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The results of the exploration missions for the gathering of legume germplasm in Monti Dauni district (southern Italy) are presented. The 52% of collected samples were common beans (Phaseolus vulgaris L.), mainly white seeded types. Information acquired in the frame of different research areas, such as passport data, GIS data, historical documents on crop cultivation in the study district, were integrated data with orthophotos and satellite images. This interdisciplinary approach allowed to obtain a multidimensional data set for the collected local varieties. A similar holistic approach permits a better knowledge of the link between the local varieties and the traditional area of cultivation, as well as the contextualization of their cultivation in both space and time. The merger of different information overcomes the limits derived from the use of a single methodology and might support local communities and policy-makers in the planning of effective actions aimed to safeguard the autochthonous germplasm.

Genotyping by sequencing (GBS) was used to analyze relationships among cowpea and asparagus bean landraces from southern Italy and to assess the utility of this technology to study taxonomy in a wider panel, including V. unguiculata cultigroups, subspecies, and other Vigna species. The analysis of SNPs derived from GBS highlighted that, among the cowpea landraces, the African samples were separated from the other material, while, for the Italian landraces, a certain clustering depending on seed color/pattern was observed in the dendrogram. When examining the V. unguiculata species complex, a clear separation between the two groups of wild subspecies, i.e., the allogamous wild perennials and the perennial out/inbreds, could be observed, the former representing the more ancestral wild progenitors of V. unguiculata. The species V. vexillata appeared more closely related to V. unguiculata than to the other Vigna species analyzed.

Cowpea (Vigna unguiculata (L.) Walp.) is a major tropical pulse in a number of countries and especially in sub-Saharan Africa where this species was domesticated. Although as niche product, a remarkable number of cowpea landraces are cultivated in the Mediterranean basin, primarily for human consumption of the grains and fresh pods. In this study, within the frame of a regional project devoted to the safeguard and management of local horticultural genetic resources, some germplasm explorations were carried out in Apulia region (southern Italy) to collect, among other crops, cowpea landraces still present in the area and promote on-farm conservation of this traditional material. A second objective was to investigate the efficiency of SSR markers in detecting genetic polymorphism in cowpea landraces from a restricted geographical area. In total, 13 samples of V. unguiculata were gathered, 12 of cultivar group unguiculata and 1 of cultivar group sesquipedalis. Genetic diversity and phylogenetic relationships among 13 Apulian accessions, 9 samples from various provenances and 1 var. spontanea accession were evaluated using SSR molecular markers. A set of 19 SSR primer pairs successfully identified closely related accessions and were able to separate the different cultivar groups of V. unguiculata.

There is an increasing attention towards common bean due to its health benefits, prevention to human diseases and as ingredient for functional or fortified foods. Phosphorus, an essential element for plant growth, is mainly stored in seeds as phytic acid (Phy). Phy is negatively associated with mineral bioavailability, but, at the same time, is a natural antioxidant. Accumulation and partitioning of phosphorus were analysed in seeds of ten Italian common bean landraces for three subsequent growing seasons. Some important seed quality traits were also evaluated. For comparative purposes, the landrace harvests of two growing locations were analysed. A wide variation of total and phytic phosphorus contents was recorded among the landraces. Moreover, P accumulation and partitioning between Phy and inorganic P, as well as seed quality traits, resulted strongly affected by growing location. Statistically significant increases of Phy levels were recorded for harvests obtained outside the traditional area of cultivation. These results highlight how the cultivation of a landrace outside of its traditional area will appreciably affect harvest quality.

The common bean (Phaseolus vulgaris L.) is one of the main legumes worldwide and represents a valuable source of nutrients. Independent domestication events in the Americas led to the formation of two cultivated genepools, namely Mesoamerican and Andean, to which European material has been brought back. In this study, Italian common bean landraces were analyzed for their genetic diversity and structure, using single nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing (GBS) technology. After filtering, 11,866 SNPs were obtained and 798 markers, pruned for linkage disequilibrium, were used for structure analysis. The most probable number of subpopulations (K) was two, consistent with the presence of the two genepools, identified through the phaseolin diagnostic marker. Some landraces were admixed, suggesting probable hybridization events between Mesoamerican and Andean material. When increasing the number of possible Ks, the Andean germplasm appeared to be structured in two or three subgroups. The subdivision within the Andean material was also observed in a principal coordinate analysis (PCoA) plot and a dendrogram based on genetic distances. The Mesoamerican landraces showed a higher level of genetic diversity compared to the Andean landraces. Calculation of the fixation index (FST) at individual SNPs between the Mesoamerican and Andean genepools and within the Andean genepool evidenced clusters of highly divergent loci in specific chromosomal regions. This work may help to preserve landraces of the common bean from genetic erosion, and could represent a starting point for the identification of interesting traits that determine plant adaptation.

Genetic relationships, agronomic, nutritional and technological traits of ten Italian landraces, two improved lines and two cultivars of lentil (Lens culinaris Medik.) were investigated using a multi-disciplinary approach. Seed storage proteins, used as biochemical markers, were able to detect polymorphisms with variability mainly related to the polypeptide abundance. Microsatellite (SSR) molecular markers provided very useful information on genetic variation and relationships among landraces, with polymorphic fragments able to discriminate all the accessions. Lentil landraces were grouped in different clusters and sub-clusters principally on the basis of their geographical origin. The highest levels of genetic diversity were observed for lentils from ‘Castelluccio di Norcia’, ‘Colliano’ and ‘Villalba’. Field trials, performed in two locations of Southern Italy, revealed a high influence of location on yield. Comparing performances at both tested locations, the best landraces were ‘Linosa’ and ‘Valle di Nevola’ suggesting that these have the highest adaptability. Technological and nutritional data together with the agronomic ones evidenced that ‘Linosa’ lentil is the best landrace, however also ‘San Gerardo’ deserves some attention.

This study was carried out to estimate the level of diversity existing within some common bean landraces still cultivated in Nebrodi mountains, North-western area of Sicily. The multidisciplinary approach adopted to reach this goal involved the characterisation of collected material through morphological, biochemical and molecular marker analyses. The nutritional quality of seeds was also investigated in view of the proposition of the best landraces as niche products. Results showed that those bean landraces retain a considerable level of heterogeneity. The use of both biochemical and molecular markers showed that all landraces clustered into two main groups, corresponding to the Andean and Mesoamerican gene pools. Our results suggest that the best strategy for preserving the diversity of common bean from a restricted area such as Nebrodi mountains, necessitates of a deep knowledge of germplasm to avoid the loss of precious genetic resources or, on the contrary, the safeguard of populations genetically redundant.

The primary structure of the double-headed Bowman-Birk inhibitor (BBI) family and the antitryptic activity were investigated in cultivated and wild Phaseolus species. Two BBI types were identified; the first one inhibits trypsin and chymotrypsin (tc-BBI), the second one elastase and trypsin (et-BBI). Only tc-BBI was found in P. lunatus and P. parvulus, while none of BBI types, identified in this study, was found in P. leptostachyus. The deduced amino acid sequences revealed some polymorphisms within both tc-BBI and et-BBI binding loops that could affect the inhibitory activity. The trypsin inhibitor content showed a high variation with the lowest value recorded in P. lepthostachyus and the highest one observed in P. oligospermus. Southern blot analysis confirmed the absence of both BBI types in P. leptostachyus and suggests that in P. coccineus, P. dumosus and P. costaricensis, the two genes were clustered in a narrow genomic region of 1.3kbp.

A BAC library from common bean has been used in order to isolate the entire multigene Bowman-Birk serine protease inhibitor family and to study its genome organization. Using a previously isolated trypsin/chymotrypsin inhibitor nucleotide sequence as probe, two positive BAC clones were identified. The P2B8 BAC clone, of about 135 kbp and containing the complete BBI family, was chosen and partially sequenced. Our results confirm that a small multigene family codes for three double-headed inhibitors named: tc-BBI-1, tc-BBI-2 and et-BBI. They contain the binding loop trypsin/chymotrypsin (tc-BBI-1 and tc-BBI-2) and the elastase/trypsin one (et-BBI), respectively. Genes coding for tc-BBI-1 and et-BBI, were found to be very close to each other and arranged in a head to head fashion. Southern blot hybridisation on genomic DNA digested with PstI enzyme suggests that all three genes are present in a fragment of 19 kbp. Northern blot analyses on RNA isolated from various common bean organs showed that the expression of tc-BBI-1 and et-BBI was restricted to the developing cotyledons.

This study was undertaken to estimate the level of variation among and within 33 local populations belonging to seven Italian common bean landraces, by analysing the polymorphism of seed storage proteins, simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) molecular markers. The nutritional seed quality of the landraces studied was also investigated. Results showed that the landraces retain a considerable level of heterogeneity. Use of both biochemical and molecular approaches provided a picture of the genetic diversity of each landrace. Cluster analyses based on Nei's genetic distances and Jaccard's similarity index as defined by SSR and AFLP markers, respectively, showed that all populations clustered into two groups corresponding to the Andean and the Mesoamerican gene pools. Knowledge of the genetic structure of a landrace is fundamental in elaborating strategies, which involve the local farmers, allowing us to improve and, at the same time, safeguard the genetic integrity of landrace genetic resources.

Digoxigenated synthetic oligonucleotides complementary tosimple repetitive DNA sequences were used to detect polymorphisms inPhaseolus lunatus L. (Lima bean)genotypes. Twenty two accessions, including wild and cultivatedrepresentatives of the Mesoamerican and Andean gene pools andintermediate forms were included in this study. It could be shownthat five repetitive motifs, namely (AAT)5,(ATG)5,(GACA)4,(GATA)4,(GGAT)4, were able to give clear-cutbands when used as probes and to reveal high levels of variation. Thehighest resolving power was revealed by the(AAT)5 probe, which was able to detect eveninter-individual polymorphisms. Therefore, oligonucleotidefingerprinting has proved to be a useful tool to analyse geneticdiversity in Lima bean. Phylogenetic reconstruction based onJACCARD's similarity index confirmed the existence of two majorgene pools and the presence of a minor group of genetically separatedintermediate genotypes.

Lectin-related polypeptides are a class of defence proteins found in seeds of Phaseolus species. In Lima bean (P. lunatus), these proteins and their genes have been well characterized in the Andean morphotype, which represents one of the two gene pools of this species. To study the molecular evolution of the lectin family in Lima bean we characterized the polypeptides belonging to this multigene family and cloned the genes belonging to the Mesoamerican gene pool. The latter gene pool contains components similar to those of the Andean pool, namely: an amylase inhibitor-like (AIL), an arcelin-like (ARL) lectin and the less abundant Lima bean lectin (LBL). These proteins originate from an ancestor gene of the lectin type which duplicated to yield the lectin gene and the progenitor of ARL and AIL. In this species. ARL represents an evolutionary intermediate form that precedes AIL. Phylogenetic analysis supports an Andean origin for Lima bean. The molecular evolutionary studies were extended to the genes of common bean and demonstrated that true lectin genes and the ancestor of lectin-related genes are the result of a duplication event that occurred before speciation. Lima and common bean followed different evolutionary pathways and in the latter species a second duplication event occurred that gave rise, in Mesoamerican wild genotypes, to arcelin genes.

Variation of the lectin and the two lectin-related proteins, AIL (α-amylase inhibitor-like) and ARL (arcelin-like) was examined in wild and cultivated accessions of Lima bean (Phaseolus lunatus L.) using electrophoresis of total seed proteins, immunoblot and RFLP analysis of lectin-related genes. Results confirm that divergence of the two major Lima bean gene pools, Andean and Mesoamerican, also apply to this protein family. All three members of the family are present in both gene pools, with differences in size, abundance and composition between gene pools, giving the possibility to distinguish Andean from Mesoamerican lectin pattern types. Both patterns show some variants, such as lack of lectin or its presence as an abundant protein. The observed variation reflects, at least in part, into genomic polymorphism. The presence of arcelin- and α-amylase-related proteins in Lima bean could represent a tool to increase our knowledge in the evolution of the lectin family in Phaseolus species.

In this chapter, we lead the reader through several topics related to common bean germplasm, including crop dissemination across the Old World, landraces developed by farmers, characterization and safeguard of germplasm. These topics are attracting a great deal of attention, especially in recent years, when the international community has become increasingly aware of the relevance of safeguarding plant genetic resources. Early studies were mainly aimed at performing exploratory analyses of agro-morphological traits, with the goal of improving yield and pest resistance. Moreover, phaseolin variation was studied to check the distribution of the two common bean gene pools. The recent development of DNA-based markers has allowed significant improvement in knowledge on genetic variation within the European germplasm, as well as the capacity to trace its divergence from the American germplasm. The different strategies applied to safeguard the European germplasm are described together with their drawbacks.

Phaseolin is the most abundant storage protein of bean seeds. To modify its amino-acidic composition by protein engineering, for the improvement of its nutritional value, regions which could be modified without detrimental effects on structural features of the protein must be identified. Data presented here, on the characterisation of the major storage protein of lima bean (Phaseolus lunatus L.) seeds, a phaseolin-like glycoprotein, provide good indications on one of such region. Phaseolus lunatus phaseolin consists of four major oligomers containing two subunit classes. Polypeptides of one class show a molecular mass ranging from 38.5 kDa to 32 kDa, while the molecular mass of polypeptides belonging to the other class ranges from 27 kDa to 21 kDa. The subunits originate from the cleavage of precursor forms, with molecular masses of 58 kDa and 54 kDa, which are still present — in residual amounts — in the mature protein. Comparison of their N-terminal sequences with those of the subunits demonstrate that cleavage occurs in a region of the molecule that instead remains uncleaved in phaseolins of the other species. Since this region can accommodate such a drastic modification, we suggest it could be a good candidate for in vitro manipulation.

Ectendomycorrhizae of Arbutus unedo L. were synthesized in the laboratory and were observed over the first 80 days of development. After 40 days some roots showed many hyphae running around enlarged tips, on the 60th day short roots had assumed a clavate form, and on the 80th day mycorrhizae could be observed with the naked eye. The laboratory-synthesized ectendomycorrhizae had a cruciform structure and were smooth, shiny, and pale orange-brown. They presented a loose sheath and a Hartig net. The first layer of cortical root cells was invaded by the fungus. Running hyphae and vesicles of Glomus microcarpum invaded A. unedo roots, but no root invasion was observed in the presence of five other vesicular–arbuscular mycorrhizal fungal species. The ericoid mycorrhizal fungus Hymenoscyphus ericae caused a darkening of cortical cells and root tips and the death of many seedlings. The correspondence between specific plant species and particular kinds of mycorrhizae and the possibility of intracellular colonization by nonspecific fungi are also discussed.

Genes for lectin, a component of legume storage proteins, were identified and characterised in two lentil cultivars ( Lens culinaris ssp. culinaris) and six wild relatives. In each taxon no differences were found among the two or three lectin clones sequenced, while differences were observed among lectin genes isolated from the different taxa. All of the clones analysed contained an insert of 828 bp and showed a high similarity with the nucleotide sequence of Pisum sativum seed lectin, PSL1. The deduced amino acid lectin sequences in all taxa were 275 amino acids long, and their multiple alignment showed that most of the variation among them occurred in regions which are not important for metal- and sugar-binding. The data from Southern blot analysis indicated the presence of only one lectin gene in all Lens taxa except L. tomentosus. Phylogenetic analyses carried out on the lectin sequences showed the existence of two main clusters and clearly indicated that L. nigricans falls outside the two groups.

Variation of phaseolin, the major storage protein in Phaseolus vulgaris L., was analysed in seeds of a germplasm collection from the Mediterranean area. Results showed a number of subvariants differing from the reference patterns for the presence, absence or faintness of single polypeptides. Maximum concentration of variation was found in the Cyprus material. Sources and causes of variation are discussed.

The origin of the molecular heterogeneity of phaseolin was investigated by studying, both in vivo and in vitro, the synthesis and processing of four different banding types of phaseolin in five cultivars of Phaseolus vulgaris L. The results demonstrate: I) Newly-synthesized (unprocessed) phaseolin in all cultivars is composed of three major components. These differ between cultivars, both in charge and Mr. II) The processing of these precursors is highly conserved and consists of the co-translational cleavage of a signal peptide, two glycosylation steps in the endoplasmic reticulum and a further modification inside the protein bodies to give the mature form. III) Some of the molecular heterogeneity of each phaseolin banding type is due to a different extent of glycosylation of its polypeptide components.

3H-thymidine incorporation and DNA-polymerase activity during early hours of wheat embryo germination at two viability levels have been studied. The patterns of two biosynthetic activities, as well as the dependence of DNA synthesis on protein synthesis, indicated the presence of a delay in the early phase of imbibition of the aged embryos with respect to viable germs.

Fusicoccin inhibits the germination of pea (Pisum sativum L. cv Progress 9) seeds by decreasing the growth of the embryonal axis and by stimulating the fresh weight increase of the cotyledons. The growth of isolated embryonal axes in the presence of sucrose and KCl is stimulated by fusicoccin. The effect of fusicoccin on the seeds is not counteracted by sucrose and KCl. Fusicoccin promotes preferentially in the cotyledons a hyperpolarization of the transmembrane electric potential and an increase in the uptake capacity, suggesting the reinforcement of the sink strength of the cotyledons in comparison with the one of the embryonal axis and therefore the inhibition of translocation from the cotyledons of some substance necessary for the growth of the embryonal axis.

Phaseolin variability was analyzed in 86 wild Phaseolus lunatus accessions, representing a wide geographical distribution from Mexico to Argentina, to determine geographical patterns of variability. The collection can be divided into two major groups. One group consists of accessions from Mexico, Central America, Cuba, Columbia, Argentina showing two Mesoamerican (M) phaseolin pattern types, already described in cultivated materials, as well as a variety of additional banding patterns. This suggests that at least two genotypes in the Mesoamerican gene pool were involved in a domestication process and spread throughout the world. The other group, consisting of wild Andean (A) forms from Northern Peru and belonging to the Andean gene pool, showed, among others, two A pattern types comparable to those found in cultivated large-seeded accessions.

Protease inhibitors (PIs) are widely distributed in plant kingdom and are present in significant amounts in pulse seeds. PIs are grouped in families and superfamilies or clans. The enzyme specificity and inhibitory activity of each inhibitor type depend on the amino acidic residues constituting the reactive binding loop. PIs interfere with the digestive process inhibiting the activity of enzymes such that hydrolyse the proteins into more short peptides. For this reason, they are considered antinutritional compounds and produce adverse effects in insects and animals eating raw seeds. Kunitz trypsin inhibitors (KTIs) and Bowman-Birk inhibitors (BBIs) are the predominant PIs in legume seeds. As consequence of the legume nutritional relevance at worldwide level, both these inhibitors elicited a very high number of studies. BBIs are the prominent PI type present in Phaseolus spp. seeds. They consist of small proteins with molecular mass around 8-9 kDa with approximately 100 amino acids and a high number of cysteine residues. BBIs are double headed inhibitors being able to bind at the same time two identical or different proteinases (trypsin/trypsin, trypsin/chymotrypsin or elastase/trypsin). In common bean (Phaseolus vulgaris L.), BBIs are encoded by a small multigene family composed by three double-headed inhibitors. Genome analysis has showed that all three BBIs clustered at a single locus on chromosome 4. Although, micro-heterogeneity has been described within and among cultivated and wild Phaseolus species, the deduced amino acidic sequences result highly conserved. Moreover, the gene sequences of BBIs has a high similarity with those of other pulses. BBI purified by some Phaseolus species showed high stability to heat and maintenance of activity over a broad range of pH. Conversely, the inhibitory activity of whole seed is significantly reduced (over 80%) by cooking. Otherwise, the BBI content shows a high variation within and among wild and cultivated Phaseolus spp. with higher values mainly associated to the wild species. Drought stress suffered by plant during the vegetative growth stage produces an appreciable increase of BBI level in common bean seeds

Genetic variation inPhaseolus lunatus (Lima bean) was investigated at isozyme and DNA levels. Sixty cultivated accessions, including representatives of the Mesoamerican and Andean gene pools and intermediate types, were analyzed for variability at 17 isozyme loci. Some accessions were also examined for restriction fragment length polymorphism (RFLP) at the rDNA level. These data were used to construct two dendrograms showing clear separation in two distinct groups corresponding to each of the gene pools and an intermediate one probably representing a transitional group.