A Survey of MicroRNA Length Variants Contributing to miRNome Complexity in Peach (Prunus Persica L.)

MicroRNAs (miRNAs) are short non-coding RNA molecules produced from hairpin structures and involved in gene expression regulation with major roles in plant development and stress response. Although each annotated miRNA in miRBase (www.mirbase.org) is a single defined sequence with no further details on possible variable sequence length, isomiRs – namely the population of variants of miRNAs coming from the same precursors – have been identified in several species and could represent a way of broadening the regulatory network of the cell. Next-gen-based sequencing makes it possible to comprehensively and accurately assess the entire miRNA repertoire including isomiRs. The aim of this work was to survey the complexity of the peach miRNome by carrying out Illumina high-throughput sequencing of miRNAs in three replicates of five biological samples arising from a set of different peach organs and/or phenological stages. Three hundred-ninety-two isomiRs (miRNA and miRNA*-related) corresponding to 26 putative miRNA coding loci, have been highlighted by mirDeep-P and analyzed. The presence of the same isomiRs in different biological replicates of a sample and in different tissues demonstrates that the generation of most of the detected isomiRs is not random. The degree of mature sequence heterogeneity is very different for each individual locus. Results obtained in the present work can thus contribute to a deeper view of the miRNome complexity and to better explore the mechanism of action of these tiny regulators

Characterization of the Resistance to Powdery Mildew and Leaf Rust Carried by the Bread Wheat Cultivar Victo

Leaf rust and powdery mildew are two important foliar diseases in wheat. A recombinant inbred line (RIL) population, obtained by crossing two bread wheat cultivars ('Victo' and 'Spada'), was evaluated for resistance to the two pathogens at seedling stage. Upon developing a genetic map of 8726 SNP loci, linkage analysis identified three resistance Quantitative Trait Loci (QTLs), with 'Victo' contributing the resistant alleles to all loci. One major QTL (QPm.gb-7A) was detected in response to Blumeria graminis on chromosome 7A, which explained 90% of phenotypic variation (PV). The co-positional relationship with known powdery mildew (Pm) resistance loci suggested that a new source of resistance was identified in T. aestivum. Two QTLs were detected in response to Puccinia triticina: a major gene on chromosome 5D (QLr.gb-5D), explaining a total PV of about 59%, and a minor QTL on chromosome 2B (QLr.gb-2B). A positional relationship was observed between the QLr.gb-5D with the known Lr1 gene, but polymorphisms were found between the cloned Lr1 and the corresponding 'Victo' allele, suggesting that QLr.gb-5D could represent a new functional Lr1 allele. Lastly, upon anchoring the QTL on the T. aestivum reference genome, candidate genes were hypothesized on the basis of gene annotation and in silico gene expression analysis

Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein

<p>Abstract</p> <p>Background</p> <p>Reactive oxygen species (ROS) are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli.</p> <p>Results</p> <p>To investigate this role further, we exposed wild type <it>Arabidopsis thaliana </it>plants and the double mutant <it>npq1lut2 </it>to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (¹O₂) accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by ¹O₂which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in <it>npq1lut2 </it>is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the <it>flu </it>mutant was the only biological system adopted to define the regulation of gene expression by ¹O₂. In this work, we propose the use of mutants accumulating ¹O₂by mechanisms different from those activated in <it>flu </it>to better identify ROS signalling.</p> <p>Conclusions</p> <p>We propose that the lack of zeaxanthin and lutein leads to ¹O₂accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to ¹O₂accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are discussed.</p

Comparative transcriptome analysis of the interaction between Actinidia chinensis var. chinensis and Pseudomonas syringae pv. Actinidiae in absence and presence of acibenzolar-S-methyl

Background: Since 2007, bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has become a pandemic disease leading to important economic losses in every country where kiwifruit is widely cultivated. Options for controlling this disease are very limited and rely primarily on the use of bactericidal compounds, such as copper, and resistance inducers. Among the latter, the most widely studied is acibenzolar-S-methyl. To elucidate the early molecular reaction of kiwifruit plants (Actinidia chinensis var. chinensis) to Psa infection and acibenzolar-S-methyl treatment, a RNA seq analysis was performed at different phases of the infection process, from the epiphytic phase to the endophytic invasion on acibenzolar-S-methyl treated and on non-treated plants. The infection process was monitored in vivo by confocal laser scanning microscopy. Results: De novo assembly of kiwifruit transcriptome revealed a total of 39,607 transcripts, of which 3360 were differentially expressed during the infection process, primarily 3 h post inoculation. The study revealed the coordinated changes of important gene functional categories such as signaling, hormonal balance and transcriptional regulation. Among the transcription factor families, AP2/ERF, MYB, Myc, bHLH, GATA, NAC, WRKY and GRAS were found differentially expressed in response to Psa infection and acibenzolar-S-methyl treatment. Finally, in plants treated with acibenzolar-S-methyl, a number of gene functions related to plant resistance, such as PR proteins, were modulated, suggesting the set-up of a more effective defense response against the pathogen. Weighted-gene coexpression network analysis confirmed these results. Conclusions: Our work provides an in-depth description of the plant molecular reactions to Psa, it highlights the metabolic pathway related to acibenzolar-S-methyl-induced resistance and it contributes to the development of effective control strategies in open field

THE STUDY OF THREE GRAPEVINE CLONES TO UNCOVER THE GENETIC TRAITS RESPONSIBLE FOR THE LOW SUSCEPTIBILITY TO FLAVESCENCE DORéE

Flavescence dorée (FD) is one of the most destructive grapevine yellows diseases and a quarantine pest in the European Community. It is caused by phytoplasmas, which are transmitted in vineyard by the leafhopper Scaphoideus titanus. Inter and intraspecific differences in susceptibility to FD have already been observed among grapevine varieties and clones of the same variety. Grapevine varieties and clones completely resistant to FD have not been uncovered yet, however these differences suggest the presence of genetic traits in grapevine related to high or low susceptibility to FD. Cultivated grapevines are clonally propagated and the genome of each cultivar is preserved, except for the accumulation of mutations over time that can generate distinguishable clones with several notable phenotypes. The differences between the genome of clones of the same variety are less than those between different varieties; thus, in order to highlight the genetic features responsible for the different phenotypes, the study of the genomes of clones with different susceptibility to FD can be a valid technique to achieve the goal. The aim of this work is to find out the genetic traits responsible for the different susceptibility to FD among three Chardonnay clones, analyzing the diversity in their genomes and transcriptomic profiles. The clones were sequenced by accurate whole genome techniques, Hi-Fi reads sequencing on PacBio platform coupled with Illumina, and then the genomes were de novo assembled. Moreover, two of them were experimentally infected in field by means of the insect vector and the transcriptomic profiles in the early stage of FD infection were analyzed. In particular, the clones were compared in absence of the disease and the vector, in presence of the healthy vector and with the FD infective vector. The results obtained from the preliminary genome comparison showed higher similarity between the three clones than compared with the reference genome from variety Pinot noir, while the analysis to identify the genomic differences among clones are still in progress. The transcriptomic profiles showed interesting differences in some pathways expressed in presence of healthy vectors, while the clones shared a similar expression profile before being in contact with the vectors as well as after the challenge with the FD infective vectors. These findings might suggest the involvement of antibiosis mechanisms in the partial resistance of the specific Chardonnay clone to FD. The results obtained, and those that will be achieved in future, are useful for new breeding programs and clonal selection. Grapevine plants more resistant to FD will permit to decrease the insecticides used to control the disease in vineyards. Moreover, the knowledge of the molecular and metabolic mechanisms involved in scarcely susceptible clones could pave the way for the development, for example, of biostimulants capable to induce such defenses even in the most susceptible plants

Transcriptomic and proteomic analyses of a pale-green durum wheat mutant shows variations in photosystem components and metabolic deficiencies under drought stress

Background: Leaf pigment content is an important trait involved in environmental interactions. In order to determine its impact on drought tolerance in wheat, we characterized a pale-green durum wheat mutant (Triticum turgidum L. var. durum) under contrasting water availability conditions. Results: The pale-green mutant was investigated by comparing pigment content and gene/protein expression profiles to wild-type plants at anthesis. Under well-watered (control) conditions the mutant had lower levels of chlorophylls and carotenoids, but higher levels of xanthophyll de-epoxidation compared to wild-type. Transcriptomic analysis under control conditions showed that defense genes (encoding e.g. pathogenesis-related proteins, peroxidases and chitinases) were upregulated in the mutant, suggesting the presence of mild oxidative stress that was compensated without altering the net rate of photosynthesis. Transcriptomic analysis under terminal water stress conditions, revealed the modulation of antioxidant enzymes, photosystem components, and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle, indicating that the mutant was exposed to greater oxidative stress than the wild-type plants, but had a limited capacity to respond. We also compared the two genotypes under irrigated and rain-fed field conditions over three years, finding that the greater oxidative stress and corresponding molecular changes in the pale-green mutant were associated to a yield reduction. Conclusions: This study provides insight on the effect of pigment content in the molecular response to drought. Identified genes differentially expressed under terminal water stress may be valuable for further studies addressing drought resistance in wheat.A. Peremarti is supported by the AGROTECNIO Foundation. The support of the Efficient Use of Water Program of IRTA is acknowledged. This study was partially funded by MICINN (Spain) under projects AGL2009- 11187, AGL2012-37217 and RTA2009-00085-00, and by MIUR (Italy) under the project ISCOCEM. AP, DV, CR authors are part of the Centre CONSOLIDER INGENIO 2010 on Agrigenomics funded by the Spanish Ministry of Education and Science

Genetic markers associated to arbuscular mycorrhizal colonization in durum wheat

In this work we investigated the variability and the genetic basis of susceptibility to arbuscular mycorrhizal (AM) colonization of wheat roots. The mycorrhizal status of wild, domesticated and cultivated tetraploid wheat accessions, inoculated with the AM species Funneliformis mosseae, was evaluated. In addition, to detect genetic markers in linkage with chromosome regions involved in AM root colonization, a genome wide association analysis was carried out on 108 durum wheat varieties and two AM fungal species (F. mosseae and Rhizoglomus irregulare). Our findings showed that a century of breeding on durum wheat and the introgression of Reduced height (Rht) genes associated with increased grain yields did not select against AM symbiosis in durum wheat. Seven putative Quantitative Trait Loci (QTLs) linked with durum wheat mycorrhizal susceptibility in both experiments, located on chromosomes 1A, 2B, 5A, 6A, 7A and 7B, were detected. The individual QTL effects (r2) ranged from 7 to 16%, suggesting a genetic basis for this trait. Marker functional analysis identified predicted proteins with potential roles in host-parasite interactions, degradation of cellular proteins, homeostasis regulation, plant growth and disease/defence. The results of this work emphasize the potential for further enhancement of root colonization exploiting the genetic variability present in wheat

Transcriptional profiling in response to terminal drought stress reveals differential responses along the wheat genome

Background: Water stress during grain filling has a marked effect on grain yield, leading to a reduced endosperm cell number and thus sink capacity to accumulate dry matter. The bread wheat cultivar Chinese Spring (CS), a Chinese Spring terminal deletion line (CS_5AL-10) and the durum wheat cultivar Creso were subjected to transcriptional profiling after exposure to mild and severe drought stress at the grain filling stage to find evidences of differential stress responses associated to different wheat genome regions. Results: The transcriptome analysis of Creso, CS and its deletion line revealed 8,552 non redundant probe sets with different expression levels, mainly due to the comparisons between the two species. The drought treatments modified the expression of 3,056 probe sets. Besides a set of genes showing a similar drought response in Creso and CS, cluster analysis revealed several drought response features that can be associated to the different genomic structure of Creso, CS and CS_5AL-10. Some drought-related genes were expressed at lower level (or not expressed) in Creso (which lacks the D genome) or in the CS_5AL- 10 deletion line compared to CS. The chromosome location of a set of these genes was confirmed by PCR-based mapping on the D genome (or the 5AL-10 region). Many clusters were characterized by different level of expression in Creso, CS and CS_AL-10, suggesting that the different genome organization of the three genotypes may affect plant adaptation to stress. Clusters with similar expression trend were grouped and functional classified to mine the biological mean of their activation or repression. Genes involved in ABA, proline, glycine-betaine and sorbitol pathways were found up-regulated by drought stress. Furthermore, the enhanced expression of a set of transposons and retrotransposons was detected in CS_5AL-10. Conclusion: Bread and durum wheat genotypes were characterized by a different physiological reaction to water stress and by a substantially different molecular response. The genome organization accounted for differences in the expression level of hundreds of genes located on the D genome or controlled by regulators located on the D genome. When a genomic stress (deletion of a chromosomal region) was combined with low water availability, a molecular response based on the activation of transposons and retrotransposons was observed

Segmental duplications are hot spots of copy number variants affecting barley gene content

Copy number variants (CNVs) are pervasive in several animal and plant genomes and contribute to shaping genetic diversity. In barley, there is evidence that changes in gene copy number underlie important agronomic traits. The recently released reference sequence of barley represents a valuable genomic resource for unveiling the incidence of CNVs that affect gene content and identifying sequence features associated with CNV formation. Using exome sequencing and read count data, we detected 16,605 deletions and duplications that affect barley gene content by surveying a diverse panel of 172 cultivars, 171 landraces, 22 wild relatives and other 32 uncategorized domesticated accessions. The quest for segmental duplications (SDs) in the reference sequence revealed many low-copy repeats, most of which overlap predicted coding sequences. Statistical analyses revealed that the incidence of CNVs increases significantly in SD-rich regions, indicating that these sequence elements act as hot spots for the formation of CNVs. This study delivers a comprehensive genome-wide study of CNVs affecting barley gene content and implicates SDs in the molecular mechanisms that lead to the formation of this class of CNVs