Shaping the biology of citrus: II. Genomic determinants of domestication

We performed genomic analyses on species and varieties of the genus Citrus to identify several determinants of domestication, based on the pattern of pummelo [Citrus maxima (Burr. f) Merr] and mandarin (Citrus reticulata Blanco) admixture into the ancestral genome, as well as population genetic tests at smaller scales. Domestication impacted gene families regulating pivotal components of citrus flavor (such as acidity) because in edible mandarin varieties, chromosome areas with negative Tajimas values were enriched with genes associated with the regulation of citric acid. Detection of sweeps in edible mandarins that diverged from wild relatives indicated that domestication reduced chemical defenses involving cyanogenesis and alkaloid synthesis, thus increasing palatability. Also, a cluster of SAUR genes in domesticated mandarins derived from the pummelo genome appears to contain candidate genes controlling fruit size. Similarly, conserved stretches of pure mandarin areas were likely important as well for domestication, as, for example, a fragment in chromosome 1 that is involved in the apomictic reproduction of most edible mandarins. Interestingly, our results also support the hypothesis that various genes subject to selective pressure during evolution or derived from whole genome duplication events later became potential targets of domestication

MELOGEN: an EST database for melon functional genomics

<p>Abstract</p> <p>Background</p> <p>Melon (<it>Cucumis melo </it>L.) is one of the most important fleshy fruits for fresh consumption. Despite this, few genomic resources exist for this species. To facilitate the discovery of genes involved in essential traits, such as fruit development, fruit maturation and disease resistance, and to speed up the process of breeding new and better adapted melon varieties, we have produced a large collection of expressed sequence tags (ESTs) from eight normalized cDNA libraries from different tissues in different physiological conditions.</p> <p>Results</p> <p>We determined over 30,000 ESTs that were clustered into 16,637 non-redundant sequences or unigenes, comprising 6,023 tentative consensus sequences (contigs) and 10,614 unclustered sequences (singletons). Many potential molecular markers were identified in the melon dataset: 1,052 potential simple sequence repeats (SSRs) and 356 single nucleotide polymorphisms (SNPs) were found. Sixty-nine percent of the melon unigenes showed a significant similarity with proteins in databases. Functional classification of the unigenes was carried out following the Gene Ontology scheme. In total, 9,402 unigenes were mapped to one or more ontology. Remarkably, the distributions of melon and Arabidopsis unigenes followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. Bioinformatic analyses primarily focused on potential precursors of melon micro RNAs (miRNAs) in the melon dataset, but many other genes potentially controlling disease resistance and fruit quality traits were also identified. Patterns of transcript accumulation were characterised by Real-Time-qPCR for 20 of these genes.</p> <p>Conclusion</p> <p>The collection of ESTs characterised here represents a substantial increase on the genetic information available for melon. A database (MELOGEN) which contains all EST sequences, contig images and several tools for analysis and data mining has been created. This set of sequences constitutes also the basis for an oligo-based microarray for melon that is being used in experiments to further analyse the melon transcriptome.</p

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Muestreo y análisis del transcriptoma de melón "cucumis melo L."

El melón es uno de los cultivos más importantes del mundo. Las infecciones por virus son una de las principales causas de pérdidas en la producción, mayoritariamente las causadas por virus de RNA. El muestreo de los transcritos de RNA (transcriptoma) que se generan a partir del genoma proporciona un abordaje funcional al contenido genético de una especie. En esta Tesis se aporta un análisis de dos porciones del transcriptoma de melón: la que corresponde a transcritos que codifican proteínas mediante un microarray de DNA, y la que corresponde a pequeños RNAs no codificantes mediante secuenciación masiva. Se ha analizado el transcriptoma en respuesta a la infección por el virus del mosaico de la sandía en un genotipo de melón resistente y otro susceptible al virus. Además, se ha optimizado un método de síntesis de cDNA para hibridaciones en microarray. Se ha optado por presentar la Tesis en modo de compendio de tres publicaciones. Palabras clave: melón, genotecas, cDNA, microarray, pirosecuenciación, ESTs, pequeños RNAs, virus, transcriptómica Melon is one of the most important horticultural crops worldwide. Plant viral diseases cause serious economic losses by reducing yield and quality. Sampling of the complete set of cellular RNA transcripts (transcriptome) has been used as an effective method for dissecting genetic content of many species and for analyzing several biological processes. In this Thesis, two parts of the melon transcriptome have been analyzed: protein-coding transcripts by means of a DNA microarray, and small RNAs by means of pyrosequencing. Transcriptomic changes in response to Watermelon mosaic virus infection have been monitored in a resistant and a susceptible melon genotype. In addition, a method for cDNA synthesis for microarray hybridizations has been optimized. Results are presented as a compilation of three publications. key words: melon, cDNA libraries, cDNA, microarray, pyrosequencing, ESTs, small RNAs, virus, transcriptomi

Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes

12 páginas, 6 figuras, 1 tabla, 3 figuras suplementarias -- PAGS nros. 203-214Plant virus infection involves the production of viral small RNAs (vsRNAs) with the potential to associate with distinct Argonaute (AGO)-containing silencing complexes and mediate diverse silencing effects on RNA and chromatin. We used multiplexed, high-throughput pyrosequencing to profile populations of vsRNAs from plants infected with viruses from different genera. Sense and antisense vsRNAs of 20 to 24 nucleotides (nts) spread throughout the entire viral genomes in an overlapping configuration; virtually all genomic nucleotide positions were represented in the data set. We present evidence to suggest that every genomic position could be a putative cleavage site for vsRNA formation, although viral genomes contain specific regions that serve as preferential sources of vsRNA production. Hotspots for vsRNAs of 21-, 22-, and 24-nt usually coincide in the same genomic regions, indicating similar target affinities among Dicer-like (DCL) enzymes. In the light of our results, the overall contribution of perfectly base paired double-stranded RNA and imperfectly base paired structures within single-stranded RNA to vsRNA formation is discussed. Our census of vsRNAs extends the current view of the distribution and composition of vsRNAs in virus-infected plants, and contributes to a better understanding of vsRNA biogenesisThis wok was supported by PhD fellowships from the Comunidad de Madrid (to L.D.) and Ministerio de Educación y Ciencia (MEC, to D.G.I.) and by Grant BIO2006-13107 from the MEC, SpainPeer reviewe

Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant

12 Pág.Translation initiation factors are universal determinants of plant susceptibility to RNA viruses, but the underlying mechanisms are poorly understood. Here, we show that a sequence in the 3' untranslated region (3'-UTR) of a viral genome that is responsible for overcoming plant eIF4E-mediated resistance (virulence determinant) functions as a 3' cap-independent translational enhancer (3'-CITE). The virus/plant pair studied here is Melon necrotic spot virus (MNSV) and melon, for which a recessive resistance controlled by melon eIF4E was previously described. Chimeric viruses between virulent and avirulent isolates enabled us to map the virulence and avirulence determinants to 49 and 26 nucleotides, respectively. The translational efficiency of a luc reporter gene flanked by 5'- and 3'-UTRs from virulent, avirulent and chimeric viruses was analysed in vitro, in wheatgerm extract, and in vivo, in melon protoplasts, showing that: (i) the virulence determinant mediates the efficient cap-independent translation in vitro and in vivo; (ii) the avirulence determinant was able to promote efficient cap-independent translation in vitro, but only when eIF4E from susceptible melon was added in trans, and, coherently, only in protoplasts of susceptible melon, but not in the protoplasts of resistant melon; (iii) these activities required the 5'-UTR of MNSV in cis. Thus, the virulence and avirulence determinants function as 3'-CITEs. The activity of these 3'-CITEs was host specific, suggesting that an inefficient interaction between the viral 3'-CITE of the avirulent isolate and eIF4E of resistant melon impedes the correct formation of the translation initiation complex at the viral RNA ends, thereby leading to resistance.This work was supported by grants from Ministerio de Educación y Ciencia (Spain) (AGL2006-08069/AGR) and Fundación Séneca (Región de Murcia, Spain) (05702/PI/07). DGI was a recipient of a predoctoral fellowship from Ministerio de Educación y Ciencia (Spain). We want to thank Christian Clepet (Unité de Recherche en Génomique Végétale, Evry Cedex, France) for his help in purifying Cm-eIF4E, A. Rakotondrafara for her help with the luciferase assays, and Mari Carmen Montesinos and Blanca Gosalvez for their excellent technical assistance.Peer reviewe

Shaping the biology of citrus: I. Genomic determinants of evolution

We performed genomic analyses on wild species of the genus Citrus to identifymajor determinants of evolution. The most notable effect occurred on the pathogen-defense genes, as observed in many other plant genera. The gene space was also characterized by changes in gene families intimately related to relevant biochemical properties of citrus fruit, such as pectin modifying enzymes, HDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase) genes, and O-methyltransferases. Citrus fruits are highly abundant on pectins and secondary metabolites such as terpenoids and flavonoids, the targets of these families. Other gene types under positive selection, expanded through tandem duplications and retained as triplets from whole genome duplications, codified for purple acid phosphatases and MATE-efflux proteins. Although speciation has not been especially rapid in the genus, analyses of selective pressure at the codon level revealed that the extant species evolved from the ancestral citrus radiation show signatures of pervasive adaptive evolution and is therefore potentially responsible for the vast phenotypic differences observed among current species. by changes in gene families intimately related to relevant biochemical properties of citrus fruit, such as pectin modifying enzymes, HDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase) genes, and O-methyltransferases. Citrus fruits are highly abundant on pectins and secondarymetabolites such as terpenoids and flavonoids, the targets of these families. Other gene types under positive selection, expanded through tandem duplications and retained as triplets from whole genome duplications, codified for purple acid phosphatases and MATE-efflux proteins. Although speciation has not been especially rapid in the genus, analyses of selective pressure at the codon level revealed that the extant species evolved from the ancestral citrus radiation show signatures of pervasive adaptive evolution and is therefore potentially responsible for the vast phenotypic differences observed among current species

Virus-infected melon plants emit volatiles that induce gene deregulation in neighboring healthy plants

It is well described that viral infections stimulate the emission of plant volatiles able to recruit viral vectors thereby promoting virus spread. In contrast, much less is known on the effects that emitted volatiles may have on the metabolism of healthy neighboring plants, which are potential targets for new infections through vector transmission. Watermelon mosaic virus (WMV) (genus Potyvirus, family Potyviridae) is an aphid-transmitted virus endemic in cucurbit crops worldwide. We have compared gene expression profiles of WMV-infected melon plants with those of healthy or healthy-but-cohabited-with-infected plants. Pathogenesis-related (PR) and small heat shock protein encoding genes were deregulated in cohabited plants, and PR deregulation depended on the distance to the infected plant. The signaling was short distance in the experimental conditions used, and cohabiting had a moderate effect on the plant susceptibility to WMV. Static headspace experiments showed that benzaldehyde and g-butyrolactone were significantly over-emitted by WMV-infected plants. Altogether, our data suggest that perception of a volatile signal encoded by WMV-infected tissues triggers a response to prepare healthy tissues or/and healthy neighboring plants for the incoming infections.Ministry of Science, Innovation and Universities Grant/Award Number: BES-2016-077826 Región de Murcia, Consejería de Empleo Universidades y Empresa; RIS3MUR program Grant/Award Number: 2I16SA000057 Ministry of Economy, Industry and Competitiveness Grant/Award Number: PTQ-15-07646 Grant/Award Number: PTQ-16-0836

CitrusGenome: Applying User Centered Design for Evaluating the Usability of Genomic User Interfaces

Several tools have been developed to extract knowledge from the vast amount of data in genomics. The success of the knowledge extraction process depends to arge extent on how easy to learn and use are the tools for bioinformaticians. User interface design is neglected frequently in the genomic tool development process. As a result, user interfaces contain usability problems that make knowledge extraction a complex task. User-Centered Design (UCD) is a design approach that can be applied to improve the usability of interfaces. A fundamental principle of UCD is to design the UI based on users’ knowledge, their needs, objectives, and tasks to ensure that the resulting user interface meets the real user needs. We apply the UCD approach to design, evaluate and improve CitrusGenome, a tool that enables bioinformaticians to extract knowledge from genomic data. Following the UCD process, we first conduct user research to define user needs by applying UCD techniques such as interviews and task analysis. Then, we design a user interface that meets those user needs by using GenomIUm. GenomIUm is a systematic method that guides the design process of user interfaces in the genomic domain. We have performed two UCD iterations and, after each iteration, the user interfaces were validated by bioinformaticians. Some usability problems were found in each iteration. Therefore, we refined the user interface by solving the usability problems and incorporating such solutions into the final design. Finally, bioinformaticians using the refined user interface reported a reduction in the complexity of extracting knowledge from genomic data. We conclude that UCD techniques, together with GenomIUm, can be a useful strategy to design user interfaces that are easier to learn and use in the genomic domai

Applying User Centred Design to Improve the Design of Genomic User Interfaces

The genomic domain is a complex data environment that has grown exponentially. Several tools have been developed to extract knowledge from this immense amount of data. Knowledge extraction processes depend to a large extent on how easy and intuitive are the user interfaces of the tools that are used by bioinformaticians. However, genomic tools have frequently ignored the design process of their User Interfaces. Consequently, they have important usability problems that complicates knowledge extraction. User Centered Design (UCD) is a design approach that can be used to improve the usability of genomic tools. It consists on putting the user and its real needs at the center of the design process. Improving the usability of these tools will facilitate knowledge extraction. This paper reports the application of the UCD approach to design a tool that improves knowledge extraction processes in a real world-use case. From a general perspective, UCD consists of “user research” and “design solutions”. The first one was carried out by conducting UCD techniques, including user interviews and task analysis. The second one was carried out by applying GenomIUm, a pattern-based method that guides the design process of genomic user interfaces. As a fundamental part in the UCD approach, the generated user interfaces were validated by expert bioinformaticians who reported that the complexity of extracting knowledge from genomic data was reduced. We conclude that UCD techniques together with GenomIUm can be a useful strategy to design more usable user interfaces in the genomic domain