A method for obtaining flexible broccoli varieties for sustainable agriculture.

BACKGROUND: The use of high inputs in agriculture resulted in few varieties (hybrids and pure lines) used in all agricultural systems. Also varieties of vegetables, including broccoli, for organic and low-input agriculture, are almost exclusively hybrids, since there are very few specific breeding programs and varieties for sustainable agriculture systems. A strategy to overcome this issue is the adoption of specific breeding programs for developing heterogeneous varieties (i.e. synthetics, open pollinated varieties, composite cross populations and mixtures). In fact, heterogeneous varieties are able to evolve and adapt to specific agro-climatic conditions. The aim of this study was to develop a method (an Evolutionary Breeding Program, EBP) for obtaining heterogeneous varieties of broccoli and test its efficiency in developing highly diverse varieties, as needed in sustainable agriculture. A synthetic variety originated from a landrace was multiplied in different environments for 3 cycles and morpho-phenological and genetic diversity of the derived populations were assessed. RESULTS: The presented results are the first and unique indication about the efficiency of a short-time EBP for an allogamous species like broccoli. Few morphological changes were observed among varieties multiplied in different environments with different agro-climatic conditions. This could be probably due to the initial genetic diversity of the landrace from which the populations were selected and also to the great plasticity of the crop. However, SSR data highlighted a genetic differentiation among populations multiplied for two/three years across Europe and in Central Italy, that was not so evident when considering morphological data only. CONCLUSIONS: Few years of multiplication in different environments resulted in genetically differentiated broccoli populations that nonetheless preserved the original genetic diversity and productivity level and appear to evolve in relationship to different environments: the applied EBP is useful for developing heterogeneous materials for sustainable agriculture.

Short-Term Local Adaptation of Historical Common Bean (Phaseolus vulgaris L.) Varieties and Implications for In Situ Management of Bean Diversity.

Recognizing both the stakes of traditional European common bean diversity and the role farmers' and gardeners' networks play in maintaining this diversity, the present study examines the role that local adaptation plays for the management of common bean diversity in situ. To the purpose, four historical bean varieties and one modern control were multiplied on two organic farms for three growing seasons. The fifteen resulting populations, the initial ones and two populations of each variety obtained after the three years of multiplication, were then grown in a common garden. Twenty-two Simple Sequence Repeat (SSR) markers and 13 phenotypic traits were assessed. In total, 68.2% of tested markers were polymorphic and a total of 66 different alleles were identified. FST analysis showed that the genetic composition of two varieties multiplied in different environments changed. At the phenotypic level, differences were observed in flowering date and leaf length. Results indicate that three years of multiplication suffice for local adaptation to occur. The spatial dynamics of genetic and phenotypic bean diversity imply that the maintenance of diversity should be considered at the scale of the network, rather than individual farms and gardens. The microevolution of bean populations within networks of gardens and farms emerges as a research perspective.

Terroir is a key driver of seed-associated microbial assemblages.

Seeds have evolved in association with diverse microbial assemblages that may influence plant growth and health. However, little is known about the composition of seed-associated microbial assemblages and the ecological processes shaping their structures. In this work, we monitored the relative influence of the host genotypes and terroir on the structure of the seed microbiota through metabarcoding analysis of different microbial assemblages associated to five different bean cultivars harvested in two distinct farms. Overall, few bacterial and fungal operational taxonomic units (OTUs) were conserved across all seed samples. The lack of shared OTUs between samples is explained by a significant effect of the farm site on the structure of microbial assemblage, which explained 12.2% and 39.7% of variance in bacterial and fungal diversity across samples. This site-specific effect is reflected by the significant enrichment of 70 OTUs in Brittany and 88 OTUs in Luxembourg that lead to differences in co-occurrence patterns. In contrast, variance in microbial assemblage structure was not explained by host genotype. Altogether, these results suggest that seed-associated microbial assemblage is determined by niche-based processes and that the terroir is a key driver of these selective forces.

Investigating the genetic basis of salt-tolerance in common bean: a genome-wide association study at the early vegetative stage.

Salinity poses a significant challenge to global crop productivity, affecting approximately 20% of cultivated and 33% of irrigated farmland, and this issue is on the rise. Negative impact of salinity on plant development and metabolism leads to physiological and morphological alterations mainly due to high ion concentration in tissues and the reduced water and nutrients uptake. Common bean (Phaseolus vulgaris L.), a staple food crop accounting for a substantial portion of consumed grain legumes worldwide, is highly susceptible to salt stress resulting in noticeable reduction in dry matter gain in roots and shoots even at low salt concentrations. In this study we screened a common bean panel of diversity encompassing 192 homozygous genotypes for salt tolerance at seedling stage. Phenotypic data were leveraged to identify genomic regions involved in salt stress tolerance in the species through GWAS. We detected seven significant associations between shoot dry weight and SNP markers. The candidate genes, in linkage with the regions associated to salt tolerance or harbouring the detected SNP, showed strong homology with genes known to be involved in salt tolerance in Arabidopsis. Our findings provide valuable insights onto the genetic control of salt tolerance in common bean and represent a first contribution to address the challenge of salinity-induced yield losses in this species and poses the ground to eventually breed salt tolerant common bean varieties.

In situ occurrence and protection of crop wild relatives in Italian sites of natura 2000 network: Insights from a data-driven approach.

Aim of this work is to evaluate the in situ status of different crop wild relative species in Italy by analysing the geographic distribution of their populations and to suggests possible strategies to improve their future conservation. The work has been focused on different species of the Allium, Beta, Brassica, Secale and Triticum genera that are of priority at European and global levels for the economic importance of the related crops, the level of threat, as well as the potential for use. Using information available in the Italian National Geoportal, geographical distribution and the overall percentage of populations occurring in Natura 2000 sites was initially analysed. In addition, due to the economic importance of the genus and species distribution in Italy, Brassica glabrescens, B. insularis, B. macrocarpa, B. montana, B. procumbens, B. rupestris, B. villosa were the object of additional analyses based on more detailed occurrence data, retrieved from multiple databases, and including land cover/land use and in situ and ex situ density analyses. Geographical distribution data were retrieved for 1,996 in situ populations belonging to 60 crop wild relative species: Allium (43), Brassica (11), Triticum (4), Beta (1) and Secale (1). Percentages of population occurring in Natura 2000 sites are quite different when the different species are considered; this also applies to Brassica species in most need of protection. Results of land cover/land use analysis showed that Brassica populations outside Natura 2000 areas mainly occur in anthropized sites while those within Natura 2000 mainly in sites characterised by natural and seminatural conditions. Areas where genetic reserves could be instituted and that could be the target of future Brassica resources collection missions are also suggested. Based on a large dataset of punctual geographical distribution data of population occurrences across the territory, this research shows that, in Italy, crop wild relatives in situ are in a quite precarious condition especially when species in most need of protection are considered. Our data also highlight the role of Natura 2000 Network in favouring in situ protection of these precious resources in Europe.

Effectiveness of in situ and ex situ conservation of crop diversity. What a Phaseolus vulgaris L. landrace case study can tell us.

The effectiveness of in situ (on-farm) and ex situ conservation strategies to maintain total genetic diversity was assessed in a threatened Phaseolus vulgaris L. landrace. Farmer seed lots (subpopulations) were sampled initially and then after in situ and ex situ multiplication (two locations). The number of plants used in the ex situ multiplications (120) was much larger than that normally used in germplasm bank procedures and the farmer seed lots were kept separate. In situ, the landrace was multiplied by each farmer with the usual population size. Eighty plants from the initial population, the in situ and the two ex situ multiplications were individually tested using 26 microsatellite markers. Most of the genetic parameters showed a consistent decline in the ex situ populations compared with the in situ population, with a notable loss of less frequent alleles. The differentiation among the farmer subpopulations increased when the multiplication took place outside of the adaptation area. Although 120 plants were multiplied in each ex situ cycle, a bottleneck effect was present. In addition, tests for neutrality detected three loci that are involved in pathogen response and are potentially under selective effects. The diversity conservation and the management practices of autogamous landrace crops are discussed.

European landrace diversity for common bean biofortification: a genome-wide association study.

Mineral deficiencies represent a global challenge that needs to be urgently addressed. An adequate intake of iron and zinc results in a balanced diet that reduces chances of impairment of many metabolic processes that can lead to clinical consequences. In plants, bioavailability of such nutrients is reduced by presence of compounds such as phytic acid, that can chelate minerals and reduce their absorption. Biofortification of common bean (Phaseolus vulgaris L.) represents an important strategy to reduce mineral deficiencies, especially in areas of the world where this crop plays a key role in the diet. In this study, a panel of diversity encompassing 192 homozygous genotypes, was screened for iron, zinc and phytate seed content. Results indicate a broad variation of these traits and allowed the identification of accessions reasonably carrying favourable trait combinations. A significant association between zinc seed content and some molecular SNP markers co-located on the common bean Pv01 chromosome was detected by means of genome-wide association analysis. The gene Phvul001G233500, encoding for an E3 ubiquitin-protein ligase, is proposed to explain detected associations. This result represents a preliminary evidence that can foster future research aiming at understanding the genetic mechanisms behind zinc accumulation in beans.

Genome-Wide Association Study Reveals Candidate Genes for Flowering Time Variation in Common Bean (Phaseolus vulgaris L.).

The common bean is one of the most important staples in many areas of the world. Extensive phenotypic and genetic characterization of unexplored bean germplasm are still needed to unlock the breeding potential of this crop. Dissecting genetic control of flowering time is of pivotal importance to foster common bean breeding and to develop new varieties able to adapt to changing climatic conditions. Indeed, flowering time strongly affects yield and plant adaptation ability. The aim of this study was to investigate the genetic control of days to flowering using a whole genome association approach on a panel of 192 highly homozygous common bean genotypes purposely developed from landraces using Single Seed Descent. The phenotypic characterization was carried out at two experimental sites throughout two growing seasons, using a randomized partially replicated experimental design. The same plant material was genotyped using double digest Restriction-site Associated DNA sequencing producing, after a strict quality control, a dataset of about 50 k Single Nucleotide Polymorphisms (SNPs). The Genome-Wide Association Study revealed significant and meaningful associations between days to flowering and several SNP markers; seven genes are proposed as the best candidates to explain the detected associations.

A methodological approach to identify agro-biodiversity hotspots for priority in situ conservation of plant genetic resources.

Agro-biodiversity is seriously threatened worldwide and strategies to preserve it are dramatically required. We propose here a methodological approach aimed to identify areas with a high level of agro-biodiversity in which to set or enhance in situ conservation of plant genetic resources. These areas are identified using three criteria: Presence of Landrace diversity, Presence of wild species and Agro-ecosystem ecological diversity. A Restrictive and an Additive prioritization strategy has been applied on the entire Italian territory and has resulted in establishing nationwide 53 and 197 agro-biodiversity hotspots respectively. At present the strategies can easily be applied at a European level and can be helpful to develop conservation strategies everywhere.

Warm Season Grain Legume Landraces From the South of Europe for Germplasm Conservation and Genetic Improvement.

Currently, there is a high concern from consumers regarding food quality, with emphasis on the origin of food sources. We here review the current situation of beans (Phaseolus spp.) and cowpea (Vigna unguiculata (L.) Walp.) landraces in the South of Europe focusing on morpho-agronomic and genetic diversity and physiological adaptation to the different agrosystems, including the symbiotic association with rhizobia. Despite the reduction in the production and consumption of grain legumes in Southern Europe, the variability of common bean, runner bean and cowpea landraces in this region is adequately preserved ex situ in germplasm banks and in breeder collections in Portugal, Spain, Italy and Greece; however, on-farm (in situ) conservation in isolated areas mainly in gardens and small fields for farmers own consumption and local markets is not guaranteed. This variability can be used for the genetic improvement of varieties, which will result in environmental-friendly improved legumes for a sustainable production in the South of Europe as well as in other regions of the World.

Soil Selenium (Se) Biofortification Changes the Physiological, Biochemical and Epigenetic Responses to Water Stress in Zea mays L. by Inducing a Higher Drought Tolerance.

Requiring water and minerals to grow and to develop its organs, Maize (Zea mays L.) production and distribution is highly rainfall-dependent. Current global climatic changes reveal irregular rainfall patterns and this could represent for maize a stressing condition resulting in yield and productivity loss around the world. It is well known that low water availability leads the plant to adopt a number of metabolic alterations to overcome stress or reduce its effects. In this regard, selenium (Se), a trace element, can help reduce water damage caused by the overproduction of reactive oxygen species (ROS). Here we report the effects of exogenous Se supply on physiological and biochemical processes that may influence yield and quality of maize under drought stress conditions. Plants were grown in soil fertilized by adding 150 mg of Se (sodium selenite). We verified the effects of drought stress and Se treatment. Selenium biofortification proved more beneficial for maize plants when supplied at higher Se concentrations. The increase in proline, K concentrations and nitrogen metabolism in aerial parts of plants grown in Se-rich substrates, seems to prove that Se-biofortification increased plant resistance to water shortage conditions. Moreover, the increase of SeMeSeCys and SeCys2 forms in roots and aerial parts of Se-treated plants suggest resistance strategies to Se similar to those existing in Se-hyperaccumulator species. In addition, epigenetic changes in DNA methylation due to water stress and Se treatment were also investigated using methylation sensitive amplified polymorphism (MSAP). Results suggest that Se may be an activator of particular classes of genes that are involved in tolerance to abiotic stresses. In particular, PSY (phytoene synthase) gene, essential for maintaining leaf carotenoid contents, SDH (sorbitol dehydrogenase), whose activity regulates the level of important osmolytes during drought stress and ADH (alcohol dehydrogenase), whose activity plays a central role in biochemical adaptation to environmental stress. In conclusion, Se-biofortification could help maize plants to cope with drought stress conditions, by inducing a higher drought tolerance.

Enhancing Legume Ecosystem Services through an Understanding of Plant-Pollinator Interplay.

Legumes are bee-pollinated, but to a different extent. The importance of the plant-pollinator interplay (PPI), in flowering crops such as legumes lies in a combination of the importance of pollination for the production service and breeding strategies, plus the increasing urgency in mitigating the decline of pollinators through the development and implementation of conservation measures. To realize the full potential of the PPI, a multidisciplinary approach is required. This article assembles an international team of genebank managers, geneticists, plant breeders, experts on environmental governance and agro-ecology, and comprises several sections. The contributions in these sections outline both the state of the art of knowledge in the field and the novel aspects under development, and encompass a range of reviews, opinions and perspectives. The first three sections explore the role of PPI in legume breeding strategies. PPI based approaches to crop improvement can make it possible to adapt and re-design breeding strategies to meet both goals of: (1) optimal productivity, based on an efficient use of pollinators, and (2) biodiversity conservation. The next section deals with entomological aspects and focuses on the protection of the "pest control service" and pollinators in legume crops. The final section addresses general approaches to encourage the synergy between food production and pollination services at farmer field level. Two basic approaches are proposed: (a) Farming with Alternative Pollinators and (b) Crop Design System.

Molecular polymorphism related to flowering trait variation in a Phaseolus vulgaris L. collection.

The aim of this study was to investigate the flowering variation and the molecular polymorphism in key regulatory genes that control flowering in a Phaseolus vulgaris L. collection of 94 accessions from Europe and the Americas. The analysis of variance revealed that the difference in days-to-flowering between accessions was significant, with European accessions characterized by flowering precocity. Population structure analysis corroborated previous data on the genetic distinction between the Andean and Mesoamerican gene pools. A low level of admixture was detected. Genomic sequences of 15 gene fragments were obtained. About 7.0 kb per accession were sequenced and a total of 48 nucleotide substitutions identified. A Mixed Linear Model analysis, including population structure and kinship, was used to identify marker-trait associations. Haplotype tagging single nucleotide polymorphisms (htSNPs) associated with the studied traits were detected: in PvVRN1 and PvPHYB with days-to-flowering, in PvMYB29 with number of flower buds per inflorescence and in PvTFL1z and PvFCA with inflorescence length. The two genes associated with days-to-flowering control belong to the photoperiod and vernalization pathways. In particular, the PvVRN1 gene appears to play an important role in regulating the adaptation process of common bean.

European Phaseolus coccineus L. landraces: population structure and adaptation, as revealed by cpSSRs and phenotypic analyses.

Relatively few studies have extensively analysed the genetic diversity of the runner bean through molecular markers. Here, we used six chloroplast microsatellites (cpSSRs) to investigate the cytoplasmic diversity of 331 European domesticated accessions of the scarlet runner bean (Phaseolus coccineus L.), including the botanical varieties albiflorus, bicolor and coccineus, and a sample of 49 domesticated and wild accessions from Mesoamerica. We further explored the pattern of diversity of the European landraces using 12 phenotypic traits on 262 individuals. For 158 European accessions, we studied the relationships between cpSSR polymorphisms and phenotypic traits. Additionally, to gain insights into the role of gene flow and migration, for a subset of 115 accessions, we compared and contrasted the results obtained by cpSSRs and phenotypic traits with those obtained in a previous study with 12 nuclear microsatellites (nuSSRs). Our results suggest that both demographic and selective factors have roles in the shaping of the population genetic structure of the European runner bean. In particular, we infer the existence of a moderate-to-strong cytoplasmic bottleneck that followed the expansion of the crop into Europe, and we deduce multiple domestication events for this species. We also observe an adaptive population differentiation in the phenology across a latitudinal gradient, which suggests that selection led to the diversification of the runner bean in Europe. The botanical varieties albiflorus, bicolor and coccineus, which are based solely on flower colour, cannot be distinguished based on these cpSSRs and nuSSRs, nor according to the 12 quantitative traits.

Evidence for introduction bottleneck and extensive inter-gene pool (Mesoamerica x Andes) hybridization in the European common bean (Phaseolus vulgaris L.) germplasm.

Common bean diversity within and between Mesoamerican and Andean gene pools was compared in 89 landraces from America and 256 landraces from Europe, to elucidate the effects of bottleneck of introduction and selection for adaptation during the expansion of common bean (Phaseolus vulgaris L.) in Europe. Thirteen highly polymorphic nuclear microsatellite markers (nuSSRs) were used to complement chloroplast microsatellite (cpSSRs) and nuclear markers (phaseolin and Pv-shatterproof1) data from previous studies. To verify the extent of the introduction bottleneck, inter-gene pool hybrids were distinguished from "pure" accessions. Hybrids were identified on the basis of recombination of gene pool specific cpSSR, phaseolin and Pv-shatterproof1 markers with a Bayesian assignments based on nuSSRs, and with STRUCTURE admixture analysis. More hybrids were detected than previously, and their frequency was almost four times larger in Europe (40.2%) than in America (12.3%). The genetic bottleneck following the introduction into Europe was not evidenced in the analysis including all the accessions, but it was significant when estimated only with "pure" accessions, and five times larger for Mesoamerican than for Andean germplasm. The extensive inter-gene pool hybridization generated a large amount of genotypic diversity that mitigated the effects of the bottleneck that occurred when common bean was introduced in Europe. The implication for evolution and the advantages for common bean breeding are discussed.

Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2012 - 31 March 2012.

This article documents the addition of 171 microsatellite marker loci and 27 pairs of single nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bombus pauloensis, Cephalorhynchus heavisidii, Cercospora sojina, Harpyhaliaetus coronatus, Hordeum vulgare, Lachnolaimus maximus, Oceanodroma monteiroi, Puccinia striiformis f. sp. tritici, Rhea americana, Salmo salar, Salmo trutta, Schistocephalus solidus, Sousa plumbea and Tursiops aduncus. These loci were cross-tested on the following species: Aquila heliaca, Bulweria bulwerii, Buteo buteo, Buteo swainsoni, Falco rusticolus, Haliaeetus albicilla, Halobaena caerulea, Hieraaetus fasciatus, Oceanodroma castro, Puccinia graminis f. sp. Tritici, Puccinia triticina, Rhea pennata and Schistocephalus pungitii. This article also documents the addition of 27 sequencing primer pairs for Puffinus baroli and Bulweria bulwerii and cross-testing of these loci in Oceanodroma castro, Pelagodroma marina, Pelecanoides georgicus, Pelecanoides urinatrix, Thalassarche chrysostoma and Thalassarche melanophrys.

Genetic diversity, structure and marker-trait associations in a collection of Italian tomato (Solanum lycopersicum L.) landraces.

The study of phenotypic and genetic diversity in landrace collections is important for germplasm conservation. In addition, the characterisation of very diversified materials with molecular markers offers a unique opportunity to define significant marker-trait associations of biological and agronomic interest. Here, 50 tomato landraces (mainly collected in central Italy), nine vintage and modern cultivars, and two wild outgroups were grown at two locations in central Italy and characterised for 15 morpho-physiological traits and 29 simple sequence repeat (SSR) loci. The markers were selected to include a group of loci in regions harbouring reported quantitative trait loci (QTLs) that affect fruit size and/or shape (Q-SSRs) and a group of markers that have not been mapped or shown to have a priori known linkage (NQ-SSRs). As revealed by univariate and multivariate analyses of morphological data, the landraces grouped according to vegetative and reproductive traits, with emphasis on fruit size, shape and final destination of the product. Compared to the low molecular polymorphism reported in tomato modern cultivars, our data reveal a high level of molecular diversity in landraces. Such diversity has allowed the inference of the existence of a genetic structure that was factored into the association analysis. As the proportion of significant associations is higher between the Q-SSR subset of markers and the subset of traits related to fruit size and shape than for all of the other combinations, we conclude that this approach is valid for establishing true-positive marker-trait relationships in tomato.

On-going on-farm microevolutionary processes in neighbouring cowpea landraces revealed by molecular markers.

A knowledge of existing levels of diversity is fundamental for planning in situ (on-farm) conservation activities. Three neighbouring cowpea landraces (LRs) currently cultivated in central Italy were studied by amplified fragment length polymorphism (AFLP) and selectively amplified microsatellite polymorphic locus (SAMPL) markers to determine the distribution of genetic variation within and among them. The three LRs studied, although relatively similar, are highly different from one another as shown by the significance of the Fisher exact test for the genic differentiation and the absence of genotype sharing among them. Data obtained from the AFLP and SAMPL markers separately and their combined data revealed a relatively high level of diversity still present within the LRs. The more efficient SAMPL technique was better at discriminating between the plants than the AFLP markers. The three LRs studied appear to be structured as a metapopulation in which a substantial differentiation is maintained at the subpopulation level. A complex interaction of factors (drift, LR isolation, farmer selection, migration within LRs) explains the observed pattern of diversity. The results suggest that the best strategy for maintaining diversity in the area is to preserve each of the LRs observed on the farms from which it came.

Efficiency of three PCR-based markers in assessing genetic variation among cowpea (Vigna unguiculata subsp. unguiculata) landraces.

The main objective of this study was to investigate the efficiency of RAPD, AFLP, and SAMPL marker systems in detecting genetic polymorphism in cowpea landraces (Vigna unguiculata subsp. unguiculata (L.) Walp.) that probably share a similar genetic pool. A second objective was to determine the level of diversity among landraces from a restricted area, to define the most appropriate strategy of on-farm conservation. Each marker system was able to discriminate among the materials analysed, but a clear distinction between all the local varieties was only obtained with AFLP and SAMPL markers. The average diversity index was quite similar for each marker system, but owing to the differences in the effective multiplex ratio values the marker index was higher for the AFLP and SAMPL systems than for the RAPD system. The AFLP and SAMPL techniques appear to be more useful than the RAPD technique in the analysis of limited genetic diversity among the cowpea landraces tested. The significant correlations of SAMPL similarity and cophenetic matrices with those of the other markers, and the lower number of primer combinations required, indicate that this technique is the most valuable. The low genetic similarity detected among landraces suggests that all the cowpea landraces should be maintained on the respective farms from which they came.