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Background: The increased attention in agri-food quality and safety has led to the progress of DNA-based tools aimed to fight adulteration issues. Among the various molecular approaches, those ones based on molecular markers and DNA barcoding have been adequately validated, whereas new tools such as droplet digital PCR (ddPCR), isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated (Cas) (CRISPR/Cas) system are starting to overtake the performances of the former ones and to be applied in the agri-food sector. Scope and approach: Herein, an overview of the recent advances and exploitations of the DNA-based techniques for the authenticity and traceability of fresh and processed plant-derived food, feed and medicinal products is provided, including research about the monitoring of contaminant and allergen presence. Moreover, the potentials and flaws of these molecular tools are also discussed. Key finding and conclusions: DNA-based technologies represent valuable tools for preventing agri-food frauds and adulteration of several vegetable products present on the market such as spices, extra virgin olive oil, wine, cocoa, and medicinal plants. The application of these methods can contribute to the protection of consumers and all stakeholders involved in the agri-food production and distribution.

Modern agriculture's greatest challenge lies in climate change, as the latter has significant impacts on agricultural systems through global warming, altered rainfall patterns, and increased frequency and intensity of extreme events. Maize, as a globally significant crop, is projected to be highly susceptible to climate change across Europe. To establish a more effective cropping system and develop resilient genotypes, existing market hybrids are inadequate due to their limited genetic diversity. In contrast, maize landraces - adapted to diverse agroecological conditions - can offer invaluable indigenous germplasm as a promising foundation for future genetic enhancements. The present research aims to achieve two objectives: a comprehensive study and genotyping of 28 Italian landraces, and the identification of genetic markers likely to be associated with environmental variables. Selected landraces from Lombardia, Emilia Romagna, Trentino-Alto Adige, Veneto, Toscana, Valle d’Aosta and Friuli Venezia Giulia were analysed using the GBS technique. Subsequent population studies were conducted based on the sequencing data, revealing 12 ancestral populations in the admixture analysis. Together with prominent and well-defined populations, including Nostrano Val Tidone, Châtillon and Entrebin, there are completely admixed groups comprising varieties from Trentino, Emilia Romagna, and Toscana, making it challenging to identify a singular reference population. The high degree of genetic fragmentation is reflected in the phylogenetic tree, which does not follow regional patterns but clearly distinguishes individual varieties. Notably, the Ottofile Mantovano variety stands out as the most distinct from all others, aligning with its unique field phenotype. In the PCA analysis as well, individual populations exhibit clear differentiation, although specific varietal groups are not discernible. Subsequently, a comprehensive analysis was conducted to investigate local adaptation in

While there is a rich collection of maize germplasm from Italy, it lacks genetic resources from the Aosta Valley, an isolated mountain region where landraces have been preserved in the absence of modern germplasm introductions. These local materials, which are still cultivated mainly at household level, can have high importance from a genetic and historical point of view. In the present study, five landraces named, after the collecting sites, Arnad, Arnad-Crest, Chatillon, Entrebin and Perloz, were sampled in Aosta Valley and subjected to historic, morphologic and genetic characterization. This study provided evidence for the landraces' long presence in Aosta Valley, a significant genetic variability and differentiation among the investigated landraces. Globally, 67 different alleles were detected ranging from 4 for markers phi127 and p-bnlg176 to 10 for phi031, with a mean of 6.7 alleles per locus. Observed heterozygosity levels were comprised from 0.16 to 0.51 and are generalkly lower than expected heterozigosity supporting fixation at some loci. STRUCTURE analysis revealed clear separation between accessions revealing the presence of four ancestral populations. This may be explained by the long reproductive isolation experienced by these materials. Finally, morphological observations confirm the high diversity between landraces revealing that they generally have flint kernels, variable color from yellow to dark red (Chatillon) while Perloz showed kernels with an apical beak. The present work confirms the importance of mountain areas in conserving biodiversity and increases the rich Italian maize germplasm with materials well adapted to marginal areas. Such new genetic variability may be used to breed new materials for more resilient agriculture.

Climate change poses a formidable challenge to modern agriculture, exerting notable effects on agricultural systems through global warming, altered rainfall patterns, and increased occurrence of extreme events. Maize, being a vital crop worldwide, is projected to face substantial vulnerability to climate change across Europe. Addressing this concern necessitates the development of a more resilient cropping system and the cultivation of genotypes that can withstand these challenges. However, current maize hybrids exhibit limited genetic diversity, making them inadequate for such endeavours. In contrast, maize landraces, which have adapted to various agroecological conditions, harbour valuable indigenous germplasm that holds promise for future genetic improvements. The primary objectives of this research are twofold: to conduct a comprehensive study and genotype analysis of 28 Italian landraces and to identify genetic markers associated with environmental factors. In the spring of 2022, carefully selected landracesfrom Lombardia, Emilia Romagna, Trentino-Alto Adige, Veneto, Toscana, Valle d’Aosta and Friuli Venezia Giulia were cultivated at the experimental farm of Università Cattolica del Sacro Cuore in Piacenza. Phenotypic characterization followed the UPOV protocol, with the apex of the final leaf sampled from five individuals per variety. Genotyping was performed using the GBS technique, and subsequent population studies based on sequencing data unveiled 12 ancestral populations in the admixture analysis. Noteworthy populations, including Nostrano Val Tidone, Châtillon, and Entrebin, displayed distinct and well-defined genetic profiles. Conversely, there were completely admixed groups consisting of varieties from Trentino, Emilia Romagna, and Tuscany, which posed challenges in identifying a singular reference population. The high degree of genetic fragmentation was evident in the phylogenetic tree structure, which did not conform to regional patterns but effecti

Despite the historical cultivation of maize (Zea mays L.) being well documented in Valle d’Aosta, no landrace from this mountain region was reported in the survey of maize carried out in 1949-1950. These materials are still cultivated and have high importance from a genetic and historical point of view. Recently, 5 landraces from Valle d’Aosta have been collected and subjected to historic, morphologic and genetic characterization. These landraces were named after the sampling location as it follows: Arnad, Arnad-Crest, Chatillon, Entrebin and Perloz. This study proved landraces’ long presence on Valle d’Aosta territory through information and photographs found in local archives. Moreover, the genetic characterization, which involved the use of SSR markers, highlighted a significant genetic variability and differentiation among the investigated landraces. This may be explained by the long reproductive isolation experienced by these materials. Finally, morphological observations confirm the high diversity between landraces revealing that they generally have flint kernels, variable colour from yellow to dark red (Chatillon) while Perloz showed kernels with an apical beak, suggesting the belonging to the “Rostrata” group. The present work confirms the importance of mountain areas in conserving agrobiodiversity and the richness of the Italian maize germplasm with materials well adapted to marginal areas. Such rich genetic variability may be characterized searching for useful traits for a more resilient agriculture.

The exploitation of heterosis is one of the leading activities in maize breeding. Here, we developed an innovative recombinant intercross (RIX) population by crossing pairs of multi-parental maize recombinant inbred lines (RILs) to evaluate the heterotic response in a heterozygous multi-parental population. Field phenotyping was performed on 400 RIX genotypes considering 11 agronomic traits and the resistance to Fusarium Ear Rot (FER) in two consecutive years. Thanks to the high level of allelic diversity available in parental genomes, a broad range of phenotypic diversity was observed. The broad-sense heritability (H2) was high for all traits (H2=0.48 to 0.89) illustrating a strong genetic basis. The heterotic response was computed based on RIL values as mid and best parent heterosis showing different magnitudes for different traits suggesting effects from partial dominance to over-dominance. GWAS detected 55 significant loci associated with agronomic traits across all chromosomes, with allelic effect ranging from 0.28 to 10.90 indicating the contribution of various sized QTLs. Several pleiotropic QTLs on chromosomes 8 and 3 were detected, indicating that these loci may contain valuable genes for multiple traits. RIX collection showed a moderate heritability of FER resistance and 7 putative QTL were associated to this trait on chromosomes 8 and 5. The allelic effect estimates indicated the presence of minor effect QTL with relatively small additive effects on disease resistance. Our findings confirm the usefulness of the RIX population to decipher heterotic loci in maize and support utilizing this resource in future to accelerate crop improvement.

The exploitation of heterosis is key in modern maize breeding to capture the superior performance of heterozygous genotypes. Here, we developed a recombinant intercross (RIX) population in maize by crossing pairs of multiparental MAGIC recombinant inbred lines (RILs) to evaluate the heterosis across mosaics of eight maize haplotypes. Field phenotyping was performed on 400 RIX genotypes considering 11 agronomic traits as well as the resistance to Fusarium Ear Rot (FER), caused by Fusarium verticillioides (Sacc.) Nirenberg, in 2018 and 2019 in Piacenza, northern Italy. Phenotypic data showed a broad range of diversity in production and phenology traits thanks to the high level of allelic diversity available in parental genomes. The heterotic response of agronomic traits was computed based on RIL values as mid parent heterosis (MPH) and best parent heterosis (BPH). Both heterosis measures showed different magnitudes for different traits, with higher level of heterosis in yield and lower in flowering time, suggesting effects from partial dominance to over-dominance. No correlation was observed between phenotypic performance and heterozygosity level of RIXs for most of the agronomic traits. A preliminary quantitative trait locus (QTL) mapping detected a number of significant associations with agronomic traits across all chromosomes. The RIX collection showed a moderate heritability of FER resistance, and QTL were associated to this trait as well. The allelic effect estimates by our mapping model indicated the presence of minor effect QTL with relatively small additive effects on disease resistance in both years. Our findings confirm the usefulness of the RIX population to decipher heterotic loci in maize and support utilizing this resource in future to accelerate crop improvement.

During 1949-1950 in Italy begun a formal investigation to characterize maize (Zea mays L.) cultivation. In 1954, started a project for the sampling of all Italian maize landraces; this work ended with the collection of 562 different accessions collected in all regions with the exception of Valle d’Aosta, even if historical cultivation of maize in this Region is well documented. In Italy maize landraces have been extensively grown until the mid of the XX century when the cultivation of hybrid took place due to their significant agronomic performances. Despite that, being Valle d’Aosta a mountain region where intensive maize cultivation never started, it was possible to preserve the presence of some landraces. These local materials, which are still cultivated, mainly at domestic level, have high importance from a genetic and historical point of view. Recently, 5 maize landraces from Valle d’Aosta and 2 landraces from the adjacent Canavese (Piedmont) have been collected and subjected to historic, morphologic and genetic characterization. These landraces were named after the sampling location as it follows: Arnad, Arnad-Crest, Chatillon, Entrebin, Perloz, Bianco Canavese, and Rostrato Canavese. Firstly, on these 6 varieties the historic characterization has been carried out. Information and photographs have been searched in local archives and this was crucial to prove their long presence in all the sampling sites under study. From this historic reconstruction, the variety Entrebin resulted as the one that is better historically characterized. To study the variability and differentiation of landraces from Valle d’Aosta, the genetic characterization was performed by the means of 10 SSR markers tested on 20 samples from each landrace. This study highlighted a significant genetic variability among the landraces and, especially, a good level of differentiation between the accessions under investigation. This last result may be explained by the long reproductive isolation exp

Beaked corn represents one of the most characteristics and neglected group of Italian maize landraces. These genotypes, classified in the “Rostrata” group, were mostly grown in northern Italy, on the left bank of the Pò river, until the end of the Second World War and then subjected to strong genetic erosion because of the subsequent introduction of improved genotypes and hybrids of US origin. These materials are experiencing a revival period for cultivation, commercial exploitation and an increased number of farmers is seeking particular genotypes for the production of niche products. In Valchiavenna (Sondrio, Lombardia, Italy) maize cultivation has historical importance for polenta preparation and is characterized by the presence of at least three accessions of “Mais Rostrato Val Chiavenna”, one conserved since 1982 in germplasm bank (here named as R17_BG) and two in situ (here named as R17_M; R17_T), with distinctive morphological traits at both ear and plant level. In the present study, these accessions have been characterized at the morphological and genetic level with 10 SSR markers and compared to other landraces of the Rostrata group. SSR analysis revealed from 3 to 11 alleles per locus evidencing a good level of heterosis and absence of allele fixation for landraces. Both phylogenetic and STRUCTURE analysis evidence that the three “Mais Rostrato Val Chiavenna” are different entities with a distinct genetic origin. Historical investigation revealed that the morphology most close to the “original” Mais Rostrato di Val Chiavenna is that of R17_T, dating back to the XIX century. These observations, coupled with morphological and genetic results may corroborate that R17_T corresponds to the original “Mais Rostrato Val Chiavenna”.

Fusarium verticillioides is one of the most relevant fungal species in maize responsible for ear, stalk and seedling rot, as well as the fumonisin contamination of kernels. Plant lipoxygenases (LOX) synthesize oxylipins that play a crucial role in the regulation of defense mechanisms against pathogens and influence the outcome of pathogenesis. To better uncover the role of these signaling molecules in maize resistance against F. verticillioides, the functional characterization of the 9‐LOX gene, ZmLOX4, was carried out in this study by employing mutants carrying Mu insertions in this gene (named as UFMulox4). In this regard, the genotyping of five UFMulox4 identified the mutant UFMu10924 as the only one having an insertion in the coding region of the gene. The impact of ZmLOX4 mutagenesis on kernel defense against F. verticillioides and fumonisin accumulation were investigated, resulting in an increased fungal susceptibility compared to the inbred lines W22 and Tzi18. Moreover, the expression of most of the genes involved in the LOX, jasmonic acid (JA) and green leaf volatiles (GLV) pathways, as well as LOX enzymatic activity, decreased or were unaffected by fungal inoculation in the mutant UFMu10924. These results confirm the strategic role of ZmLOX4 in controlling defense against F. verticillioides and its influence on the expression of several LOX, JA and GLV genes.

Species of the genus Crocus are found over a wide range of climatic areas. In natural hab-itats, these geophytes diverge in the flowering strategies. This variability was assessed by analyzing the flowering traits of the Spanish collection of wild crocuses, preserved in the Bank of Plant Germplasm of Cuenca. Plants of the seven Spanish species were analyzed both in their natural environments (58 native populations) and in common garden experiments (112 accessions). Differences among species observed in the native habitats were maintained under uniform environmental conditions, suggesting a genetic basis for flowering mechanisms. Two eco-morphological types, au-tumn-and spring-flowering species, share similar patterns of floral induction and differentiation period in summer. The optimal temperature for this process was 23 °C for both types. Unlike Irano-Turanian crocuses, spring-flowering Spanish species do not require low winter temperatures for flower elongation. Hysteranthous crocuses flower in autumn prior to leaf elongation. We conclude that the variability in flowering traits in crocuses is related to the genetic and environmental regulation of flower primordia differentiation and elongation prior to emergence above the soil surface. The elucidation of the physiological differences between eco-morphological types of crocuses: synanthous with cold requirements and synanthous and hysteranthous without cold requirements, unlocks a new approach to the flowering evolution of geophytes in Mediterranean regions. Crocus species can serve both as a new model in the study of the molecular basis of hysteranthy and for the purposes of developing the molecular markers for desirable flowering traits.

Sorghum [Sorghum bicolor (L.) Moench] is a subsistence crop and the main food for populations in arid or semiarid regions and it is appreciated for the production of gluten-free products, forages, raw materials for industrial transformation and packaging. The end-use of different sorghum purposes having various plant or kernel characteristics require specific breeding programs to develop the desired ideotype. Sorghum grains can be classified according to kernel color, tannins and polyphenols content: white, yellow, red, brown, and black. White sorghum is characterized by a low level of total phenolic content and tannins. The advantage of using white sorghum is: increased protein digestibility, nutritional composition and consumer acceptance similar to other cereals. A collection of 117 white grain sorghums was characterized using 10 SSRs and preliminary agronomic observations were made for main traits. SSR analysis revealed from 10 to 33 alleles per locus.Observed heterozygosity was lower than expected according to the reproduction system of sorghum. Phylogenetic analysis revealed 6 main groups of genotypes. Only one group is constituted by genotypes with the same geographical origin (Egypt) while other groups are admixtures of different countries. The principal coordinate analysis revealed good correspondence between genetic profiles and groups evidenced by similar agronomic performances.

Il progetto RI.COL.MA (RIcupero, Caratterizzazione, COLtivazione del Mais Antico) è finanziato dal PSR 2014-2020 della Regione Emilia-Romagna. Obiettivo generale di RI.COL.MA è censire, recuperare, conservare e caratterizzare dal punto di vista morfologico, genetico, agronomico e tecnologico le varietà di mais tradizionali presenti in Emilia-Romagna, abbandonate da tempo dalle grandi colture e che rappresentano fonti di variabilità genetica per caratteristiche qualitative della granella e per adattamento all’ambiente. Le informazioni ottenute saranno rese disponibili attraverso attività di disseminazione e formazione che permetteranno alle aziende che intendono utilizzare le varietà tradizionali di mais di sviluppare nuove filiere produttive e, nello stesso tempo, di conservare il patrimonio storico e culturale alla base della biodiversità maidicola. Il progetto vede coinvolte l’Università Cattolica del Sacro Cuore di Piacenza, l’Università degli Studi di Pavia, il CRPV (Centro Ricerche Produzioni Vegetali) e otto aziende agricole emiliano-romagnole.

Il progetto RI.COL.MA (RIcupero, Caratterizzazione, COLtivazione del Mais Antico) è finanziato dal PSR 2014-2020 della regione Emilia-Romagna. Obiettivo generale di RI.COL.MA è censire, recuperare, conservare e caratterizzare dal punto di vista morfologico, genetico, agronomico e tecnologico le varietà di mais tradizionali presenti in Emilia Romagna, abbandonate da tempo dalle grandi colture e che rappresentano fonti di variabilità genetica per caratteristiche qualitative della granella e per ’adattamento all’ambiente. Le informazioni ottenute saranno rese disponibili attraverso attività di disseminazione e formazione che permetteranno alle aziende che intendono utilizzare le varietà tradizionali di mais di sviluppare nuove filiere produttive e, nello stesso tempo, di conservare il patrimonio storico e culturale alla base della biodiversità maidicola. Il progetto vede coinvolte l’Università Cattolica del Sacro Cuore di Piacenza, l’Università degli Studi di Pavia, il Centro Ricerche Produzioni Vegetali, e 8 aziende agricole emiliano-romagnole.

Wine is frequently reported as one of the most adulterated agro-food products worldwide. Among the traceability methods available, DNA is of particular interest providing the possibility to recognize uniquely the wine production cultivar/cultivars. Several studies carried out in controlled conditions (laboratory level or small production wineries) support the use of DNA in wine traceability, but the situation can change completely when moving from controlled to uncontrolled realities. In the present study, the entire production chain, in a large cooperative Italian winery, was followed, for a monovarietal (Pinot noir PDO) and a polyvarietal (Rosso Oltrepò TGI) production. Results support the feasibility of DNA traceability from grape delivering to the whole fermentation process and through the most common oenological operations as racking and filtration. The application of most aggressive methods (as the thermovinification process) can increase DNA degradation reducing but not hampering the possibility to apply DNA for traceability purposes. A different situation concerns the storage of wine in tanks, despite the controlled temperature and light conditions, or in bottles where DNA degradation continues strongly influencing the possibility to apply traceability.

The Multi-parent Advanced Generation Inter-Cross (MAGIC) maize population is a formidable tool for QTL mapping and for producing segregant hybrids to study heterosis; a fundamental aspect of maize success. The MAGIC collection was used as inbred source for recombinant intercrosses (RIX) development. RIXs are in-fact hybrid genotypes which are derived by crossing between pairs of MAGIC RILs; each RIX having known genomic contribution by its two parental RILs. The resulting RIX population offers some unique properties to facilitate the genetic analysis of complex traits, and this population, combined with the genomic resources available in maize, provides a powerful resource to study the genetic basis of agronomic traits and resistance to Fusarium Ear Rot (FER) in heterozygous genotypes, filling the gap from inbred lines to hybrids. Hence, this study was conducted on a set of 400 RIXs that was phenotyped in two consecutive years for agronomic, yield and FER resistance traits. High level of phenotypic diversity and trait distribution closed to normality indicates that the mode of inheritance of quantitative traits was continuous and should be influenced by many genes. After the GWAS analyses, 55 significant putative QTL associated to different traits were found. Several overlapping QTL were observed for highly correlated agronomic traits suggesting the possible presence of a pleiotropic effect influencing multiple linked traits at once. Heritability for FER was moderate and seven putative QTL for resistance were identified on chromosomes 5 and 8. The QTLs identified as associated to relevant agronomic, yield and disease-resistance traits may be used to harbor potential candidate genes for agronomic performance and Fusarium resistance. These findings represent the basis for future research by providing additional layers of information about heterosis and allowing a finer characterization of QTL to contribute to maize productivity and stability.

Maize is mainly affected by two fungal pathogens, Fusarium verticillioides and Aspergillus flavus, causing Fusarium ear rot (FER) and Aspergillus ear rot (AER), respectively. Both fungi are of concern to stakeholders as they affect crop yield and quality, contaminating maize grains with the mycotoxins fumonisins and aflatoxins. The easiest strategy to prevent pre-harvest contamination by F. verticillioides and A. flavus is to develop maize hybrids resistant to FER and AER, as well as to their associated mycotoxins. The objective of this investigation was to test 46 F1 hybrids, originated from different Italian, US and Canadian breeding groups, for these important traits and their agronomic performances. All hybrids were planted and artificially inoculated with toxigenic strains of F. verticillioides and A. flavus at two locations in 2017, and the best performing 17 out of 46 were also tested in 2018. Ear rots were present in all hybrids in 2017 and 2018, with percentages ranging from 6.50 to 49.50%, and 5.50 to 45.53%, for FER and AER, respectively. Seven hybrids (PC8, PC15, PC9, PC11, PC14, PC34 and PC17) presented the lowest levels of both diseases considering the overall locations and growing seasons, and three of these (PC8, PC11 and PC14) were also amongst the least mycotoxin contaminated hybrids in 2017. The inbred lines used in hybrid production may provide additional sources of resistance suitable in breeding programs targeting multiple pathogens and their mycotoxins.

Fusarium verticillioides, which causes ear, kernel and stem rots, has been reported as the most prevalent species on maize worldwide. Kernel infection by F. verticillioides results in reduced seed yield and quality as well as fumonisin contamination, and may affect seedling traits like germination rate, entire plant seedling length and weight. Maize resistance to Fusarium is a quantitative and complex trait controlled by numerous genes with small effects. In the present work, a Genome Wide Association Study (GWAS) of traits related to Fusarium seedling rot was carried out in 230 lines of a maize association population using 226,446 SNP markers. Phenotypes were scored on artificially infected kernels applying the rolled towel assay screening method and three traits related to disease response were measured in inoculated and not-inoculated seedlings: plant seedling length (PL), plant seedling weight (PW) and germination rate (GERM). Overall, GWAS resulted in 42 SNPs significantly associated with the examined traits. Two and eleven SNPs were associated with PL in inoculated and not-inoculated samples, respectively. Additionally, six and one SNPs were associated with PW and GERM traits in not-inoculated kernels, and further nine and thirteen SNPs were associated to the same traits in inoculated kernels. Five genes containing the significant SNPs or physically closed to them were proposed for Fusarium resistance, and 18 out of 25 genes containing or adjacent to significant SNPs identified by GWAS in the current research co-localized within QTL regions previously reported for resistance to Fusarium seed rot, Fusarium ear rot and fumonisin accumulation. Furthermore, linkage disequilibrium analysis revealed an additional gene not directly observed by GWAS analysis. These findings could aid to better understand the complex interaction between maize and F. verticillioides.

The worldwide demand for cocoa is rising but, in the supply chain, several cases of adulteration, usually of fine cocoa with bulk cocoa, are reported while industry requires cocoa stocks with constant aromas and quality. In cocoa chain, adulteration and lots misidentification can happen along the entire chain from the bean to the production of liquor. The availability of a DNA traceability system able to recognize the different cocoa varieties is something expected to have a big impact on cocoa sector. Several studies carried out at the laboratory level support the use of DNA in traceability but, clearly, the situation can change completely when moving from the lab to real cases. In the present paper, samples were recovered from big lots of beans and liquors provided by an Italian big brand. Two different cocoa types labelled as “Nacional” and “CCN51” from Ecuador have been examined; DNA was extracted from both beans and cocoa liquor and successfully amplified and examined by using cocoa reference SSR markers. Allele profiles revealed that “Nacional” is an admixture of several genotypes as confirmed also by single beans and leaves profiling. Contrary to Nacional, analyses suggest that CCN51 from Ecuador is characterised by a single diploid genotype. The comparison of CCN51 alleles with data present in databases revealed that misidentification is present also for this modern vegetatively propagated variety and that in different countries are present different CCN51. The production of artificial mixtures demonstrates that DNA markers are able to identify even small traces of unwanted genotypes. Finally, the results suggest that, while for Nacional DNA traceability is not feasible, because of the multigenotype nature of the variety, for CCN51 traceability is possible. However, before doing this, it is necessary to precisely define the genetic identity of CCN51 by comparing accessions coming from the different countries where it is present and cultivated.

Polyphenols from five pigmented sorghum (PS) flours were in vitro evaluated as possible modulators of starch digestibility. White sorghum (WS) flour was used as control. Untargeted metabolomics depicted the phenolic composition of raw and cooked flours (obtained through heating at 100 °C for 30 min in water) highlighting differences in flavonoids and phenolic acids. Raw PS flours were characterized by greater tannin and kafirin contents when compared to WS, and, after cooking, PS flours had greater resistant starch (from 4.2 to 21.4 g /100 g dry matter), and lower starch hydrolysis index (HI) with respect to cooked WS. Multivariate statistics showed that flavonoids characterizing PS were the most discriminant compounds during the in vitro digestion. In addition, kafirin and total tannins content (on raw ingredients) along with the anthocyanin profiles (on cooked samples) were negative correlated with HI. Therefore, PS flours might be good candidates for the formulation of functional foods.

In this chapter, we report a possible alternative use of epigenetics by applying methylation-sensitive amplified fragment length polymorphisms (MS-AFLP) to saffron traceability. Saffron is the most expensive plant-derived product in the world and one of the most frequently adulterated. One of the most frequent adulteration is by adding to saffron stigmas different parts of the saffron flower itself to increase volumes. While DNA is the same in all the parts of the plant, the epigenetic state can vary according to the organ and/or tissue of origin, making it possible to discriminate the stigmas from the other parts of saffron flower. In the subsequent method, the protocol to carry out a MS-AFLP analysis of saffron DNA methylation patterns is described.

Fungal infection by Fusarium verticillioides is cause of substantial reductions in maize yield and grain quality worldwide. Developing natural resistance in maize genotypes is an effective way to achieve sustainable control of F. verticillioides in the field, and breeding for resistance may be accelerated by identifying genes and loci responsible for natural disease resistance. Significant advances have been made in the development of transcriptomic, genetic and genomic information for maize, F. verticillioides moulds, and their interactions over recent years. Several quantitative trait loci (QTL) and single-nucleotide polymorphism markers for resistance to Fusarium deriving from QTL mapping and genome-wide association studies have been described in three different maize populations: 1. Bi-parental population; 2. Association mapping panel; 3. Multi-parent Advanced Generation Inter Crosses (MAGIC). To guide the identification of candidate genes within the identified QTL, transcriptomic and sequencing information have been exploited. Promising candidate genes associated with disease resistance and pathogen related-mechanisms at the Fusarium resistant loci have been identified on maize chromosomes 4, 5 and 7. Many of the identified candidates genes offer hints to key metabolic pathways that may have a significant effect on reducing Fusarium infection. Measuring Fusarium resistance in open field could confirm and support their direct use in maize breeding either through crosses or genome editing approaches.

The project aims at the characterization and conservation of regional maize landraces. Genetic material, consisting of 32 accessions, was retrieved both from recent surveys in Emilia Romagna, 11 accessions, and from the germplasm bank of CREA-MAC (Bergamo), 21 accessions. In particular, these last samples derive from the whole Italian collection of maize landraces carried out in the 50’s by Brandolini and Fenaroli. In the first two years of the project, we carried out the landrace census and field characterization of the different materials according to UPOV guidelines and prepared, for each accession, a descriptive sheet. For each landrace, 100 seeds were sown and, from each plant, leaf samples were collected for DNA analysis. Approximately 2,000 plants, around 60 plants per landrace, were sampled. Each landrace is maintained in field by controlled randomintermating. For genetic analyses, 80 SSR markers were tested to identify the 10 most polymorphic to be used for the genetic characterization of all samples. . In parallel to these activities, an agronomic trial has been set up with the 32 landraces on four replicates to investigate production potential and resistance to mycotoxigenic fungi, with a special focus on Fusarium verticillioides. Considering that landraces can be exploited in conditions of low input agriculture, one of the objectives of the project is the identification of the best performing landraces suitable for cultivation nowadays (with a particular interest at mountain and disadvantaged areas). With this purpose, seeds, obtained from the propagation carried out in the first year, were distributed to partner farms and fields have been set-up in different hilly and mountainous areas of the region Emilia Romagna. These fields are presently underway with the double aim to evaluate the landraces in low input conditions and to propagate seeds for the future cultivation. The work was supported by PSR 2014- 2020, Emilia Romagna Region.

Saffron is a high-quality and expensive spice being widely subjected to adulteration. An UHPLC-ESI/QTOF-MS metabolomic-based approach was therefore used to investigate the discrimination potential between adulterated (added with different percentage of other parts of the flower) and authentic saffron, as well as to trace its geographical origin. Both unsupervised (hierarchical clustering) and supervised OPLS-DA multivariate statistics allowed discriminating authentic saffron from styles added of other floral components, as well as PDO (Protected Designation of Origin) vs non PDO saffron samples according to their chemical fingerprints. The proposed markers were then validated through ROC curves. Anthocyanins and glycosidic flavonols were the best markers of the styles' adulteration. However, other flavonoids (mainly free flavonols and flavones), together with protocatechuic aldehyde and isomeric forms of hydroxybenzoic acid, were also validated as markers for the discrimination of PDO vs non PDO saffron samples. This work outlines the potential of untargeted metabolomics based on UHPLC-ESI/QTOF mass spectrometry for saffron authenticity and traceability.

Fungal infection by Fusarium verticillioides is cause of prevalent maize disease leading to substantial reductions in yield and grain quality worldwide. Maize resistance to the fungus may occur at different developmental stages, from seedling to maturity. The breeding of resistant maize genotypes may take advantage of the identification of quantitative trait loci (QTL) responsible for disease resistance already commenced at seedling level. The Multi-parent Advance Generation Intercross (MAGIC) population was used to conduct high-definition QTL mapping for Fusarium seedling rot (FSR) resistance using rolled towel assay. Infection severity level, seedling weight and length were measured on 401 MAGIC maize recombinant inbred lines (RILs). QTL mapping was performed on reconstructed RIL haplotypes. One-fifth of the MAGIC RILs were resistant to FSR and 10 QTL were identified. For FSR, two QTL were detected at 2.8 Mb and 241.8 Mb on chromosome 4, and one QTL at 169.6 Mb on chromosome 5. Transcriptomic and sequencing information generated on the MAGIC founder lines was used to guide the identification of eight candidate genes within the identified FSR QTL. We conclude that the rolled towel assay applied to the MAGIC maize population provides a fast and cost-effective method to identify QTL and candidate genes for early resistance to F. verticillioides in maize.

Climate change is aggravating the incidence of several maize diseases worldwide. Fusarium verticillioides is a prevalent fungal pathogen affecting all growth stages of maize, triggering several diseases including seedling rot and ear rot. The breeding of resistant maize genotypes is the most effective way to achieve sustainable control of Fusarium infection and may take advantage of the identification of genes and loci responsible for natural disease resistance. Significant advances have been made in the development of transcriptomic, genetic and genomic information for maize, F. verticillioides moulds, and their interactions over recent years. Several quantitative trait loci (QTL) and single-nucleotide polymorphism markers for resistance to Fusarium deriving from QTL mapping and genome-wide association studies have been described in three different maize populations: 1. Bi-parental population; 2. Association mapping panel; 3. Multi-parent Advanced Generation Inter Crosses (MAGIC). To guide the identification of candidate genes within the identified QTL, transcriptomic and sequencing information have been exploited. Promising candidate genes associated with disease resistance and pathogen related-mechanisms at the Fusarium resistant loci have been identified on maize chromosomes 4, 5 and 7. Many of the identified candidates genes offer hints to key metabolic pathways that may have a significant effect on reducing Fusarium infection. Measuring Fusarium resistance in open field could confirm and support their direct use in maize breeding either through crosses or genome editing approaches.

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide-resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F 2 or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.