Functional conservation of the grapevine candidate gene INNER NO OUTER for ovule development and seed formation.

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed.

Re.Ger.O.P.: An Integrated Project for the Recovery of Ancient and Rare Olive Germplasm.

The olive tree is one of the most important economic, cultural, and environmental resources for Italy, in particular for the Apulian region, where it shows a wide diversity. The increasing attention to the continuous loss of plant genetic diversity due to social, economic and climatic changes, has favored a renewed interest in strategies aimed at the recovery and conservation of these genetic resources. In the frame of a project for the valorization of the olive Apulian biodiversity (Re.Ger.O.P. project), 177 minor genotypes were recovered in different territories of the region. They were submitted to morphological, molecular, technological and phytosanitary status analysis in comparison with reference cultivars, then they were propagated and transferred in an ex situ field. All the available information was stored in an internal regional database including photographic documentation and geographic position. The work allowed obtaining information about the genetic diversity of Apulian germplasm, to clarify cases of homonymy and synonymy, to check the sanitary status, and to identify candidate genotypes useful both to set up breeding programs and to enrich the panel of olive cultivars available to farmers for commercial exploitation.

GBS-derived SNP catalogue unveiled wide genetic variability and geographical relationships of Italian olive cultivars.

Information on the distribution of genetic variation is essential to preserve olive germplasm from erosion and to recover alleles lost through selective breeding. In addition, knowledge on population structure and genotype-phenotype associations is crucial to support modern olive breeding programs that must respond to new environmental conditions imposed by climate change and novel biotic/abiotic stressors. To further our understanding of genetic variation in the olive, we performed genotype-by-sequencing on a panel of 94 Italian olive cultivars. A reference-based and a reference-independent SNP calling pipeline generated 22,088 and 8,088 high-quality SNPs, respectively. Both datasets were used to model population structure via parametric and non parametric clustering. Although the two pipelines yielded a 3-fold difference in the number of SNPs, both described wide genetic variability among our study panel and allowed individuals to be grouped based on fruit weight and the geographical area of cultivation. Multidimensional scaling analysis on identity-by-state allele-sharing values as well as inference of population mixtures from genome-wide allele frequency data corroborated the clustering pattern we observed. These findings allowed us to formulate hypotheses about geographical relationships of Italian olive cultivars and to confirm known and uncover novel cases of synonymy.

Genetic flow among olive populations within the Mediterranean basin.

BACKGROUND: The olive tree is a typical crop of the Mediterranean basin where it shows a wide diversity, accounting for more than 2,600 cultivars. The ability to discriminate olive cultivars and determine their genetic variability is pivotal for an optimal exploitation of olive genetic resources. METHODS: We investigated the genetic diversity within 128 olive accessions belonging to four countries in the Mediterranean Basin (Italy, Algeria, Syria, and Malta), with the purpose of better understanding the origin and spread of the olive genotypes across Mediterranean Basin countries. Eleven highly polymorphic simple sequence repeat (SSR) markers were used and proved to be very informative, producing a total of 179 alleles. RESULTS: Cluster analysis distinguished three main groups according to their geographical origin, with the current sample of Maltese accessions included in the Italian group. Phylogenetic analysis further differentiated Italian and Maltese olive accessions, clarifying the intermediate position of Maltese accessions along the x/y-axes of principal coordinate analysis (PCoA). Model-based and neighbor clustering, PCoA, and migration analysis suggested the existence of two different gene pools (Algerian and Syrian) and that the genetic exchange occurred between the Syrian, Italian and Maltese populations. DISCUSSION: The close relationship between Syrian and Italian and Maltese olives was consistent with the historical domestication and migration of olive tree from the North Levant to eastern Mediterranean basin. This study lays the foundations for a better understanding of olive genetic diversity in the Mediterranean basin and represents a step toward an optimal conservation and exploitation of olive genetic resources.

Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques.

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

A new high-resolution melting assay for genotyping Alternaria species causing citrus brown spot.

BACKGROUND: Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. To set up effective control strategy, the accurate detection and identification of the species responsible for the diseases is crucial. However, characterization based on morphology and/or multilocus genetic approaches is time consuming, requires great expertise and sometimes is not conclusive. Therefore, the set-up of a rapid and efficient DNA-based assay might be of paramount importance. High-resolution melting (HRM) analysis represents an interesting tool for the uncovering of nucleotide variations as small as one base difference and, as such, relevant to species characterization. RESULTS: In the present investigation, an HRM assay based on the Alternaria barcoding region OPA1-3 was set up. Specimen strains of the main citrus-associated Alternaria species and morphotypes generated distinct and normalized profiles, allowing their differentiation when HRM-tested. Moreover, when the assay was used to screen an Alternaria collection from citrus fruit and leaves, it distributed the 180 isolates in three independent clusters, readily and consistently resolved. Isolates were identified as belonging to the species Alternaria alternata and the species complex A. arborescens. Within A. alternata, the morphotypes alternata (77% of the collection) and limoniasperae (17% of the collection) were present. CONCLUSIONS: Although further validation experiments will be performed to optimize the assay for a diagnostic use, this HRM approach might represent a rapid, sensitive and specific method for the detection and identification of Alternaria spp. responsible for citrus brown spot disease. © 2018 Society of Chemical Industry.

Screening Auxin Response, In Vitro Culture Aptitude and Susceptibility to Agrobacterium-Mediated Transformation of Italian Commercial Durum Wheat Varieties.

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like durum wheat requires the identification and optimization of factors affecting T-DNA delivery and plant regeneration. The purpose of this research was to compare the behavior of diverse durum wheat genotypes during in vitro culture and Agrobacterium tumefaciens-mediated transformation, using immature embryos as explants. Apart from plant genotype, two of the main influencing factors for a successful genetic transformation have been examined here, i.e., auxin source (Dicamba and 2,4-D) and duration of the pre-culture period (one, seven and 21 days). The addition of Dicamba to the media in combination with seven days pre-cultivation resulted in a general enhancement of T-DNA delivery for most of the analyzed cultivars, as revealed by ß-glucuronidase (GUS) histochemical assay. Although all genotypes were able to produce calli, significant differences were detected in regeneration and transformation efficiencies, since only two (Karalis and Neolatino) out of 14 cultivars produced fertile transgenic plants. The estimated transformation efficiencies were 6.25% and 1.66% for Karalis and Neolatino, respectively, and χ² analysis revealed the stable integration and segregation of the gus transgene in T1 and T2 progenies. This research has demonstrated that, among the influencing factors, genotype and auxin type play the most important role in the success of durum wheat transformation.

A Rapid Assay to Detect Toxigenic Penicillium spp. Contamination in Wine and Musts.

Wine and fermenting musts are grape products widely consumed worldwide. Since the presence of mycotoxin-producing fungi may greatly compromise their quality characteristics and safety, there is an increasing need for relatively rapid "user friendly" quantitative assays to detect fungal contamination both in grapes delivered to wineries and in final products. Although other fungi are most frequently involved in grape deterioration, secondary infections by Penicillium spp. are quite common, especially in cool areas with high humidity and in wines obtained by partially dried grapes. In this work, a single-tube nested real-time PCR approach-successfully applied to hazelnut and peanut allergen detection-was tested for the first time to trace Penicillium spp. in musts and wines. The method consisted of two sets of primers specifically designed to target the ß-tubulin gene, to be simultaneously applied with the aim of lowering the detection limit of conventional real-time PCR. The assay was able to detect up to 1 fg of Penicillium DNA. As confirmation, patulin content of representative samples was determined. Most of analyzed wines/musts returned contaminated results at >50 ppb and a 76% accordance with molecular assay was observed. Although further large-scale trials are needed, these results encourage the use of the newly developed method in the pre-screening of fresh and processed grapes for the presence of Penicillium DNA before the evaluation of related toxins.

Evolution and perspectives of cultivar identification and traceability from tree to oil and table olives by means of DNA markers.

In recent years, an increasing number of typicality marks has been awarded to high-quality olive oils produced from local cultivars. In this case, quality control requires effective varietal checks of the starting materials. Moreover, accurate cultivar identification is essential in vegetative-propagated plants distributed by nurseries and is a pre-requisite to register new cultivars. Food genomics provides many tools for cultivar identification and traceability from tree to oil and table olives. The results of the application of different classes of DNA markers to olive with the purpose of checking cultivar identity and variability of plant material are extensively discussed in this review, with special regard to repeatability issues and polymorphism degree. The characterization of olive germplasm from all countries of the Mediterranean basin and from less studied geographical areas is described and innovative high-throughput molecular tools to manage reference collections are reviewed. Then the transferability of DNA markers to processed products - virgin olive oils and table olives - is overviewed to point out strengths and weaknesses, with special regard to (i) the influence of processing steps and storage time on the quantity and quality of residual DNA, (ii) recent advances to overcome the bottleneck of DNA extraction from processed products, (iii) factors affecting whole comparability of DNA profiles between fresh plant materials and end-products, (iv) drawbacks in the analysis of multi-cultivar versus single-cultivar end-products and (v) the potential of quantitative polymerase chain reaction (PCR)-based techniques. © 2016 Society of Chemical Industry.

Jasmonic acid-isoleucine formation in grapevine (Vitis vinifera L.) by two enzymes with distinct transcription profiles.

The plant hormone jasmonic acid (JA) is essential for stress responses and the formation of reproductive organs, but its role in fruit development and ripening is unclear. Conjugation of JA to isoleucine is a crucial step in the JA signaling pathway since only JA-Ile is recognized by the jasmonate receptor. The conjugation reaction is catalyzed by JA-amido synthetases, belonging to the family of Gretchen Hagen3 (GH3) proteins. Here, in vitro studies of two grapevine (Vitis vinifera L. cv Shiraz) GH3 enzymes, VvGH3-7 and VvGH3-9, demonstrated JA-conjugating activities with an overlapping range of amino acid substrates, including isoleucine. Expression studies of the corresponding genes in grape berries combined with JA and JA-Ile measurements suggested a primary role for JA signaling in fruit set and cell division and did not support an involvement of JA in the ripening process. In response to methyl JA (MeJA) treatment, and in wounded and unwounded (distal) leaves, VvGH3-9 transcripts accumulated, indicating a participation in the JA response. In contrast, VvGH3-7 was unresponsive to MeJA and local wounding, demonstrating a differential transcriptional regulation of VvGH3-7 and VvGH3-9. The transient induction of VvGH3-7 in unwounded, distal leaves was suggestive of the involvement of an unknown mobile wound signal.

Traceability of Italian Protected Designation of Origin (PDO) table olives by means of microsatellite molecular markers.

The aim of this work was to develop a DNA microsatellite-based method of analysis to allow traceability of the three Italian Protected Designation of Origin (PDO) table olives in comparison with fruits of another seven highly diffused table olive cultivars. The analyses were carried out by using 16 primer pairs, with a mean of five different alleles detected per primer set, and power of discrimination from 0.56 to 0.90. Allelic error rates in the range of 0-3.8% were observed. By combining data from the most reliable and highly informative microsatellites (DCA3, DCA16, DCA17, DCA18, UDO-043, and GAPU101), it was possible to identify the PDO fruits over the panel of 10 cultivars, with the probability of a chance match between different cultivars as low as 10(-9) and with 0.5% error rate. The amplification profile is independent of environmental and processing conditions and is helpful to verify the authenticity of PDO samples.