NexGenEx-Tom: A gene expression platform to investigate the functionalities of the tomato genome

BACKGROUND: Next Generation Sequencing technologies (NGS) unexpectedly pushed forward the capability of solving genome organization and of widely depicting gene expression. However, although the flourishing of tools to process the NGS data, versatile and user-friendly computational environments for integrative and comparative analyses of the results from the increasing amount of collections are still required. The gene expression of tomato tissues has been widely investigated in the years, thanks to both EST sequencing and different microarray platforms. However, the resulting collections are heterogeneous in terms of experimental approaches, genotypes and conditions, making the data far from representing a gene expression atlas for the species. Therefore, the recent release of NGS transcriptome collections from several tissues and stages from physiological conditions for specific tomato genotypes provides a relevant resource to be appropriately exploited to address key questions on gene expression patterns, such as those related to fruit ripening and development in tomato. The organization of the results from the processed collections in web accessible environments, enriched with tools for their exploration, may represent a precious opportunity for the scientific research in tomato and a reference example for similar efforts. DESCRIPTION: Here we present the architecture and the facilities of NexGenEx-, a web based platform that offers processed NGS transcriptome collections and enables immediate analyses of the results. The platform allows gene expression investigations, profiling and comparisons, and exploits different resources. Specifically, we present here the platform partition NexGenEx-Tom, dedicated to the organization of results from tomato NGS based transcriptomes. CONCLUSION: In the current version, NexGenEx-Tom includes processed and normalized NGS expression data from three collections covering several tissue/stages from different genotypes. Beyond providing a user-friendly interface, the platform was designed with the aim to easily be expanded to include other NGS based transcriptome collections. It can also integrate different genome releases, possibly from different cultivars or genotypes, but even from different species. The platform is proposed as an example effort in tomato, and is described as a profitable approach for the exploitation of these challenging and precious datasets

BIOINFORMATICS STRATEGIES FOR GENOMICS: EXAMPLES AND APPROACHES FOR TOMATO

My PhD is funded by the Solanaceae Pollen thermotolerance – Initial Training Network (SPOT-ITN) in the frame of the European Marie Curie Actions. The consortium aims to investigate fundamental and applied aspects contributing to the protection of pollen at increased environmental temperatures, deciphering the underlying of pollen development and its response to heat stress, starting from analyses on Tomato. Obviously, the findings are supposed to be a guideline, and the procedures to be applicable to other plants in the future. In the light of the SPOT-ITN project objectives, and to provide a comprehensive bioinformatics infrastructure to support extensive genomics analyses in tomato, we collected, processed and integrated different resources; and organized them into dedicated databases with appropriate query user interfaces. This bioinformatics effort required the design of the most adequate software to reconcile the manifold resources from different cell information levels (genomics, transcriptomics, epigenomics). This is fundamental for data integration and analysis. The development of appropriate tools to mine the data from the “omics” approaches employed to trace the pollen development and the heat stress response has also been necessary to the project. In this thesis, the main efforts undertaken and the analyses conducted on the basis of such resources with the strategies and approaches developed are reported in details

pATsi: Paralogs and singleton genes from Arabidopsis thaliana

Arabidopsis thaliana is widely accepted as a model species in plant biology. Its genome, due to its small size and diploidy, was the first to be sequenced among plants, making this species also a reference for plant comparative genomics. Nevertheless, the evolutionary mechanisms that shaped the Arabidopsis genome are still controversial. Indeed, duplications, translocations, inversions, and gene loss events that contributed to the current organization are difficult to be traced. A reliable identification of paralogs and single-copy genes is essential to understand these mechanisms. Therefore, we implemented a dedicated pipeline to identify paralog genes and classify single-copy genes into opportune categories. PATsi, a web-accessible database, was organized to allow the straightforward access to the paralogs organized into networks and to the classification of single-copy genes. This permits to efficiently explore the gene collection of Arabidopsis for evolutionary investigations and comparative genomics

Identification of an SCPL gene controlling anthocyanin acylation in carrot (Daucus carota L.) root

Anthocyanins are natural health promoting pigments that can be produced in large quantities in some purple carrot cultivars. Decoration patterns of anthocyanins, such as acylation, can greatly influence their stability and biological properties and use in the food industry as nutraceuticals and natural colorants. Despite recent advances made toward understanding the genetic control of anthocyanin accumulation in purple carrot, the genetic mechanism controlling acylation of anthocyanin in carrot root have not been studied yet. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify the genetic factor conditioning the accumulation of non-acylated (Cy3XGG) versus acylated (Cy3XFGG and Cy3XSGG) cyanidin derivatives, in three carrot populations. Segregation and mapping analysis pointed to a single gene with dominant effect controlling anthocyanin acylation in the root, located in a 576kb region containing 29 predicted genes. Orthologous and phylogenetic analyses enabled the identification of a cluster of three SCPL-acyltransferases coding genes within this region. Comparative transcriptome analysis indicated that only one of these three genes, DcSCPL1, was always expressed in association with anthocyanin pigmentation in the root and was co-expressed with DcMYB7, a gene known to activate anthocyanin biosynthetic genes in carrot. DcSCPL1 sequence analysis, in root tissue containing a low level of acylated anthocyanins, demonstrated the presence of an insertion causing an abnormal splicing of the 3rd exon during mRNA editing, likely resulting in the production of a non-functional acyltransferase and explaining the reduced acylation phenotype. This study provides strong linkage-mapping and functional evidences for the candidacy of DcSCPL1 as a primary regulator of anthocyanin acylation in carrot storage root.Fil: Curaba, Julien. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados UnidosFil: Bostan, Hamed. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados UnidosFil: Cavagnaro, Pablo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria La Consulta; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Departamento de Producción Agropecuaria. Cátedra de Horticultura y Floricultura; ArgentinaFil: Senalik, Douglas A.. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Mengist, Molla Fentie. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados UnidosFil: Zhao, Yunyang. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados UnidosFil: Simon, Philipp W.. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Iorizzo, Massimo. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados Unido

CMD: A Database to Store the Bonding States of Cysteine Motifs with Secondary Structures

Computational approaches to the disulphide bonding state and its connectivity pattern prediction are based on various descriptors. One descriptor is the amino acid sequence motifs flanking the cysteine residue motifs. Despite the existence of disulphide bonding information in many databases and applications, there is no complete reference and motif query available at the moment. Cysteine motif database (CMD) is the first online resource that stores all cysteine residues, their flanking motifs with their secondary structure, and propensity values assignment derived from the laboratory data. We extracted more than 3 million cysteine motifs from PDB and UniProt data, annotated with secondary structure assignment, propensity value assignment, and frequency of occurrence and coefficiency of their bonding status. Removal of redundancies generated 15875 unique flanking motifs that are always bonded and 41577 unique patterns that are always nonbonded. Queries are based on the protein ID, FASTA sequence, sequence motif, and secondary structure individually or in batch format using the provided APIs that allow remote users to query our database via third party software and/or high throughput screening/querying. The CMD offers extensive information about the bonded, free cysteine residues, and their motifs that allows in-depth characterization of the sequence motif composition

High-Density Linkage Map Construction and QTL Identification in a Diploid Blueberry Mapping Population

Genotyping by sequencing approaches have been widely applied in major crops and are now being used in horticultural crops like berries and fruit trees. As the original and largest producer of cultivated blueberry, the United States maintains the most diverse blueberry germplasm resources comprised of many species of different ploidy levels. We previously constructed an interspecific mapping population of diploid blueberry by crossing the parent F1#10 (Vaccinium darrowii Fla4B × diploid V. corymbosum W85–20) with the parent W85–23 (diploid V. corymbosum). Employing the Capture-Seq technology developed by RAPiD Genomics, with an emphasis on probes designed in predicted gene regions, 117 F1 progeny, the two parents, and two grandparents of this population were sequenced, yielding 131.7 Gbp clean sequenced reads. A total of 160,535 single nucleotide polymorphisms (SNPs), referenced to 4,522 blueberry genome sequence scaffolds, were identified and subjected to a parent-dependent sliding window approach to further genotype the population. Recombination breakpoints were determined and marker bins were deduced to construct a high density linkage map. Twelve blueberry linkage groups (LGs) consisting of 17,486 SNP markers were obtained, spanning a total genetic distance of 1,539.4 cM. Among 18 horticultural traits phenotyped in this population, quantitative trait loci (QTLs) that were significant over at least 2 years were identified for chilling requirement, cold hardiness, and fruit quality traits of color, scar size, and firmness. Interestingly, in 1 year, a QTL associated with timing of early bloom, full bloom, petal fall, and early green fruit was identified in the same region harboring the major QTL for chilling requirement. In summary, we report here the first high density bin map of a diploid blueberry mapping population and the identification of several horticulturally important QTLs

A Cluster of MYB Transcription Factors Regulates Anthocyanin Biosynthesis in Carrot (Daucus carota L.) Root and Petiole

Purple carrots can accumulate large quantities of anthocyanins in their roots and –in some genetic backgrounds- petioles, and therefore they represent an excellent dietary source of antioxidant phytonutrients. In a previous study, using linkage analysis in a carrot F2 mapping population segregating for root and petiole anthocyanin pigmentation, we identified a region in chromosome 3 with co-localized QTL for all anthocyanin pigments of the carrot root, whereas petiole pigmentation segregated as a single dominant gene and mapped to one of these “root pigmentation” regions conditioning anthocyanin biosynthesis. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify candidate genes controlling anthocyanin pigmentation in the carrot root and petiole. Fine mapping was performed in four carrot populations with different genetic backgrounds and patterns of pigmentation. The regions controlling root and petiole pigmentation in chromosome 3 were delimited to 541 and 535 kb, respectively. Genome wide prediction of transcription factor families known to regulate the anthocyanin biosynthetic pathway coupled with orthologous and phylogenetic analyses enabled the identification of a cluster of six MYB transcription factors, denominated DcMYB6 to DcMYB11, associated with the regulation of anthocyanin biosynthesis. No anthocyanin biosynthetic genes were present in this region. Comparative transcriptome analysis indicated that upregulation of DcMYB7 was always associated with anthocyanin pigmentation in both root and petiole tissues, whereas DcMYB11 was only upregulated with pigmentation in petioles. In the petiole, the level of expression of DcMYB11 was higher than DcMYB7. DcMYB6, a gene previously suggested as a key regulator of carrot anthocyanin biosynthesis, was not consistently associated with pigmentation in either tissue. These results strongly suggest that DcMYB7 is a candidate gene for root anthocyanin pigmentation in all the genetic backgrounds included in this study. DcMYB11 is a candidate gene for petiole pigmentation in all the purple carrot sources in this study. Since DcMYB7 is co-expressed with DcMYB11 in purple petioles, the latter gene may act also as a co-regulator of anthocyanin pigmentation in the petioles. This study provides linkage-mapping and functional evidence for the candidacy of these genes for the regulation of carrot anthocyanin biosynthesis

Whole-genome re-sequencing of two Italian tomato landraces reveals sequence variations in genes associated with stress tolerance, fruit quality and long shelf-life traits

Tomato is a high value crop and the primary model for fleshy fruit development and ripening. Breeding priorities include increased fruit quality, shelf life and tolerance to stresses. To contribute towards this goal, we re-sequenced the genomes of Corbarino (COR) and Lucariello (LUC) landraces, which both possess the traits of plant adaptation to water deficit, prolonged fruit shelf-life and good fruit quality. Through the newly developed pipeline Reconstructor, we generated the genome sequences of COR and LUC using datasets of 65.8M and 56.4M of 30–150bp paired-end reads, respectively. New contigs including reads that could not be mapped to the tomato reference genome were assembled, and a total of 43, 054 and 44, 579 gene loci were annotated in COR and LUC. Both genomes showed novel regions with similarity to Solanum pimpinellifolium and Solanum pennellii. In addition to small deletions and insertions, 2, 000 and 1, 700 single nucleotide polymorphisms (SNPs) could exert potentially disruptive effects on 1, 371 and 1, 201 genes in COR and LUC, respectively. A detailed survey of the SNPs occurring in fruit quality, shelf life and stress tolerance related-genes identified several candidates of potential relevance. Variations in ethylene response components may concur in determining peculiar phenotypes of COR and LUC

Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum)

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this cro

Multi-omics data integration provides insights into the post-harvest biology of a long shelf-life tomato landrace

In this study we investigated the transcriptome and epigenome dynamics of the tomato fruit during post-harvest in a landrace belonging to a group of tomatoes (Solanum lycopersicum L.) collectively known as "Piennolo del Vesuvio", all characterized by a long shelf-life. Expression of protein-coding genes and microRNAs as well as DNA methylation patterns and histone modifications were analysed in distinct post-harvest phases. Multi-omics data integration contributed to the elucidation of the molecular mechanisms underlying processes leading to long shelf-life. We unveiled global changes in transcriptome and epigenome. DNA methylation increased and the repressive histone mark H3K27me3 was lost as the fruit progressed from red ripe to 150 days post-harvest. Thousands of genes were differentially expressed, about half of which were potentially epi-regulated as they were engaged in at least one epi-mark change in addition to being microRNA targets in ~5% of cases. Down-regulation of the ripening regulator MADS-RIN and of genes involved in ethylene response and cell wall degradation was consistent with the delayed fruit softening. Large-scale epigenome reprogramming that occurred in the fruit during post-harvest likely contributed to delayed fruit senescence