NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock's eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages-"zero sign of disease" and "evident sign of infection". Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. 'Koroneiki' showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in 'Koroneiki', while 'Nocellara del Belice' was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.

Diversity Assessment and DNA-Based Fingerprinting of Sicilian Hazelnut (Corylus avellana L.) Germplasm.

The characterization of plant genetic resources is a precondition for genetic improvement and germplasm management. The increasing use of molecular markers for DNA-based genotype signature is crucial for variety identification and traceability in the food supply chain. We collected 75 Sicilian hazelnut accessions from private and public field collections, including widely grown varieties from the Nebrodi Mountains in north east Sicily (Italy). The germplasm was fingerprinted through nine standardized microsatellites (SSR) for hazelnut identification to evaluate the genetic diversity of the collected accessions, validating SSR discrimination power. We identified cases of homonymy and synonymy among acquisitions and the unique profiles. The genetic relationships illustrated by hierarchical clustering, structure, and discriminant analyses revealed a clear distinction between local and commercial varieties. The comparative genetic analysis also showed that the Nebrodi genotypes are significantly different from the Northern Italian, Iberian, and Turkish genotypes. These results highlight the need and urgency to preserve Nebrodi germplasm as a useful and valuable source for traits of interest employable for breeding. Our study demonstrates the usefulness of molecular marker analysis to select a reference germplasm collection of Sicilian hazelnut varieties and to implement certified plants' production in the supply chain.

Identifying conserved genes involved in crop tolerance to cold stress.

Low temperature is a limiting factor for crop productivity in tropical and subtropical climates. Cold stress response in plants involves perceiving and relaying the signal through a transcriptional cascade composed of different transduction components, resulting in altered gene activity. We performed a meta-analysis of four previously published datasets of cold-tolerant and cold-sensitive crops to better understand the gene regulatory networks and identify key genes involved in cold stress tolerance conserved across phylogenetically distant species. Re-analysing the raw data with the same bioinformatics pipeline, we identified common cold tolerance-related genes. We found 236 and 242 commonly regulated genes in sensitive and tolerant genotypes, respectively. Gene enrichment analysis showed that protein modifications, hormone metabolism, cell wall, and secondary metabolism are the most conserved pathways involved in cold tolerance. Upregulation of the abiotic stress (heat and drought/salt) related genes [heat shock N -terminal domain-containing protein, 15.7kDa class I-related small heat shock protein-like, DNAJ heat shock N -terminal domain-containing protein, and HYP1 (HYPOTHETICAL PROTEIN 1)] in sensitive genotypes and downregulation of the abiotic stress (heat and drought/salt) related genes (zinc ion binding and pollen Ole e 1 allergen and extensin family protein) in tolerant genotypes was observed across the species. Almost all development-related genes were upregulated in tolerant and downregulated in sensitive genotypes. Moreover, protein-protein network analysis identified highly interacting proteins linked to cold tolerance. Mapping of abiotic stress-related genes on analysed species genomes provided information that could be essential to developing molecular markers for breeding and building up genetic improvement strategies using CRISPR/Cas9 technologies.

Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus.

A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material.

Transcriptomic Analysis of the Pistacia vera (L.) Fruits Enable the Identification of Genes and Hormone-Related Gene Linked to Inflorescence Bud Abscission.

Pistacia vera (L.) is an alternate bearing species. The tree produces axillary inflorescence buds every year. Still, they abscise in "ON" overloaded shoots, causing a limited production in the following "OFF" year, causing a significant and unfavorable production fluctuation. In this work, we carried out de novo discovery and transcriptomic analysis in fruits of "ON" and "OFF" shoots of the cultivar Bianca. We also investigated whether the fruit signaling pathway and hormone biosynthesis directly or indirectly linked to the premature fall of the inflorescence buds causing alternate bearing. We identified 1536 differentially expressed genes (DEGs) in fruits of "ON" vs. "OFF" shoots, which are involved primarily in sugar metabolism, plant hormone pathways and transcription factors. The premature bud abscission linked to the phenomenon is attributable to a lack of nutrients (primarily sugar) and the possible competition between the same branches' sinks (fruits vs. inflorescence buds). Hormone pathways are involved as a response to signals degradation and remobilization of carbon and nutrients due to the strengthening of the developing embryos. Genes of the secondary metabolism and transcription factors are also involved in tailoring the individual branches response to the nutritional stress and sink competition. Crosstalk among sugar and various hormone-related genes, e.g., ethylene, auxin, ABA and cytokinin, were determined. The discovery of putative biomarkers like callose synthase 5, trehalose-6-phosphate synthase, NAD(P)-linked oxidoreductase and MIOX2, Jasmonate, and salicylic acid-related genes can help to design precision farming practices to mitigate the alternate bearing phenomenon to increase farming profitability. The aim of the analysis is to provide insight into the gene expression profiling of the fate of "ON" and "OFF" fruits associated with the alternate bearing in the pistachio.

Algerian Olive Germplasm and Its Relationships with the Central-Western Mediterranean Varieties Contributes to Clarify Cultivated Olive Diversification.

Olive tree with its main final product, olive oil, is an important element of Mediterranean history, considered the emblematic fruit of a civilization. Despite its wide diffusion and economic and cultural importance, its evolutionary and phylogenetic history is still difficult to clarify. As part of the Mediterranean basin, Algeria was indicated as a secondary diversification center. However, genetic characterization studies from Maghreb area, are currently underrepresented. In this context, we characterized 119 endemic Algerian accessions by using 12 microsatellite markers with the main goal to evaluate the genetic diversity and population structure. In order to provide new insights about the history of olive diversification events in the Central-Western Mediterranean basin, we included and analyzed a sample of 103 Italian accessions from Sicily and, a set of molecular profiles of cultivars from the Central-Western Mediterranean area. The phylogenetic investigation let us to evaluate genetic relationships among Central-Mediterranean basin olive germplasm, highlight new synonymy cases to support the importance of vegetative propagation in the cultivated olive diffusion and consolidate the hypothesis of more recent admixture events occurrence. This work provided new information about Algerian germplasm biodiversity and contributed to clarify olive diversification process.

Towards a Joint International Database: Alignment of SSR Marker Data for European Collections of Cherry Germplasm.

The objective of our study was the alignment of microsatellite or simple sequence repeat (SSR) marker data across germplasm collections of cherry within Europe. Through the European Cooperative program for Plant Genetic Resources ECPGR, a number of European germplasm collections had previously been analysed using standard sets of SSR loci. However, until now these datasets remained unaligned. We used a combination of standard reference genotypes and ad-hoc selections to compile a central dataset representing as many alleles as possible from national datasets produced in France, Great Britain, Germany, Italy, Sweden and Switzerland. Through the comparison of alleles called in data from replicated samples we were able to create a series of alignment factors, supported across 448 different allele calls, that allowed us to align a dataset of 2241 SSR profiles from six countries. The proportion of allele comparisons that were either in agreement with the alignment factor or confounded by null alleles ranged from 67% to 100% and this was further improved by the inclusion of a series of allele-specific adjustments. The aligned dataset allowed us to identify groups of previously unknown matching accessions and to identify and resolve a number of errors in the prior datasets. The combined and aligned dataset represents a significant step forward in the co-ordinated management of field collections of cherry in Europe.

Gaining Insight into Exclusive and Common Transcriptomic Features Linked to Drought and Salinity Responses across Fruit Tree Crops.

The present study aimed at identifying and mapping key genes expressed in root tissues involved in drought and salinity tolerance/resistance conserved among different fruit tree species. Twenty-six RNA-Seq samples were analyzed from six published studies in five plant species (Olea europaea, Vitis riparia Michx, Prunus mahaleb, Prunus persica, Phoenix dactylifera). This meta-analysis used a bioinformatic pipeline identifying 750 genes that were commonly modulated in three salinity studies and 683 genes that were commonly regulated among three drought studies, implying their conserved role in resistance/tolerance/response to these environmental stresses. A comparison was done on the genes that were in common among both salinity and drought resulted in 82 genes, of which 39 were commonly regulated with the same trend of expression (23 were upregulated and 16 were downregulated). Gene set enrichment and pathway analysis pointed out that pathways encoding regulation of defense response, drug transmembrane transport, and metal ion binding are general key molecular responses to these two abiotic stress responses. Furthermore, hormonal molecular crosstalk plays an essential role in the fine-tuning of plant responses to drought and salinity. Drought and salinity induced a different molecular "hormonal fingerprint". Dehydration stress specifically enhanced multiple genes responsive to abscisic acid, gibberellin, brassinosteroids, and the ethylene-activated signaling pathway. Salt stress mostly repressed genes encoding for key enzymes in signaling proteins in auxin-, gibberellin-(gibberellin 2 oxidase 8), and abscisic acid-related pathways (aldehyde oxidase 4, abscisic acid-responsive element-binding protein 3). Abiotic stress-related genes were mapped into the chromosome to identify molecular markers usable for the improvement of these complex quantitative traits. This meta-analysis identified genes that serve as potential targets to develop cultivars with enhanced drought and salinity resistance and/or tolerance across different fruit tree crops in a biotechnological sustainable way.

Transcriptome Analysis of Pistacia vera Inflorescence Buds in Bearing and Non-Bearing Shoots Reveals the Molecular Mechanism Causing Premature Flower Bud Abscission.

The alteration of heavy ("ON/bearing") and light ("OFF/non-bearing") yield in pistachio (Pistacia vera L.) has been reported to result from the abscission of inflorescence buds on high yielding trees during the summer, but the regulatory mechanisms involved in this bud abscission remain unclear. The analysis provides insights into the transcript changes between inflorescence buds on bearing and non-bearing shoots, that we indicated as "ON" and "OFF", and shed light on the molecular mechanisms causing premature inflorescence bud abscission in the pistachio cultivar "Bianca" which can be related to the alternate bearing behavior. In this study, a transcriptome analysis was performed in inflorescence buds of "ON" and "OFF" shoots. A total of 14,330 differentially expressed genes (DEGs), most of which are involved in sugar metabolism, plant hormone pathways, secondary metabolism and oxidative stress pathway, were identified. Our results shed light on the molecular mechanisms underlying inflorescence bud abscission in pistachio and we proposed a hypothetical model behind the molecular mechanism causing this abscission in "ON" shoots. Results highlighted how changes in genes expressed in nutrient pathways (carbohydrates and mineral elements) in pistachio "ON" vs. "OFF" inflorescence buds triggers a cascade of events involving trehalose-6-phosphate and target of rapamycin (TOR) signaling, SnRK1 complex, hormones, polyamines and ROS which end, through programmed cell death and autophagy phenomena, with the abscission of inflorescence buds. This is the first study reporting gene expression profiling of the fate of "ON" and "OFF" inflorescence buds associated with the alternate bearing in the pistachio.

A new self-compatibility haplotype in the sweet cherry 'Kronio', S5', attributable to a pollen-part mutation in the SFB gene.

'Kronio' is a Sicilian cultivar of sweet cherry (Prunus avium), nominally with the incompatibility genotype S(5)S(6), that is reported to be naturally self-compatible. In this work the cause of its self-compatibility was investigated. Test selfing confirmed self-compatibility and provided embryos for analysis; PCR with consensus primers designed to amplify S-RNase and SFB alleles showed that the embryos were of two types, S(5)S(5) and S(5)S(6), indicating that S(6) pollen failed, but S(5) succeeded, perhaps because of a mutation in the pollen or stylar component. Stylar RNase analysis indicated active S-RNases for both S(5) and S(6). The S-RNase alleles were cloned and sequenced; and sequences encode functional proteins. Cloning and sequencing of SFB alleles showed that S(6) was normal but S(5) had a premature stop codon upstream of the variable region HVa resulting in a truncated protein. Therefore, the self-compatibility can be attributed to a pollen-part mutation of S(5), designated S(5)', the first reported case of breakdown of self-incompatibility in diploid sweet cherry caused by a natural mutation at the S-locus. The second intron of the S-RNase associated with S(5)' contained a microsatellite smaller than that associated with S(5); primers designed to amplify across this microsatellite effectively distinguished S(5) from S(5)'. Analysis of some other Sicilian cherries with these primers indicated that S(5)' is also present in the Sicilian cultivar 'Maiolina a Rappu', and this proved to be self-compatible.