The fate of duplicated genes in a polyploid plant genome.

Polyploidy is generally not tolerated in animals, but is widespread in plant genomes and may result in extensive genetic redundancy. The fate of duplicated genes is poorly understood, both functionally and evolutionarily. Soybean (Glycine max L.) has undergone two separate polyploidy events (13 and 59 million years ago) that have resulted in 75% of its genes being present in multiple copies. It therefore constitutes a good model to study the impact of whole-genome duplication on gene expression. Using RNA-seq, we tested the functional fate of a set of approximately 18 000 duplicated genes. Across seven tissues tested, approximately 50% of paralogs were differentially expressed and thus had undergone expression sub-functionalization. Based on gene ontology and expression data, our analysis also revealed that only a small proportion of the duplicated genes have been neo-functionalized or non-functionalized. In addition, duplicated genes were often found in collinear blocks, and several blocks of duplicated genes were co-regulated, suggesting some type of epigenetic or positional regulation. We also found that transcription factors and ribosomal protein genes were differentially expressed in many tissues, suggesting that the main consequence of polyploidy in soybean may be at the regulatory level.

A systems-wide comparison of red rice (Oryza longistaminata) tissues identifies rhizome specific genes and proteins that are targets for cultivated rice improvement.

BACKGROUND: The rhizome, the original stem of land plants, enables species to invade new territory and is a critical component of perenniality, especially in grasses. Red rice (Oryza longistaminata) is a perennial wild rice species with many valuable traits that could be used to improve cultivated rice cultivars, including rhizomatousness, disease resistance and drought tolerance. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development and function in this plant. RESULTS: We used an integrated approach to compare the transcriptome, proteome and metabolome of the rhizome to other tissues of red rice. 116 Gb of transcriptome sequence was obtained from various tissues and used to identify rhizome-specific and preferentially expressed genes, including transcription factors and hormone metabolism and stress response-related genes. Proteomics and metabolomics approaches identified 41 proteins and more than 100 primary metabolites and plant hormones with rhizome preferential accumulation. Of particular interest was the identification of a large number of gene transcripts from Magnaportha oryzae, the fungus that causes rice blast disease in cultivated rice, even though the red rice plants showed no sign of disease. CONCLUSIONS: A significant set of genes, proteins and metabolites appear to be specifically or preferentially expressed in the rhizome of O. longistaminata. The presence of M. oryzae gene transcripts at a high level in apparently healthy plants suggests that red rice is resistant to this pathogen, and may be able to provide genes to cultivated rice that will enable resistance to rice blast disease.

Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome.

The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP-) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP- seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP- soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed's transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP- line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFP-expressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content.

Next-generation sequencing-based transcriptomic and proteomic analysis of the common reed, Phragmites australis (Poaceae), reveals genes involved in invasiveness and rhizome specificity.

PREMISE OF THE STUDY: The common reed (Phragmites australis), one of the most widely distributed of all angiosperms, uses its rhizomes (underground stems) to invade new territory, making it one of the most successful weedy species worldwide. Characterization of the rhizome transcriptome and proteome is needed to identify candidate genes and proteins involved in rhizome growth, development, metabolism, and invasiveness. METHODS: We employed next-generation sequencing technologies including 454 and Illumina platforms to characterize the reed rhizome transcriptome and used quantitative proteomics techniques to identify the rhizome proteome. KEY RESULTS: Combining 336514 Roche 454 Titanium reads and 103350802 Illumina paired-end reads in a de novo hybrid assembly yielded 124450 unique transcripts with an average length of 549 bp, of which 54317 were annotated. Rhizome-specific and differentially expressed transcripts were identified between rhizome apical tips (apical meristematic region) and rhizome elongation zones. A total of 1280 nonredundant proteins were identified and quantified using GeLC-MS/MS based label-free proteomics, where 174 and 77 proteins were preferentially expressed in the rhizome elongation zone and apical tip tissues, respectively. Genes involved in allelopathy and in controlling development and potentially invasiveness were identified. CONCLUSIONS: In addition to being a valuable sequence and protein data resource for studying plant rhizome species, our results provide useful insights into identifying specific genes and proteins with potential roles in rhizome differentiation, development, and function.

Development and use of genic molecular markers (GMMs) for construction of a transcript map of chickpea (Cicer arietinum L.).

A transcript map has been constructed by the development and integration of genic molecular markers (GMMs) including single nucleotide polymorphism (SNP), genic microsatellite or simple sequence repeat (SSR) and intron spanning region (ISR)-based markers, on an inter-specific mapping population of chickpea, the third food legume crop of the world and the first food legume crop of India. For SNP discovery through allele re-sequencing, primer pairs were designed for 688 genes/expressed sequence tags (ESTs) of chickpea and 657 genes/ESTs of closely related species of chickpea. High-quality sequence data obtained for 220 candidate genic regions on 2-20 genotypes representing 9 Cicer species provided 1,893 SNPs with an average frequency of 1/35.83 bp and 0.34 PIC (polymorphism information content) value. On an average 2.9 haplotypes were present in 220 candidate genic regions with an average haplotype diversity of 0.6326. SNP2CAPS analysis of 220 sequence alignments, as mentioned above, provided a total of 192 CAPS candidates. Experimental analysis of these 192 CAPS candidates together with 87 CAPS candidates identified earlier through in silico mining of ESTs provided scorable amplification in 173 (62.01%) cases of which predicted assays were validated in 143 (82.66%) cases (CGMM). Alignments of chickpea unigenes with Medicago truncatula genome were used to develop 121 intron spanning region (CISR) markers of which 87 yielded scorable products. In addition, optimization of 77 EST-derived SSR (ICCeM) markers provided 51 scorable markers. Screening of easily assayable 281 markers including 143 CGMMs, 87 CISRs and 51 ICCeMs on 5 parental genotypes of three mapping populations identified 104 polymorphic markers including 90 markers on the inter-specific mapping population. Sixty-two of these GMMs together with 218 earlier published markers (including 64 GMM loci) and 20 other unpublished markers could be integrated into this genetic map. A genetic map developed here, therefore, has a total of 300 loci including 126 GMM loci and spans 766.56 cM, with an average inter-marker distance of 2.55 cM. In summary, this is the first report on the development of large-scale genic markers including development of easily assayable markers and a transcript map of chickpea. These resources should be useful not only for genome analysis and genetics and breeding applications of chickpea, but also for comparative legume genomics.

Prediction of novel miRNAs and associated target genes in Glycine max.

BACKGROUND: Small non-coding RNAs (21 to 24 nucleotides) regulate a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. Among these, the most conserved classes are microRNAs (miRNAs) and small interfering RNAs (siRNAs), both of which are produced by RNase III-like enzymes called Dicers. Many plant miRNAs play critical roles in nutrient homeostasis, developmental processes, abiotic stress and pathogen responses. Currently, only 70 miRNA have been identified in soybean. METHODS: We utilized Illumina's SBS sequencing technology to generate high-quality small RNA (sRNA) data from four soybean (Glycine max) tissues, including root, seed, flower, and nodules, to expand the collection of currently known soybean miRNAs. We developed a bioinformatics pipeline using in-house scripts and publicly available structure prediction tools to differentiate the authentic mature miRNA sequences from other sRNAs and short RNA fragments represented in the public sequencing data. RESULTS: The combined sequencing and bioinformatics analyses identified 129 miRNAs based on hairpin secondary structure features in the predicted precursors. Out of these, 42 miRNAs matched known miRNAs in soybean or other species, while 87 novel miRNAs were identified. We also predicted the putative target genes of all identified miRNAs with computational methods and verified the predicted cleavage sites in vivo for a subset of these targets using the 5' RACE method. Finally, we also studied the relationship between the abundance of miRNA and that of the respective target genes by comparison to Solexa cDNA sequencing data. CONCLUSION: Our study significantly increased the number of miRNAs known to be expressed in soybean. The bioinformatics analysis provided insight on regulation patterns between the miRNAs and their predicted target genes expression. We also deposited the data in a soybean genome browser based on the UCSC Genome Browser architecture. Using the browser, we annotated the soybean data with miRNA sequences from four tissues and cDNA sequencing data. Overlaying these two datasets in the browser allows researchers to analyze the miRNA expression levels relative to that of the associated target genes. The browser can be accessed at http://digbio.missouri.edu/soybean_mirna/.

SSWAP: A Simple Semantic Web Architecture and Protocol for semantic web services.

BACKGROUND: SSWAP (Simple Semantic Web Architecture and Protocol; pronounced "swap") is an architecture, protocol, and platform for using reasoning to semantically integrate heterogeneous disparate data and services on the web. SSWAP was developed as a hybrid semantic web services technology to overcome limitations found in both pure web service technologies and pure semantic web technologies. RESULTS: There are currently over 2400 resources published in SSWAP. Approximately two dozen are custom-written services for QTL (Quantitative Trait Loci) and mapping data for legumes and grasses (grains). The remaining are wrappers to Nucleic Acids Research Database and Web Server entries. As an architecture, SSWAP establishes how clients (users of data, services, and ontologies), providers (suppliers of data, services, and ontologies), and discovery servers (semantic search engines) interact to allow for the description, querying, discovery, invocation, and response of semantic web services. As a protocol, SSWAP provides the vocabulary and semantics to allow clients, providers, and discovery servers to engage in semantic web services. The protocol is based on the W3C-sanctioned first-order description logic language OWL DL. As an open source platform, a discovery server running at http://sswap.info (as in to "swap info") uses the description logic reasoner Pellet to integrate semantic resources. The platform hosts an interactive guide to the protocol at http://sswap.info/protocol.jsp, developer tools at http://sswap.info/developer.jsp, and a portal to third-party ontologies at http://sswapmeet.sswap.info (a "swap meet"). CONCLUSION: SSWAP addresses the three basic requirements of a semantic web services architecture (i.e., a common syntax, shared semantic, and semantic discovery) while addressing three technology limitations common in distributed service systems: i.e., i) the fatal mutability of traditional interfaces, ii) the rigidity and fragility of static subsumption hierarchies, and iii) the confounding of content, structure, and presentation. SSWAP is novel by establishing the concept of a canonical yet mutable OWL DL graph that allows data and service providers to describe their resources, to allow discovery servers to offer semantically rich search engines, to allow clients to discover and invoke those resources, and to allow providers to respond with semantically tagged data. SSWAP allows for a mix-and-match of terms from both new and legacy third-party ontologies in these graphs.

Pediatric fluid resuscitation and airway management.

In summary, if the problem is volume shock, stop the loss and replace the fluid without overloading. IV fluids may be necessary to maintain perfusion pressure and administer medications. Pediatric airway anatomy requires different techniques and equipment for proper management, but all airway management starts with basic life support skills.

Making a chocolate chip: development and evaluation of a 6K SNP array for Theobroma cacao.

Theobroma cacao, the key ingredient in chocolate production, is one of the world's most important tree fruit crops, with ∼4,000,000 metric tons produced across 50 countries. To move towards gene discovery and marker-assisted breeding in cacao, a single-nucleotide polymorphism (SNP) identification project was undertaken using RNAseq data from 16 diverse cacao cultivars. RNA sequences were aligned to the assembled transcriptome of the cultivar Matina 1-6, and 330,000 SNPs within coding regions were identified. From these SNPs, a subset of 6,000 high-quality SNPs were selected for inclusion on an Illumina Infinium SNP array: the Cacao6kSNP array. Using Cacao6KSNP array data from over 1,000 cacao samples, we demonstrate that our custom array produces a saturated genetic map and can be used to distinguish among even closely related genotypes. Our study enhances and expands the genetic resources available to the cacao research community, and provides the genome-scale set of tools that are critical for advancing breeding with molecular markers in an agricultural species with high genetic diversity.

Asian Citrus Psyllid Expression Profiles Suggest Candidatus Liberibacter Asiaticus-Mediated Alteration of Adult Nutrition and Metabolism, and of Nymphal Development and Immunity.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the insect vector of the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease, or Huanglongbing (HLB). The widespread invasiveness of the psyllid vector and HLB in citrus trees worldwide has underscored the need for non-traditional approaches to manage the disease. One tenable solution is through the deployment of RNA interference technology to silence protein-protein interactions essential for ACP-mediated CLas invasion and transmission. To identify psyllid interactor-bacterial effector combinations associated with psyllid-CLas interactions, cDNA libraries were constructed from CLas-infected and CLas-free ACP adults and nymphs, and analyzed for differential expression. Library assemblies comprised 24,039,255 reads and yielded 45,976 consensus contigs. They were annotated (UniProt), classified using Gene Ontology, and subjected to in silico expression analyses using the Transcriptome Computational Workbench (TCW) (http://www.sohomoptera.org/ACPPoP/). Functional-biological pathway interpretations were carried out using the Kyoto Encyclopedia of Genes and Genomes databases. Differentially expressed contigs in adults and/or nymphs represented genes and/or metabolic/pathogenesis pathways involved in adhesion, biofilm formation, development-related, immunity, nutrition, stress, and virulence. Notably, contigs involved in gene silencing and transposon-related responses were documented in a psyllid for the first time. This is the first comparative transcriptomic analysis of ACP adults and nymphs infected and uninfected with CLas. The results provide key initial insights into host-parasite interactions involving CLas effectors that contribute to invasion-virulence, and to host nutritional exploitation and immune-related responses that appear to be essential for successful ACP-mediated circulative, propagative CLas transmission.

Comparison of potato and asian citrus psyllid adult and nymph transcriptomes identified vector transcripts with potential involvement in circulative, propagative liberibacter transmission.

The potato psyllid (PoP) Bactericera cockerelli (Sulc) and Asian citrus psyllid (ACP) Diaphorina citri Kuwayama are the insect vectors of the fastidious plant pathogen, Candidatus Liberibacter solanacearum (CLso) and Ca. L. asiaticus (CLas), respectively. CLso causes Zebra chip disease of potato and vein-greening in solanaceous species, whereas, CLas causes citrus greening disease. The reliance on insecticides for vector management to reduce pathogen transmission has increased interest in alternative approaches, including RNA interference to abate expression of genes essential for psyllid-mediated Ca. Liberibacter transmission. To identify genes with significantly altered expression at different life stages and conditions of CLso/CLas infection, cDNA libraries were constructed for CLso-infected and -uninfected PoP adults and nymphal instars. Illumina sequencing produced 199,081,451 reads that were assembled into 82,224 unique transcripts. PoP and the analogous transcripts from ACP adult and nymphs reported elsewhere were annotated, organized into functional gene groups using the Gene Ontology classification system, and analyzed for differential in silico expression. Expression profiles revealed vector life stage differences and differential gene expression associated with Liberibacter infection of the psyllid host, including invasion, immune system modulation, nutrition, and development.