Genetic analysis by DNA markers is very useful for characterization and traceability of traditional varieties and typical products. The genetic identity of products is generally referred to the cultivars and does not consider the genetic contribution of pollen from external sources. The gene flow between cultivated and wild populations is important in chestnut, because grafted cultivars, selected pollen donors, and wild trees are often located in the same areas. Indeed, the chestnut cultivars "Marrone" produce poor o none pollen.In "Multicast" and "CASTADIVA" research programmes, chestnut groves of Umbria and Lombardy regions, respectively, were characterized by genetic analysis of orchards, fruits, and wild populations. The newly started "CASTANEVAL" programme will continue the study in Lombardy region. Genetic data depict the structure of the regional cultivated chestnut. A conventional management system was found in Umbria region, with chestnut orchards composed of genetically uniform trees grafted with few cultivars. A more heterogeneous situation was found in Lombardy region, where two distinct areas were investigated. Chestnut orchards of Varese area showed a great genetic diversity, with genetically heterogeneous plantations, numerous grafted genotypes and few clonal replicates. On the contrary, in Brescia area, chestnut groves included a relatively high fraction of grafted "Marrone" plants, together with other local cultivars. The integrated analysis of mother plants, fruit embryos, and wild populations showed that the fruits from "Marrone" trees were genetically intermediate between mother plants and local wild populations, which act as pollen donor. On the opposite, the offspring from genetically heterogeneous orchards displayed a genetic profile similar to the respective mother plants. The in situ interaction between cultivated and wild chestnut confers a geographic and genetic signature to chestnut fruits. These results open perspectives to improve the characterization and traceability of chestnut productions.

Castanea sativa Mill. is a multi-functional species with a high ecological and cultural relevance. Thanks to its features, chestnut has become an essential element of subsistence for many societies in mountain and sub-mountainous areas.In this context, the principal aim of this study was to characterize the chestnut germplasm grown in pilot areas of the Lombardy Region (Municipality of Serle, province of Brescia, and the Varese Prealps, province of Varese) by integrating genetic and morphometric data analysis, in order to evaluate the regional chestnut genetic resources and promote their conservation. Twelve polymorphic microsatellite (SSRs) markers were used for genotyping 467 trees sampled between 2021 and 2022 in eight fruit tree orchards and three natural populations. Leaves and embryos were sampled for each individual to evaluate inter and intra-population gene flow. In parallel, a subset of fifty individuals from different collection sites were selected for replicated fruit morphological and nutritional analyses (specific carbohydrates, moisture, fat, protein, ash and fibers). The genetic data showed a number of clones identified as "Marrone" in Serle area. The analysis of the genetic structure, conducted with a Bayesian method and Principal Coordinates Analysis, showed different gene pools representing the two geographical areas. The combined data of mother plants and embryos from Varese showed high genetic similarity among individuals of the same area, whereas we found high genetic divergence between mother plants and embryos in Serle area. In addition, we found large inter-individual variation of several morphological traits, with the dimensional characteristics principally affected by local environmental drivers. Our results suggest that the genetic diversity and structure of the cultivated chestnut populations was shaped by different historical management systems in the two areas.

Proteases, including serine proteases, are involved in the entire life cycle of plants. Proteases are controlled by protease inhibitors (PI) to limit any uncontrolled or harmful protease activity. The role of PIs in biotic and abiotic stress tolerance is well documented, however their role in various other plant processes has not been fully elucidated. Seed development is one such area that lack detailed work on the function of PIs despite the fact that this is a key process in the life cycle of the plant. Serine protease inhibitors (SPI) such as the Bowman-Birk inhibitors and Kunitz-type inhibitors, are abundant in legume seeds and act as antinutrients in humans and animals. Their role in seed development is not fully understood and present an interesting research target. Whether lowering the levels and activity of PIs, in order to lower the antinutrient levels in seed will affect the development of viable seed, remains an important question. Studies on the function of SPI in seed development are therefore required. In this Perspective paper, we provide an overview on the current knowledge of seed storage proteins, their degradation as well as on the serine protease-SPI system in seeds and what is known about the consequences when this system is modified. We discuss areas that require investigation. This includes the identification of seed specific SPIs; screening of germplasms, to identify plants with low seed inhibitor content, establishing serine protease-SPI ratios and lastly a focus on molecular techniques that can be used to modify seed SPI activity.

I fagioli (Phaseolus vulgaris L.) sono un'importante fonte di nutrienti con effetti benefici sulla salute. Tuttavia, finora sono stati poco utilizzati per la preparazione di snack. La disponibilità di una farina di fagiolo priva di lectine attive (lec-), che non necessita di un trattamento prima dell'uso e biofortificata grazie ad un ridotto contenuto di acido fitico (lpa), che normalmente diminuisce la biodisponibilità dei cationi minerali, può aumentare l'utilizzo della farina di fagiolo per la preparazione di snack. L'obiettivo di questa ricerca è stato quello di valutare l'apprezzamento sensoriale di cracker, biscotti e una crema preparati con questa farina di fagiolo biofortificata e priva di lectine rispetto agli stessi prodotti ottenuti con farine tradizionali. I risultati evidenziano che i prodotti con farina di fagiolo sono meno apprezzati di quelli di riferimento, tuttavia la presenza di un intenso sapore umami nei prodotti con fagiolo risulta apprezzato.

Common beans (Phaseolus vulgaris L.), a staple food in many regions of the world, are a major source of dietary fibers, essential amino acid-rich proteins, some vitamins and often display a high content in essential minerals such as iron, zinc and calcium. However, these minerals are scarcely bioavailable, mainly due to the presence of phytic acid (PA) and phenolic compounds. Besides, about 60% of common beans produced worldwide are grown in regions subjected to water stress, the second most important factor that contributes to yield reduction. To address this problem, BIO-BELIEF, a multidisciplinary project consisting of a trans-continentalconsortium of European (CREA-GB and CNR-IBBA in Italy and INRAE, INSERM in France); and African partners (CIAT in Kenya and the University of Pretoria with UKZN andStellenbosch University as local South African partners) with Embrapa in Brazil and the seedcompany Blumen in Italy as partners in kind, aims to select new biofortified more droughtresilient common bean lines, in order to promote a healthy diet in a general frame of food andnutrient security. The approach, to study biofortified common beans lines exposed to drought,has, to our knowledge, never been done before and is therefore innovative. Current researchactivities and results already obtained include that 20 common bean lines derived from Europeand Africa have been already planted for seed multiplication and the germination potential ofdifferent lines, including lpa mutant lines low in phytic acid, is currently tested in twocontinents (Europe and Africa at three test sites). First results already indicate that a low phyticacid content does not significantly affect seed germination. Common bean lines are currentlyalso tested in the field and greenhouse for drought tolerance in Italy (open field and greenhouse)and in South Africa (open field under rainout shelters) by applying the leaf wilting techniquefor monitoring drought tolerance. Selected lines will be further used for introgression of thebiofortification trait into a drought-resilient genetic background and the innovative technologyof Genome Editing (GE) will also be applied in a next step to modify/silence candidate genesinvolved in phytic acid production and drought tolerance. In particular, PvMYB 60, atranscriptional repressor of anthocyanin, and PvDREB 2, a transcription factor coding for adehydration-responsive element binding protein, which are both described as negativeregulators of the drought stress response will be selected to be edited in order to develop moredrought-tolerant common bean lines. Biofortified lines will be further exploited in the next twoyears by preparing bean-based recipes, which will be characterized for their nutritional profileand micronutrient bioavailability. All identified superior lines will be finally tested byconsumers to validate any new biofortified diets for European, African and South Americanpopulations. Overall, we expect that the project will ultimately release breeding lines, with ahigh nutritional value and more drought-resilient, suitable to be grown in Europe, Africa andLatin America. For more information including a video on the BIO-BELIEF project see forexample https://www linkedin com/company/biobelief/ as well as in the two recentpublications Losa et al. (2022). Drought and heat affect common bean minerals and humandiet--What we know and where to go. Food and Energy Security 11:e351. doi:10.1002/fes3.351 and Cominelli et al. (2022). Antinutritional factors, nutritional improvement,and future food use of common beans: A perspective. Frontiers Plant Sciences 13:992169. doi:10.3389/fpls.2022.992169

L'analisi genetica pemette la caratterizzazione e tracciabilità delle produzioni locali e varietali. Le caratteristiche genetiche delle produzioni (frutti, semi e prodotti di trasformazione) vengono generalmente riferite al profilo genetico di una varietà di riferimento. Tale approccio non tiene conto del contributo pollinico esterno alla varietà, che può essere molto rilevante nelle piante auto-incompatibili o maschiosterili. Il castagno rappresenta un modello unico per studiare questo flusso genico, perché varietà coltivate, impollinatori selezionati e piante selvatiche possono coesistere. Inoltre, le varietà di "Marrone" sono spesso maschiosterili e ricevono flusso genico obbligato dagli impollinatori locali. Grazie ai progetti PSR "Multicast" e "Castadiva", aree castanicole delle regioni Umbria e Lombardia, sono state caratterizzate attraverso l'analisi genetica integrata di piante da frutto, frutti e impollinatori. I risultati hanno mostrato una gestione più convenzionale in Umbria, con castagneti da frutto geneticamente omogenei, innestati con poche varietà, diverse tra le aree castanicole. In Lombardia esiste invece una maggiore diversità genetica e gestionale con molte varietà o genotipi coesistenti all'interno di castagneti geneticamente eterogenei. In altre aree castanicole Lombarde, invece, i castagneti contengono principalmente piante innestate di "Marrone", insieme a poche altre varietà. In Umbria, l'analisi genetica integrata ha rivelato che i frutti delle varietà di "Marrone" mostrano un profilo genetico intermedio tra le piante coltivate e gli impollinatori selvatici locali. Ciò conferisce un'identità genetica e geografica alle produzioni. I risultati aprono prospettive per la caratterizzazione genetica e la tracciabilità delle produzioni. Questo approccio potrebbe integrare il concetto di "terroir", perché il territorio di coltivazione lascia una traccia genetica nelle produzioni locali, attraverso il flusso genico tra piante coltivate e selvatiche.

Over the centuries, the chestnut tree, cultivated for its fruits and timber, has become an essential element of subsistence for many societies in mountain and sub-mountainous areas, revealing its potential as a multifunctional species. Today most chestnut forests are in a state of decline and abandonment, mainly due to the depopulation of rural areas, global climate changes and recent outbreaks of exotic pests. With a view to the recovery and enhancement of chestnut genetic resources, the CASTADIVA project mainly aims to map the chestnut groves in pilot areas of the Lombardy Region (Municipality of Serle, BS and in the Varese Prealps, VA); evaluate the genetic diversity of chestnut stands, and genetically characterize local varieties. In parallel, the project includes a characterization of the fruits through morphological, pomological, qualitative and nutritional analyses. CASTADIVA will require integration of data obtained from genetic and morphometric analyses with ecological, silvicultural, phytosanitary and possibly socio-economic data, in order to identify the best practices and management criteria for the conservation and enhancement of chestnut genetic resources. Transferring information to local administrations and the agricultural stakeholder will be a final step of crucial importance. Preliminary results of genetic and nutritional analyses will be presented.

Common bean (Phaseolus vulgaris L.) is a staple food in many regions in the world. Bean seeds are a major source of dietary fibers, essential amino acid-rich proteins, some vitamins and often display a high content in essential minerals such as iron, zinc and calcium. However, these minerals are scarcely bioavailable, mainly due to the presence of phytic acid (PA) and phenolic compounds. Besides, about 60% of common beans produced worldwide are grown in regions subjected to water stress, thus after diseases, drought is the second most important factor that contributes to yield reduction. BIO-BELIEF (BIOfortification of common Bean to promote heaLthy dIEt and Food security) is a multidisciplinary project, financed in the frame of the ERA-NET FOSC call, born from the establishment of a trans-continental consortium between European, African and Brasilian partners, aims to select new biofortified and drought resilient bean lines, in order to promote a healthy diet in a general frame of food security. The project will release genetic materials with high nutritional values and develop nutritional improved and drought resilient beans suitable to be grown in Europe, Africa and Latin America (last two are regions where bean is a major staple food). BIO-BELIEF will capitalize previous works carried out by some partners that have selected biofortified lines with reduced level of PA, increased iron content and improved drought resilience. About 20 lines will be tested for seed quality in response to drought treatment in two continents. Meanwhile, the biofortification traits will be introgressed in the drought resilient genetic backgrounds. The innovative technology of Genome Editing (GE) will also be applied to explore candidate genes involved in drought resilience. The biofortified lines will be exploited by preparing bean-based recipes, which will be characterized for their nutritional profile and micronutrient bioavailability. The selected lines will be used for testing by the consumers to validate new biofortified diets for European, African and South American populations. Here we will present the preliminary results of BIO-BELIEF project.

Common bean seeds are an excellent source of protein as well as of carbohydrates, minerals, vitamins, and bioactive compounds reducing, when in the diet, the risks of diseases. The presence of bioactive compounds with antinutritional properties (e.g., phytic acid, lectins, raffinosaccharides, protease inhibitors) limits, however, the bean's nutritional value and its wider use in food preparations. In the last decades, concerted efforts have been, therefore, made to develop new common bean genotypes with reduced antinutritional compounds by exploiting the natural genetic variability of common bean and also applying induced mutagenesis. However, possible negative, or positive, pleiotropic effects due to these modifications, in terms of plant performance in response to stresses or in the resulting technological properties of the developed mutant genotypes, have yet not been thoroughly investigated. The purpose of the perspective paper is to first highlight the current advances, which have been already made in mutant bean characterization. A view will be further provided on future research directions to specifically explore further advantages and disadvantages of these bean mutants, their potential use in innovative foods and representing a valuable genetic reservoir of combinations to assess the true functional role of specific seed bioactive components directly in the food matrix.

I fagioli (Phaseolus vulgaris L.) sono una fonte importante di nutrienti con effetti benefici sulla salute umana. Tuttavia contengono lectine che limitano l'uso diretto della farina nelle preparazioni alimentari e acido fitico che riduce la biodisponibilità dei cationi minerali. Si sono valutati snack biofortificati e una crema ottenuti con una farina di fagiolo non trattata priva di lectine attive (lec-) e con ridotto contenuto di acido fitico (lpa) e si è valutato l'apprezzamento da parte dei consumatori. I prodotti con la farina lec- lpa non mostrano attività eritroagglutinante residua dovuta alle lectine; mostrano una maggiore attività residua dell'inibitore dell'?-amilasi, un ridotto indice glicemico in vitro e una maggiore biodisponibilità di ferro rispetto a quelli preparati con farina standard. I prodotti sono meno apprezzati di quelli di riferimento senza farina di fagiolo, ma la presenza di un intenso sapore umami nei prodotti con fagiolo può risultare apprezzata.

Global climate change, causing large parts of the world to become drier with longer drought periods, severely affects production of common beans (Phaseolus vulgaris L.). The bean is worldwide the most produced and consumed food grain legume in the human diet. In common beans, adapted to moderate climates, exposure to drought/heat stress not only results in significant reduction of bean yield but also the nutritional value. This review explores the contribution of common beans to food and nutrient security as well as health. Also discussed is the existing knowledge of the impact of drought/heat stress, associated with a changing climate, specifically on iron (Fe) and phytic acid (PA) that are both among the most important mineral and anti-nutritional compounds found in common beans. Further discussed is how the application of modern "omics" tools contributes in common beans to higher drought/heat tolerance as well as to higher Fe and reduced PA content. Finally, possible future actions are discussed to develop new common bean varieties with both improved drought/heat tolerance and higher mineral (Fe) content.

Common beans (Phaseolus vulgaris L.) are an important source of nutrients with beneficial effects on human health. However, they contain lectins, that limit the direct use of flour in food preparations without thermal treatment, and phytic acid, that reduces mineral cation bioavailability. The objectives of this research were: to obtain biofortified snacks and a cream using an untreated common bean flour devoid of active lectins (lec-) and with reduced content of phytic acid (lpa) and to evaluate the sensorial appreciation for these products. The main results of the present work were: the products with the lpa lec- flour did not retain residual hemagglutinating activity due to lectins; they showed higher residual ?-amylase inhibitor activity (from 2.2 to 135 times), reduced in vitro predicted glycemic index (about 5 units reduction) and increased iron bioavailability compared to the products with wild type flour; products with common bean flour were less appreciated than the reference ones without this flour, but the presence of an intense umami taste can be a positive attribute. Results confirmed that the use of the lpa lec- flour has important advantages in the preparation of safe and nutritionally improved products, and provide useful information to identify target consumers, such as children and elderly people.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for human consumption providing up to 15% of total daily calories and 36% of total daily protein in parts of Africa and the Americas. As a legume, it also has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. Wild common bean is organized in two geographically isolated and genetically differentiated wild gene pools (Mesoamerican and Andean) that diverged from a common ancestral wild population more than 100,000 years ago. From these wild gene pools, common bean was independently domesticated in Mexico and in South America nearly 8,000 years ago, and these domestication events were followed by local adaptations resulting in landraces with distinct characteristics (Schmutz et al. 2014). Domestication led to morphological changes in seed and leaf sizes, in the growth habit and photoperiod responses, variation in seed coat color and pattern that distinguish culturally adapted classes of beans. This unique example of parallel domestication is the subject of the PARDOM project that, starting from the Phaseolus replicated experiment, aims at understanding common bean genome evolution and adaptation. In the framework of the PARDOM project, we are developing TILLING-by-sequencing and genome editing technological platforms for the functional validation of candidate domestication genes in common bean. For the development of the TILLING-by-seq platform, DNA from seeds of a P. vulgaris TILLING population developed in the Mesoamerican genotype BAT93 (Porch et al. 2009; Cominelli et al. 2018) was extracted. A three-dimensional pooling system of 54 pools, each of 96 samples on average, at resolution of a population of 1728 individuals was used for NGS targeted sequencing based on custom capture probes. For the genotyping, a total of 719 genes of interest were chosen, based on the presence of one or more signals of domestication, differential expression between the Andean genotype and Mesoamerican genotype, known involvement in the phenomenon of shattering, seed development and in the cytokinin hormonal pathway. Among these genes, 27 had a complete CDS sequence coverage, whereas for the others the first 1-3 exons were covered, for a total of approximately 491Mb. The validation of candidate genes for domestication is currently in progress also via forward genetics, following the identification of target regions in coding sequences for genome editing based on CRISPR/Cas9 technology. Fifteen target candidate domestication genes have been selected, based on the presence of one or more signals of domestication. Current editing approach is directed toward MYB26, encoding a transcription factor involved in pod shattering phenotype. Given the challenges posed by common bean transformation (biolistic transgenesis), the genome editing approach is being simultaneously carried out also on soybean ( Glycine max) homologous genes.

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