Marker-Assisted Introgression of the Salinity Tolerance Locus Saltol in Temperate Japonica Rice.

BACKGROUND: Rice is one of the most salt sensitive crops at seedling, early vegetative and reproductive stages. Varieties with salinity tolerance at seedling stage promote an efficient growth at early stages in salt affected soils, leading to healthy vegetative growth that protects crop yield. Saltol major QTL confers capacity to young rice plants growing under salt condition by maintaining a low Na+/K+ molar ratio in the shoots. RESULTS: Marker-assisted backcross (MABC) procedure was adopted to transfer Saltol locus conferring salt tolerance at seedling stage from donor indica IR64-Saltol to two temperate japonica varieties, Vialone Nano and Onice. Forward and background selections were accomplished using polymorphic KASP markers and a final evaluation of genetic background recovery of the selected lines was conducted using 15,580 SNP markers obtained from Genotyping by Sequencing. Three MABC generations followed by two selfing, allowed the identification of introgression lines achieving a recovery of the recurrent parent (RP) genome up to 100% (based on KASP markers) or 98.97% (based on GBS). Lines with highest RP genome recovery (RPGR) were evaluated for agronomical-phenological traits in field under non-salinized conditions. VN1, VN4, O1 lines were selected considering the agronomic evaluations and the RPGR% results as the most interesting for commercial exploitation. A physiological characterization was conducted by evaluating salt tolerance under hydroponic conditions. The selected lines showed lower standard evaluation system (SES) scores: 62% of VN4, and 57% of O1 plants reaching SES 3 or SES 5 respectively, while only 40% of Vialone Nano and 25% of Onice plants recorded scores from 3 to 5, respectively. VN1, VN4 and O1 showed a reduced electrolyte leakage values, and limited negative effects on relative water content and shoot/root fresh weight ratio. CONCLUSION: The Saltol locus was successfully transferred to two elite varieties by MABC in a time frame of three years. The application of background selection until BC3F3 allowed the selection of lines with a RPGR up to 98.97%. Physiological evaluations for the selected lines indicate an improved salinity tolerance at seedling stage. The results supported the effectiveness of the Saltol locus in temperate japonica and of the MABC procedure for recovering of the RP favorable traits.

Pathways of flower infection and pollen-mediated dispersion of Pseudomonas syringae pv. actinidiae, the causal agent of kiwifruit bacterial canker.

Flowers can provide a protected and nutrient-rich environment to the epiphytic microflora, thus representing a sensible entry point for pathogens such as Pseudomonas syringae pv. actinidiae (Psa). This bacterium can colonize both male and female Actinidia flowers, causing flower browning and fall, and systemic invasion of the host plant, eventually leading to its death. However, the process of flower colonization and penetration into the host tissues has not yet been fully elucidated. In addition, the presence of Psa in the pollen from infected flowers, and the role of pollination in the spread of Psa requires confirmation. The present study employed a Psa strain constitutively expressing the fluorescent GFPuv protein, to visualize in vivo flower colonization. Microscopy observations were performed by means of confocal laser scanning and wide-field fluorescent microscopy, and were coupled with the study of Psa population dynamics by quantitative PCR (q-PCR). The pathogen was shown to colonize stigmata, move along the stylar furrow, and penetrate the receptacles via the style or nectarhodes. Once the receptacle was invaded, the pathogen migrated along the flower pedicel and became systemic. Psa was also able to colonize the anthers epiphytically and endophytically. Infected male flowers produced contaminated pollen, which could transmit Psa to healthy plants. Finally, pollinators (Apis mellifera and Bombus terrestris) were studied in natural conditions, showing that, although they can be contaminated with Psa, the pathogen's transmission via pollinators is contrasted by its short survival in the hive.

Plant Microbiome and Its Link to Plant Health: Host Species, Organs and Pseudomonas syringae pv. actinidiae Infection Shaping Bacterial Phyllosphere Communities of Kiwifruit Plants.

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of the bacterial canker, the most devastating disease of kiwifruit vines. Before entering the host tissues, this pathogen has an epiphytic growth phase on kiwifruit flowers and leaves, thus the ecological interactions within epiphytic bacterial community may greatly influence the onset of the infection process. The bacterial community associated to the two most important cultivated kiwifruit species, Actinidia chinensis and Actinidia deliciosa, was described both on flowers and leaves using Illumina massive parallel sequencing of the V3 and V4 variable regions of the 16S rRNA gene. In addition, the effect of plant infection by Psa on the epiphytic bacterial community structure and biodiversity was investigated. Psa infection affected the phyllosphere microbiome structures in both species, however, its impact was more pronounced on A. deliciosa leaves, where a drastic drop in microbial biodiversity was observed. Furthermore, we also showed that Psa was always present in syndemic association with Pseudomonas syringae pv. syringae and Pseudomonas viridiflava, two other kiwifruit pathogens, suggesting the establishment of a pathogenic consortium leading to a higher pathogenesis capacity. Finally, the analyses of the dynamics of bacterial populations provided useful information for the screening and selection of potential biocontrol agents against Psa.

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.

Genome-Wide Association Study for Traits Related to Plant and Grain Morphology, and Root Architecture in Temperate Rice Accessions.

BACKGROUND: In this study we carried out a genome-wide association analysis for plant and grain morphology and root architecture in a unique panel of temperate rice accessions adapted to European pedo-climatic conditions. This is the first study to assess the association of selected phenotypic traits to specific genomic regions in the narrow genetic pool of temperate japonica. A set of 391 rice accessions were GBS-genotyped yielding-after data editing-57000 polymorphic and informative SNPS, among which 54% were in genic regions. RESULTS: In total, 42 significant genotype-phenotype associations were detected: 21 for plant morphology traits, 11 for grain quality traits, 10 for root architecture traits. The FDR of detected associations ranged from 3 · 10-7 to 0.92 (median: 0.25). In most cases, the significant detected associations co-localised with QTLs and candidate genes controlling the phenotypic variation of single or multiple traits. The most significant associations were those for flag leaf width on chromosome 4 (FDR = 3 · 10-7) and for plant height on chromosome 6 (FDR = 0.011). CONCLUSIONS: We demonstrate the effectiveness and resolution of the developed platform for high-throughput phenotyping, genotyping and GWAS in detecting major QTLs for relevant traits in rice. We identified strong associations that may be used for selection in temperate irrigated rice breeding: e.g. associations for flag leaf width, plant height, root volume and length, grain length, grain width and their ratio. Our findings pave the way to successfully exploit the narrow genetic pool of European temperate rice and to pinpoint the most relevant genetic components contributing to the adaptability and high yield of this germplasm. The generated data could be of direct use in genomic-assisted breeding strategies.

Haplotype variability and identification of new functional alleles at the Rdg2a leaf stripe resistance gene locus.

The barley Rdg2a locus confers resistance to the leaf stripe pathogen Pyrenophora graminea and, in the barley genotype Thibaut, it is composed of a gene family with three highly similar paralogs. Only one member of the gene family (called as Rdg2a) encoding for a CC-NB-LRR protein is able to confer resistance to the leaf stripe isolate Dg2. To study the genome evolution and diversity at the Rdg2a locus, sequences spanning the Rdg2a gene were compared in two barley cultivars, Thibaut and Morex, respectively, resistant and susceptible to leaf stripe. An overall high level of sequence conservation interrupted by several rearrangements that included three main deletions was observed in the Morex contig. The main deletion of 13,692 bp was most likely derived from unequal crossing over between Rdg2a paralogs leading to the generation of a chimeric Morex rdg2a gene which was not associated to detectable level of resistance toward leaf stripe. PCR-based analyses of genic and intergenic regions at the Rdg2a locus in 29 H. vulgare lines and one H. vulgare ssp. spontaneum accession indicated large haplotype variability in the cultivated barley gene pool suggesting rapid and recent divergence at this locus. Barley genotypes showing the same haplotype as Thibaut at the Rdg2a locus were selected for a Rdg2a allele mining through allele re-sequencing and two lines with polymorphic nucleotides leading to amino acid changes in the CC-NB and LRR encoding domains, respectively, were identified. Analysis of nucleotide diversity of the Rdg2a alleles revealed that the polymorphic sites were subjected to positive selection. Moreover, strong positively selected sites were located in the LRR encoding domain suggesting that both positive selection and divergence at homologous loci are possibly representing the molecular mechanism for the generation of high diversity at the Rdg2a locus in the barley gene pool.

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.

Identification and mapping of the leaf stripe resistance gene Rdg1a in Hordeum spontaneum.

Leaf stripe of barley, caused by Pyrenophora graminea, is an important seed-borne disease in organically grown as well as in conventionally grown Nordic and Mediterranean barley districts. Two barley segregating populations represented by 103 recombinant inbred lines (RILs) of the cross L94 (susceptible) x Vada (resistant) and 194 RILs of the cross Arta (susceptible) x Hordeum spontaneum 41-1 (resistant) were analysed with two highly virulent leaf stripe isolates, Dg2 and Dg5, to identify loci for P. graminea resistance. A major gene with its positive allele contributed by Vada and H. spontaneum 41-1 was detected in both populations and for both pathogen isolates on chromosome 2HL explaining 44.1 and 91.8% R (2), respectively for Dg2 and Dg5 in L94 x Vada and 97.8 and 96.1% R (2), respectively for Dg2 and Dg5 in Arta x H. spontaneum 41-1. Common markers in the gene region of the two populations enabled map comparison and highlighted an overlapping for the region of the resistance locus. Since the map position of the resistance locus identified in this report is the same as that for the leaf stripe resistance gene Rdg1a, mapped earlier in Alf and derived from the 'botanical' barley line H. laevigatum, we propose that leaf stripe resistance in Vada and H. spontaneum 41-1 is governed by the same gene, namely by Rdg1a, and that Rdg1a resistance could be traced back to H. spontaneum, the progenitor of cultivated barley. PCR-based molecular markers that can be used for marker-assisted selection (MAS) of Rdg1a were identified. An Rdg1a syntenic interval with the rice chromosome arm 4L was identified on the basis of rice orthologs of EST-based barley markers. Analysis of the rice genes annotated into the syntenic interval did not reveal sequences strictly belonging to the major class (nucleotide-binding site plus leucine-rich repeat) of the resistance genes. Nonetheless, four genes coding for domains that are present in the major disease-resistance genes, namely receptor-like protein kinase and ATP/GTP-binding proteins, were identified together with a homolog of the barley powdery mildew resistance gene mlo. Three (out of five) homologs of these genes were mapped in the Rdg1a region in barley and the mlo homolog map position was tightly associated with the LOD score peak in both populations.