sunTILL: a TILLING resource for gene function analysis in sunflower

<p>Abstract</p> <p>Background</p> <p>Cultivated sunflower (<it>Helianthus annus </it>L.) is a globally important oilseed crop, subjected to intensive genetic and genomic studies. Although classical mutagenesis has successfully been applied to <it>Helianthus </it>genus in the past, we have developed the first sunflower TILLING resource.</p> <p>Results</p> <p>To balance the maximum mutation density with an acceptable plant survival rate, a 'kill curve' analysis was first conducted with different ethylmethanesulfonate (EMS) dosages and different exposure times. According to the germination rate, a treatment with 0.7% EMS for 6 h was chosen. An M₂progeny of 3,651 fertile plants was obtained. Totally, 4.79% of the whole population showed clear aberrant phenotypes. A microsatellite analysis on a representative sample of the original seed stock and mutant lines confirmed the uniformity of the genetic background of plant material. The TILLING procedure was successfully applied to sunflower genome, initially by a <it>Cel</it>I-nuclease mismatch cleavage assay coupled with a DNA-pooling level test. To investigate the efficiency of the mutagenic treatment, a pilot screening was carried out on 1,152 M₂ lines focusing on four genes, three involved in the fatty acid biosynthetic pathway and one for downy mildew resistance. A total of 9 mutant lines were identified and confirmed by sequencing; thereby, the estimated overall mutation frequency for the pilot assay resulted to be 1/475 kb.</p> <p>Conclusion</p> <p>A first TILLING population for a high throughput identification of EMS-induced point mutations in sunflower genome has been successfully obtained. This represents a powerful tool to a better understanding of gene function in sunflower.</p

Self-Incompatibility Assessment of Some Italian Olive Genotypes (Olea europaea L.) and Cross-Derived Seedling Selection by SSR Markers on Seed Endosperms

The morphology of olive flowers allows either self- or cross-pollination that could partially explain the existence of both reproductive features in this species. However, a high degree of self-incompatibility is reported for many olive genotypes, that could be an important reproductive barrier influencing olive yield. Due to the strong environmental influence, results of compatibility tests are often contradictory, making cultivar classification quite imprecise. In this study, the self-incompatibility value has been determined for four olive genotypes (Bella di Spagna, Coratina, Leccino, and Ogliarola barese) widespread in the Mediterranean basin. Moreover, the incompatibility relationships of cultivar Coratina with some suitable pollinizers (Leccino, Oliastro, and Picholine) have been studied in controlled crosses: the in vitro germination potential of progenies has been evaluated and the selection of cross-derived embryos has been indirectly performed by the molecular characterization of the corresponding endosperm. The results increase knowledge on factors affecting self-compatibility in olive. Moreover, they provide useful information to farmers about the most effective cultivars for the set-up of new olive grove or for graft planning. Finally, they provide a new strategy and procedure based on endosperm analysis by SSRs for an accurate, fast, and relatively cheap screening of embryos/seedlings

DNA markers as a tool for genetic traceability of primary product in agri-food chains

The agri-food components of the Made in Italy are well known all over the world, therefore they may significantly contribute to the Italian economy. However, also owing to a large number of cases of improper labelling, the Italian agro-food industry faces an ever-increasing competition. For this reason, there is a decline of consumers' confidence towards food production systems and safety controls. To prevent erroneous classification of products and to protect consumers from false instore information, it is important to develop and validate techniques that are able to detect mislabelling at any stage of the food-chain. This paper describes some examples of genetic traceability of primary products in some important plant food chains such as durum wheat, olive and tomato, based on DNA analysis both of raw material and of processed food (pasta, olive oil, and peeled tomato)

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 �-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 �2 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

CITRUS TRISTEZA VIRUS RESISTANCE GENE LOCUS: SMALL RNA PROFILE AND PRELIMINARY EPIGENETIC STUDIES

Small interfering RNAs (siRNAs), play a vital role in epigenetics of plant virus-host plant interactions. It has been extensively studied at both the transcriptional and post-transcriptional levels. In plants, siRNAs initiate and manage gene silencing by directing DNA methylation and/or histone methylation. In Arabidopsis, the ~24 nt siRNAs directs DNA methylation (RNA-directed DNA methylation, RdDM) and chromatin remodeling at their target loci. Recent advances in highthroughput sequencing techniques has enabled thorough exploration of small RNAs populations and allow rapid analysis of massive datasets to assemble complete full-length genome sequence for different plant species. This large database of sequence information also allows identification of genome regions specifically matched by siRNAs that likely differ among tolerant, resistant or susceptible hosts and advance epigenetic studies on diseased plants. Resistance to Citrus tristeza virus (CTV), the most severe virus affecting Citrus spp., associated with a single dominant gene locus Ctv occurring in Poncirus trifoliata while all Citrus spp. are considered susceptible. This locus contains 22 putative genes, but their regulation and mechanism for resistance remains unknown. In our study, CTV was graft-inoculated on Carrizo citrange (Poncirus trifoliata x C. sinensis (I think) ) and C. aurantium (sour orange) seedlings, and the population of siRNA characterized by high-throughput sequencing using an ILLUMINA platform. The Ctv-derived siRNA (~2% of the total short reads) were dominated in both hosts by the 24-nt. However, CTV infection caused an increase in accumulation of 24-nt siRNA sequences homologous to the Ctv gene in Carrizo but it decreased in sour orange. Distribution of the 24nt along the Ctv gene locus (282Kb) had a clearly different distribution between the two host. The predominant hot spot of siRNA in Carrizo mapped in the putative gene Ctv-20, whereas in sour orange it associated to the intergenic region between the putative genes Ctv-11 and Ctv-12, where a Copia-like retrotransposon C is located. This distribution profile was conserved for each species between CTV-infected and uninfected plants but, as previously mentioned, the frequency of the 24nt siRNAs was altered by the presence of the virus. We supposed that the different profile of 24nt between the two host in the locus ctv is due to RdDM mechanisms. To demonstrate the methylation status of the resistance locus we performed a bisulfite treatment of DNA. in which unmethylated cytosine was converted to uracile, while methylated cytosine did not react. A methylcytosines mapping was carried out on Ctv-11 and Ctv-12 sequences. By specific software were found 5 different CpG islands in the Copia-likeretrotransposon sequence and 42 primer pair were designed. The PCR analyses have been carried out using MSP and BSP primers followed by combined bisulfite restriction analysis (COBRA)

Surgical management of adult Brainstem Gliomas: a systematic review and meta-analysis

The present review aims to investigate the survival and functional outcomes in adult high-grade brainstem gliomas (BGSs) by comparing data from resective surgery and biopsy. MEDLINE, EMBASE and Cochrane Library were screened to conduct a systematic review of the literature, according to the PRISMA statement. Analysis was limited to articles including patients older than 18 years of age and those published from 1990 to September 2022. Case reports, review articles, meta-analyses, abstracts, reports of aggregated data, and reports on multimodal therapy where surgery was not the primary treatment were excluded. The ROBINS-I tool was applied to evaluate the risk of bias. Six studies were ultimately considered for the meta-analysis. The resective group was composed of 213 subjects and the bioptic group comprised 125. The analysis demonstrated a survival benefit in those patients in which an extensive resection was possible (STR HR 0.59 (95% CI 0.42, 0.82)) (GTR HR 0.63 (95% CI 0.43, 0.92)). Although surgical resection is associated with increased survival, the significantly higher complication rate makes it difficult to recommend surgery instead of biopsy for BSGs. Future investigations combining volumetric data and molecular profiles could add important data to better define the proper indication between resection and biopsy

Quantitatively Unraveling Hierarchy of Factors Impacting Virgin Olive Oil Phenolic Profile and Oxidative Stability

A single phenolic group and even a compound play different roles in the sensory properties and stability of virgin olive oil (VOO), which in turn are strongly influenced by several factors. Understanding the causes of differences in phenolic compound composition and oxidative stability (OS) in VOOs is essential for targeted and timely harvest and processing while maintaining desired oil quality. The phenolic profile and OS of two monocultivar VOOs (Oblica and Leccino) grown in two geographical sites of different altitudes (coastal plain and hilly hinterland) were analyzed throughout the ripening period over two years. Concentration of secoiridoids was 30% higher in the Oblica than in the Leccino VOOs, which in turn had significantly higher values of OS. Both cultivars had more than twice as high concentrations of the two most abundant phenolic compounds, the dialdehyde form of decarboxymethyl oleuropein aglycone and the dialdehyde form of decarboxymethyl ligstroside aglycone, and OS values in a colder growing site of higher altitude. Among the studied monocultivar VOOs, the secoiridoid group did not behave equally during ripening. The hierarchy of different influencing factors was investigated using multivariate statistics and revealed: cultivar &gt; geographical site &gt; harvest period &gt; growing season. In addition, the possibility of traceability of VOO using molecular markers was investigated by establishing SSR profiles of oils of the studied cultivars and comparing them with SSR profiles of leaves

Antioxidant Efficacy of Olive By-Product Extracts in Human Colon HCT8 Cells

The production of olive oil is accompanied by the generation of a huge amount of waste and by-products including olive leaves, pomace, and wastewater. The latter represents a relevant environmental issue because they contain certain phytotoxic compounds that may need specific treatments before the expensive disposal. Therefore, reducing waste biomass and valorizing by-products would make olive oil production more sustainable. Here, we explore the biological actions of extracts deriving from olive by-products including olive pomace (OP), olive wastewater (OWW), and olive leaf (OLs) in human colorectal carcinoma HCT8 cells. Interestingly, with the same phenolic concentration, the extract obtained from the OWW showed higher antioxidant ability compared with the extracts derived from OP and OLs. These biological effects may be related to the differential phenolic composition of the extracts, as OWW extract contains the highest amount of hydroxytyrosol and tyrosol that are potent antioxidant compounds. Furthermore, OP extract that contains a higher level of vanillic acid than the other extracts displayed a cytotoxic action at the highest concentration. Together these findings revealed that phenols in the by-product extracts may interfere with signaling molecules that cross-link several intracellular pathways, raising the possibility to use them for beneficial health effects