*) Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, Via Brecce Bianche, 60131 Ancona (Italy) **) Department of Agriculture, University of Sassari, Via E. de Nicola, 07100 Sassari (Italy) ***) Institute of Life Sciences, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà 33, 56127 Pisa (Italy) ****) Department of Biotechnology, University of Verona, 37134 Verona (Italy) *****) Scuola di Scienze Agrarie, Forestali, Alimentari ed Ambientali, Università degli Studi della Basilicata, Potenza (Italy)

Complete List of Authors: Rodriguez, Monica; Università degli Studi di Sassari, Dipartimento di Agraria Rau, Domenico; Università degli Studi di Sassari, Dipartimento di Agraria Bitocchi, Elena; Università Politecnica delle Marche, Dipartimento di Scienze Agrarie, Alimentari ed Ambientali Bellucci, Elisa; Università Politecnica delle Marche, Dipartimento di Scienze Agrarie, Alimentari ed Ambientali Biagetti, Eleonora; Università Politecnica delle Marche, Dipartimento di Scienze Agrarie, Alimentari ed Ambientali Carboni, Andrea; Consiglio per la ricerca e la sperimentazione in agricoltura (CRA-CIN), Centro di Ricerca per le Colture Industriali Gepts, Paul; University of California, Department of Plant Sciences / MS1 Nanni, Laura; Università Politecnica delle Marche, Dipartimento di Scienze Agrarie, Alimentari ed Ambientali Papa, Roberto; Università Politecnica delle Marche, Dipartimento di Scienze Agrarie, Alimentari ed Ambientali Attene, Giovanna; Università degli Studi di Sassari, Dipartimento di Agraria; Università degli Studi di Sassari, Centro per la Conservazione e Valorizzazione della Biodiversità Vegetale

In this study, new single nucleotide polymorphism (SNP) markers were developed for common bean (Phaseolus vulgaris L.) and related Phaseolus species. The applied strategy presents new and interesting aspects, such as the choice of accessions used, which was aimed at capturing a large portion of the genetic diversity present in the common bean, with particular focus on wild and domesticated materials from Mesoamerica and the identification of loci for sequencing. Indeed, the primer pairs for 34 loci were designed with the main strategy being to search for single-copy orthologous genes among the legumes (for use in other legume species and comparative analyses). The 10 remaining loci were selected as being near to domestication quantitative trait loci or detected as putatively under selection during domestication in previous studies. To provide an efficient and inexpensive genotyping platform for geneticists and breeders, we used sequence data to develop 60 new SNP markers for KASPar assay genotyping. The same sample was also genotyped with SNP markers developed for common bean in other studies for the same assay. This allowed testing for systematic bias according to the criteria chosen to select the genotypes in which the genetic diversity is surveyed during SNP discovery. Finally, we show that most of the SNP markers worked well in a set of accessions of other species belonging to the Phaseolus genus. The genetic resources developed will be very useful not only for breeding, but also for biodiversity conservation management and evolutionary studies on legumes.

Evolutionary studies in plant and animal breeding are aimed at understanding the structure and organization of genetic variations of species. We have identified and characterized a genomic sequence in Phaseolus vulgaris of 1,200 bp (PvSHP1) that is homologous to SHATTERPROOF-1 (SHP1), a gene involved in control of fruit shattering in Arabidopsis thaliana. The PvSHP1 fragment was mapped to chromosome Pv06 in P. vulgaris and is linked to the flower and seed color gene V. Amplification of the PvSHP1 sequence from the most agronomically important legume species showed a high degree of interspecies diversity in the introns within the Phaseoleae, while the coding region was conserved across distant taxa. Sequencing of the PvSHP1 sequence in a sample of 91 wild and domesticated genotypes that span the geographic distribution of this species in the centers of origin showed that PvSHP1 is highly polymorphic and, therefore, particularly useful to further investigate the origin and domestication history of P. vulgaris. Our data confirm the gene pool structure seen in P. vulgaris along with independent domestication processes in the Andes and Mesoamerica; they provide additional evidence for a single domestication event in Mesoamerica. Moreover, our results support the Mesoamerican origin of this species. Finally, we have developed three indel-spanning markers that will be very useful for bean germplasm characterization, and particularly to trace the distribution of the domesticated Andean and Mesoamerican gene pools.

Here, we present a brief overview of the main studies conducted on the common bean (Phaseolus vulgarisL.) in Europe and other countries outside its centres of origin. We focus on the proportions of the Andean and Mesoamerican gene pools, and on the inter-gene pool hybridization events. In Europe, for chloroplast microsatellites, 67% of European germplasm is of Andean origin. Within Europe, interesting trends have been seen; indeed, the majority of the Andean type is found in the three macro-areas of the Iberian Peninsula, Italy and central-northern Europe, while, in eastern and south-eastern Europe, the proportion of the Mesoamerican type increased. On a local scale, the contribution of the Mesoamerican type is always low. On other continents, various situations are seen using different markers: in China and Brazil, the Mesoamerican gene pool prevails, while in an African sample, overall, both gene pools are equally represented, with differences in individual countries. The frequency of European bean genotypes deriving from at least one hybridization event was 44% with an uneven distribution. Interestingly, hybrids tend to have intermediate seed size in comparison with ‘pure’ Andean or Mesoamerican types. On other continents, very few hybrids are found, probably because of the different marker systems used.

Evolutionary studies in plant and animal breeding are aimed at understanding the structure and organization of genetic variations of species. We have identified and characterized a genomic sequence in of 1,200 bp ( that is homologous to (), a gene involved in control of fruit shattering in The fragment was mapped to chromosome Pv06 in and is linked to the flower and seed color gene . Amplification of the sequence from the most agronomically important legume species showed a high degree of interspecies diversity in the introns within the Phaseoleae, while the coding region was conserved across distant taxa. Sequencing of the sequence in a sample of 91 wild and domesticated genotypes that span the geographic distribution of this species in the centers of origin showed that is highly polymorphic and, therefore, particularly useful to further investigate the origin and domestication history of . Our data confirm the gene pool structure seen in along with independent domestication processes in the Andes and Mesoamerica; they provide additional evidence for a single domestication event in Mesoamerica. Moreover, our results support the Mesoamerican origin of this species. Finally, we have developed three indel-spanning markers that will be very useful for bean germplasm characterization, and particularly to trace the distribution of the domesticated Andean and Mesoamerican gene pools.

Wild legumes constitute an important component of widespread pastures in the Mediterranean basin. This region is experiencing remarkable effects from climate change, and continuous monitoring of species and population dynamics is important in order to plan and enact valuable conservation programmes. Tripodion tetraphyllum (L.) Fourr. [=Anthyllis tetraphylla L.] (2n=16), belongs to the tribe Loteae (Fabaceae), and could be very important for soil protection and sward improvement in abandoned or degraded Mediterranean areas. This alternative pasture legume is very closely related to Lotus japonicus and has some important characteristics for survival of the species in difficult and overgrazed Mediterranean areas. In this study, we have investigated the molecular diversity and population structure of T. tetraphyllum from North Africa using ISSR markers and plastidial microsatellites. To date, this is the first study concerning the genetic diversity and geographic differentiation of T. tetraphyllum. Ninety genotypes from three North African countries were analysed according to ISSRs, cpSSRs and one phenotypic trait. T. tetraphyllum shows a clear geographical structure, with differentiation associated with longitudinal differences; moreover, there is a general reduction in genetic diversity from Morocco to Tunisia. With all the markers used, strong differentiation was seen among collection sites. Our data highlight a genetic diversity gradient and cline of distribution, indicating that T. tetraphyllum has extended its area of distribution from Morocco to Tunisia.

This study focuses on the expansion of Phaseolus vulgaris in Europe. The pathways of distribution of beans into and across Europe were very complex, with several introductions from the New World that were combined with direct exchanges between European and other Mediterranean countries. We have analyzed here six chloroplast microsatellite (cpSSR) loci and two unlinked nuclear loci (for phaseolin types and Pv - shatterproof1 ). We have assessed the genetic structure and level of diversity of a large collection of European landraces of P . vulgaris (307) in comparison to 94 genotypes from the Americas that are representative of the Andean and Mesoamerican gene pools. First, we show that most of the European common bean landraces (67%) are of Andean origin, and that there are no strong differences across European regions for the proportions of the Andean and Mesoamerican gene pools. Moreover, cytoplasmic diversity is evenly distributed across European regions. Secondly, the cytoplasmic bottleneck that was due to the introduction of P. vulgaris into the Old World was very weak or nearly absent. This is in contrast to evidence from nuclear analyses that have suggested a bottleneck of greater intensity. Finally, we estimate that a relatively high proportion of the European bean germplasm (about 44%) was derived from hybridization between the Andean and Mesoamerican gene pools. Moreover, although hybrids are present everywhere in Europe, they show an uneven distribution, with high frequencies in central Europe, and low frequencies in Spain and Italy. On the basis of these data, we suggest that the entire European continent and not only some of the countries therein can be regarded as a secondary diversification center for P. vulgaris . Finally, we outline the relevance of these inter-gene pool hybrids for plant breeding.

Landraces are domesticated local plant varieties that did not experience a deliberate and intensive selection during a formal breeding programme. In Europe, maize landraces are still cultivated, particularly in marginal areas where traditional farming is often practiced. Here, we have studied the evolution of flint maize landraces from central Italy over 50 years of on‐farm cultivation, when dent hybrid varieties were introduced and their use was widespread. We have compared an ‘old’ collection, obtained during the 1950s, before the introduction of hybrids, and a recent collection of maize landraces. For comparison, a sample of maize landraces from north Italy, and of improved germplasm, including hybrids and inbred lines were also used. A total of 296 genotypes were analysed using 21 microsatellites. Our results show that the maize landraces collected in the last 5–10 years have evolved directly from the flint landrace gene pool cultivated in central Italy before the introduction of modern hybrids. The population structure, diversity and linkage disequilibrium analyses indicate a significant amount of introgression from hybrid varieties into the recent landrace populations. No evidence of genetic erosion of the maize landraces was seen, suggesting that in situ conservation of landraces is an efficient strategy for preserving genetic diversity. Finally, the level of introgression detected was very variable among recent landraces, with most of them showing a low level of introgression; this suggests that coexistence between different types of agriculture is possible, with the adoption of correct practices that are aimed at avoiding introgression from undesired genetic sources.

The main aim of this study was to use an AFLP-based, large-scale screening of the whole genome of Phaseolus vulgaris L. to determine the effects of selection on the structure of the genetic diversity in wild and domesticated populations. We first used pooled DNA samples, seven each of wild and domesticated populations of P. vulgaris were studied using 2,506 AFLP markers (on average, one every 250 kb). About 10% of the markers were also analysed on individual genotypes and were used to empirically infer allelic frequencies from bulk data. In both datasets, we tested the departure from neutral expectation for each marker using an F ST -based method. Moreover, we tested with 19 AFLP primer combination a large set of accession from the three known gene pool of P. vulgaris (Andean, Mesoamerican and ancestral) in order to highlight the signature of selection under domestication within and between gene pools. The most important outcome is that a large fraction of the genome of the common bean appears to have been subjected to effects of selection during domestication. We also mapped and classified the markers obtained in individual genotypes according to their proximities to known genes and QTLs of the domestication syndrome. Most of the markers that were found to be potentially under the effects of selection were located in the proximity of previously mapped genes and QTLs related to the domestication syndrome. Overall, our results indicate that domestication appears to have affected not only target genes, but also a large portion of the genome around these genes. These “domestication islands” have probably experienced a higher level of isolation between the wild and the domesticated forms in comparison with the rest of the genome probably because of linkage to the loci selected during domestication. Thus, the regions of the genome surrounding the major domestication genes are particularly interesting to tag the introgression from wild relatives into modern cultivars. As most of the markers that are under the effects of selection are linked to known loci related to the domestication syndrome, we conclude that population genomics approaches are efficient in detecting QTLs. We also present a method based on bulk DNA samples that is effective in prescreening for a large number of markers to determine selection signatures.

The main aim of this study was to test the patterns of sequence divergence and haplotype structure at the MAT locus of Pyrenophora teres, the causal agent of barley ‘net blotch’ disease. P. teres is a heterothallic ascomycete that co-occurs in two symptomatological forms, the net form (NF) and the spot form (SF). The mating-type genes MAT1-1-1 and MAT1-2-1 were sequenced from 22 NF isolates (12 MAT1-1-1 and 10 MAT1-2-1 sequences) and 17 SF isolates (10 MAT1-1-1 and seven MAT1-2-1 sequences) collected from Sardinian barley landrace populations and worldwide. On the basis of a parsimony network analysis, the two forms of P. teres are phylogenetically separated. More than 85% of the total nucleotide variation was found between formae speciales. The two forms do not share any polymorphisms. Six diagnostic nucleotide polymorphisms were found in the MAT1-1-1 intron (1) and in the MAT1-1-1 (3) and MAT1-2-1 (2) exons. Three diagnostic non-synonymous mutations were found, one in MAT1-1-1 and two in MAT1-2-1. For comparison with P. teres sequence data, the mating-type genes from Pyrenophora graminea were also isolated and sequenced. Divergence between P. graminea and P. teres is of a similar magnitude to that between NF and SF of P. teres. The MAT genes of P. graminea were closer to those of SF than to NF, with the MAT1-2-1 SF peptide not different from the MAT1-2-1 peptide of P. graminea. Overall, these data suggest long genetic isolation between the two forms of P. teres and that hybridization is rare or absent under field conditions, with each form having some particular niche specialization. This indicates that research on resistance to P. teres should consider the two forms separately, as different species.

The genetic diversity of 66 Phaseolus genotypes was investigated, which included 14 local varieties of Phaseolus vulgaris and nine local varieties of P. coccineus, collected in Marche, central Italy. Their genetic diversity was assessed using three types of molecular marker: inter simple sequence repeats (ISSRs), nuclear gene-tagged simple sequence repeats (SSRs) and chloroplast simple sequence repeats (CpSSRs). Phaseolus vulgaris shows a higher genetic diversity than P. coccineus for the SSRs and CpSSRs, but not for the putative neutral ISSR markers. These data suggest that selection by farmers and adaptation to heterogeneous environments has maintained the diversity in landraces of the common bean. Comparing genetic diversity in Marche with that of the American controls reveals that 71% of the local P. vulgaris varieties in Marche are of Andean origin. The two gene pools of the common bean can be found on the same farm, and there is some evidence of past hybridization events between these two gene pools.

For the genus Anthyllis (Fam. Fabaceae, tribe Loteae), with few exceptions, little information is available on the genetic variation among and within species. This genus contains 20 species distributed throughout Europe, Africa, and the Mediterranean basin. The most widespread species is A. vulneraria, and over 30 intraspecies taxa have been identified based on plant morphology. To study the molecular phylogeny of the genus, the sequences of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA of 10 Anthyllis species, including 11 subspecies of A. vulneraria and three subspecies of A. montana, were obtained and analysed together with sequences of five other species of the genus obtained from GenBank. Our results suggest that the genus Anthyllis is not monophyletic and is divided in two main clades: the Anthyllis sensu strictu and the "tetraphylla clade". The former includes most of the Anthyllis species, and the latter includes three annual species more closely related to Lotus. All the taxa were also analysed according to seven chloroplast microsatellites, and these data closely confirm the results obtained with the ITS phylogeny.