confer nearly complete resistance to both root and leaf symptoms of SDS caused by F. virguliforme and provided partial resistance to three different populations of nematodes (mature female cysts were reduced by 30-50%). In the presence of Rhg4 the plants with the transgene were nearly classed as fully resistant to SCN (females reduced to 11% of the susceptible control) as well as SDS. A reduction in the rate of early seedling root development was also shown to be caused by the resistance allele of the GmRLK18-1. Field trials of transgenic plants showed an increase in foliar susceptibility to insect herbivory. CONCLUSIONS: The inference that soybean has adapted part of an existing pathogen recognition and defense cascade (H.glycines; SCN and insect herbivory) to a new pathogen (F. virguliforme; SDS) has broad implications for crop improvement. Stable resistance to many pathogens might be achieved by manipulation the genes encoding a small number of pathogen recognition proteins.

Genomic analysis of 16 Colorado human NL63 coronaviruses identifies a new genotype, high sequence diversity in the N-terminal domain of the spike gene and evidence of recombination.

This study compared the complete genome sequences of 16 NL63 strain human coronaviruses (hCoVs) from respiratory specimens of paediatric patients with respiratory disease in Colorado, USA, and characterized the epidemiology and clinical characteristics associated with circulating NL63 viruses over a 3-year period. From 1 January 2009 to 31 December 2011, 92 of 9380 respiratory specimens were found to be positive for NL63 RNA by PCR, an overall prevalence of 1 %. NL63 viruses were circulating during all 3 years, but there was considerable yearly variation in prevalence and the month of peak incidence. Phylogenetic analysis comparing the genome sequences of the 16 Colorado NL63 viruses with those of the prototypical hCoV-NL63 and three other NL63 viruses from the Netherlands demonstrated that there were three genotypes (A, B and C) circulating in Colorado from 2005 to 2010, and evidence of recombination between virus strains was found. Genotypes B and C co-circulated in Colorado in 2005, 2009 and 2010, but genotype A circulated only in 2005 when it was the predominant NL63 strain. Genotype C represents a new lineage that has not been described previously. The greatest variability in the NL63 virus genomes was found in the N-terminal domain (NTD) of the spike gene (nt 1-600, aa 1-200). Ten different amino acid sequences were found in the NTD of the spike protein among these NL63 strains and the 75 partial published sequences of NTDs from strains found at different times throughout the world.

Identification and characterization of the defensin-like gene family of grapevine.

Defensins are a class of small and diverse cysteine-rich proteins found in plants, insects, and vertebrates, which share a common tertiary structure and usually exert broad-spectrum antimicrobial activities. We used a bioinformatic approach to scan the Vitis vinifera genome and identified 79 defensin-like sequences (DEFL) corresponding to 46 genes and allelic variants, plus 33 pseudogenes and gene fragments. Expansion and diversification of grapevine DEFL has occurred after the split from the last common ancestor with the genera Medicago and Arabidopsis. Grapevine DEFL localization on the 'Pinot Noir' genome revealed the presence of several clusters likely evolved through local duplications. By sequencing reverse-transcription polymerase chain reaction products, we could demonstrate the expression of grapevine DEFL with no previously reported record of expression. Many of these genes are predominantly or exclusively expressed in tissues linked to plant reproduction, consistent with findings in other plant species, and some of them accumulated at fruit ripening. The transcripts of five DEFL were also significantly upregulated in tissues infected with Botrytis cinerea, a necrotrophic mold, suggesting a role of these genes in defense against this pathogen. Finally, three novel defensins were discovered among the identified DEFL. They inhibit B. cinerea conidia germination when expressed as recombinant proteins.