phaseomicsv - cnr · phaseomicsv varenna, 23-26 may 2007 organised by francesca sparvoli,...
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PhaseomicsV
Varenna, 23-26 May 2007
Organised by Francesca Sparvoli, Incoronata Galasso,
Istituto di Biologia e Biotecnologia Agraria, CNR, Milan, Italy
and William Broughton, Université de Genève, Genève, Switzerland
CRA Istituto Sperimentale
per l’Orticoltura
www.phaseomicsv.net
2
PhaseomicsV
Varenna, 23-26 May 2007
Wednesday, May 23
14.00 to 16.00 Registration
16,00 to 18,00 Opening Session
CHAIRPERSON: Mario Aguilar
Francesca Sparvoli, IBBA-CNR, Milan, Italy
Welcome to PHASEOMICS V
William Broughton, Univeristy of Geneva, Switzerland
Welcome to PHASEOMICS V
OPENING CONFERENCE: Roberto Papa, Università Politecnica delle Marche, Italy
“The effects of domestication on the structure of the genetic diversity
of Phaseolus vulgaris L.”
18,00 Welcome Party
CRA Istituto Sperimentale
per l’Orticoltura
www.phaseomicsv.net
3
Thursady, May 24
9,00 to 11,00 Evolutionary Genetics and Genomics
CHAIRPERSON: Matthew Blair
9,00 Matthew Blair, CIAT, Cali, Colombia
“Utilization and Discovery of Genetic Diversity in Common Bean"
9,30 Andrea Carboni, ICO-CRA, MiPAF, Bologna, Italy
"From Genomics to Breeding (and vice versa): a new strategy to associate Resistance Gene
Analogs to the root-knot Nematode resistance in Phaseolus vulgaris L." 10,00 Monica Rossi, Department of Plant Sciences, North Dakota State University, USA
“The effect of selection on loci within close proximity of domestication loci in common bean
(Phaseolus vulgaris L.)” 10,30 Luis Eduardo Servin, IBT-UNAM, Cuernavaca, Mexico
“A new phylogenetic analysis of Phaseolus species: Patterns of diversification and biogeography”
11.00 to 11,30 Coffee Break
11,30 to 12,30 Seed and pod nutritional quality
CHAIRPERSON: Roberto Bollini
11,30 Marina Carbonaro, INRAN, Rome, Italy
“Perspectives into factors affecting the nutritional quality of common bean (Phaseolus vulgaris)" 11,50 Francesca Sparvoli, Istituto di Biologia e Biotecnologia Agraria-CNR, Milan, Italy
“Identification and characterisation of a low phytic acid (lpa ) mutant in bean” 12,10 Incoronata Galasso, Istituto di Biologia e Biotecnologia Agraria-CNR, Milan, Italy
“Bowman-Birk inhibitors in common bean“
12,30 to 14,00 Lunch
14,00 to 15,20 Development of tools and biological resources
CHAIRPERSON: Georgina Hernandez
14,00 Tim Porch, USDA/ARS/TARS, Mayaguez, Puerto Rico
“EMS population development for TILLING with BAT 93”
14,30 Pietro Piffanelli, ParcoTecnologico Padano (PTP), Lodi, Italy
“FLUOTILL: a high-throughput platform for TILLING and ECOTILLING analyses”
15,00 Federico Sanchez, IBT-UNAM, Cuernavaca, Mexico
“Functional genomics in Phaseolus vulgaris: Gene silencing by RNAi and miRNA technologies” 15,20 Francisco Aragão, EMBRAPA,Brasilia, Brasil
“RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean”
15,40 Subhash Bhore, Melaka Institute of Biotechnology, Melaka, Malaysia
4
“Phaseomics: Generation and Analysis of Expressed Sequence Tags (ESTs) From Early and Late
Pod Stage Tissue of Phaseolus vulgaris L. Variety BAT93”
16,00 Antonio De Ron, Misión Biológica de Galicia,CSIC, Pontevedra, Spain
“What we know and what we do not know about European beans”
16,30 to 17,00 Coffee Break
17,00 to 18,30 Bridging science to agronomy
CHAIRPERSON: Roseline Remans
17,00 Roseline Remans, CMPG, Katholieke Universiteit Leuven, Leuven, Belgium
“Bridging science and society dynamics in crop research: common bean as a model”
17,30 Jean-Jacques Drevon, INRA, Montpellier, France
“Nodular diagnosis for integrated improvement of symbiotic nitrogen fixation in cropping
systems.”
18,00 Bruno Campion, ISPORT, Montanaso Lombardo, Lodi, Italy
“Development of bean breeding lines for food and feed uses ”
Free Evening
Friday, May 25
9,00 to 10,30 Biotic and abiotic stresses
CHAIRPERSON: Jean-Jaques Drevon
9,00 Giulia De Lorenzo, Università della Sapienza, Rome, Italy
“Molecular diversification of PGIP gene family members in Phaseolus vulgaris-I” 9,30 Renato D’Ovidio, Università della Tuscia, Viterbo, Italy
“Molecular diversification of PGIP gene family members in Phaseolus vulgaris-II” 9,50 Georgina Hernandez, CCG-UNAM, Cuernavaca, Mexico
“Functional genomic of beans under abiotic stess conditons”
10,10 Peter Pauls, Univeristy of Guelph, Guelph, Canada
“Towards Molecular Characterization of Common Bacterial Blight Resistance Genes in Phaseolus vulgaris”
10,30 to 11,00 Coffee Break
5
11,00 to 13,00 Interaction with beneficial organisms
CHAIRPERSON: William Broughton
11,00 Jos Vanderleyden, CMPG, Katholieke Universiteit Leuven, Leuven, Belgium
“The effects of Plant Growth Promoting Rhizobacteria on bean nodulation.”
11,20 Mario Aguilar, Universidad National de La Plata, La Plata, Argentina
“Early common bean response in the preferential nodulation by rhizobia”
11,40 William Broughton, Univeristy of Geneva, Switzerland
“Role of rhizobial carbohydrates in nodulation of beans”
12,00 Patricia Lariguet, Univeristy of Geneva, Switzerland
“Common bean responses to the Type Three secretion system of Rhizobium NGR234” 12,20 Jan Michiels, CMPG, Katholieke Universiteit Leuven, Leuven, Belgium
“Surface migration and quorum sensing in Rhizobium etli” 12,40 Carmen Quinto, IBT-UNAM, Cuernavaca, Mexico
“Dissecting the impaired signalling pathway in a non-nodulating Phaseolus vulgaris mutant”
13,00 to 14,00 Lunch
14,00 to 15,00 Poster Session
15,00 to 16,30 Round table on 7WP: perspectives, which future for
Phaseomics?
CHAIRPERSON: Jos Vanderleyden
15,00 William Broughton, Univeristy of Geneva, Switzerland
“Ideas for Project Proposal to FP7 call”
15,30 Open discussion
16,30 to 17,00 Coffee Break
Free afternoon
20,30 Phaseomics Dinner
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Saturday, May 26
9,00 to 10,00 Bioinformatic and dissemination
CHAIRPERSON: Francesca Sparvoli
9,00 Nancy Terryn, IPBO, Ghent, Belgium
“Comunicating and disseminating on Grain legume research”
9,30 Mario Nenno,
“Internet and Phaseolus”
10,00 to 10,30 Coffee Break
10,30 to 12,00 Quo Vadis Phaseomics – general discussion 12,00 Departures
7
The effects of domestication on the structure of the genetic diversity of
Phaseolus vulgaris L.
Roberto Papa
Università Politecnica delle Marche, Via Brecce Bianche,
60131 ANCONA, ITALY, [email protected]
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.
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 in P. vulgaris a large portion of the genome appears to have been
subjected to the effects of selection, probably because of linkage to the loci selected during
domestication. 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
very efficient in detecting QTLs. We also present a method based on bulk DNA samples that is
effective in pre-screening for a large number of markers to determine selection signatures.
8
Discovery and utilization of wild accession diversity for common bean improvement
M.W. Blair, G. Iriarte, H.F. Buendía, A. Hoyos, A. Hincapie, S. Beebe
Advanced backcross QTL analysis and wild accession diversity are being used at CIAT to improve
various characteristics of common beans. In an initial study quantitative trait loci for agronomic
performance were identified in a population of BC2F3:5 introgression lines created from the cross of
a Colombian large red-seeded commercial cultivar, ICA Cerinza, and a wild common bean
accession, G24404. A total of 157 lines were evaluated for phenological traits, plant architecture,
seed weight, yield and yield components in replicated trials in three environments in Colombia and
genotyped with microsatellite, SCAR and phaseolin markers that were used to create a genetic map
that covered all eleven linkage groups of the common bean genome with markers spaced at an
average distance of every 10.4 cM. Segregation distortion was most significant in regions
orthologous for a seed coat color locus (R-C) on linkage group b08 and two domestication
syndrome genes, one on linkage group b01 at the determinacy (fin) locus and the other on linkage
group b02 at the seed shattering (st) locus. Composite interval mapping analysis identified a total
of 41 significant QTL for the eight traits measured of which five for seed weight, two for days to
flowering and one for yield were consistent across two or more environments. QTL were located
on every linkage group with b06 showing the greatest number of independent loci. A total of 13
QTL for plant height, yield and yield components along with a single QTL for seed size showed
positive alleles from the wild parent while the remaining QTL showed positive alleles from the
cultivated parent. Some QTL co-localized with regions that had previously been described to be
important for these traits. Compensation was observed between greater pod and seed production
and smaller seed size and may have resulted from QTL for these traits being linked or pleiotropic.
Although wild beans have been used before to transfer biotic stress resistance traits, this study is the
first to attempt to simultaneously obtain a higher yield potential from wild beans and to analyze this
trait with single-copy markers. The wild accession was notable for being from a unique center of
diversity and for contributing positive alleles for yield and other traits to the introgression lines
showing the potential that advanced backcrossing has in common bean improvement. Future
studies will concentrate on improving combining ability across gene pools and transferring nutrition
quality traits from landraces to commercial cultivars.
9
The effect of selection on loci within close proximity of domestication loci in common bean
(Phaseolus vulgaris l.)
M. Rossi
1,2, S. Mamidi
2,3, E. Bellucci
1, M.D. Mcconnell
2,3, R.K. Lee
2,3, R. Papa
1, P.E. Mcclean
2,3
1Dipartimento di Scienze degli Alimenti, Facoltà di Agraria, Università Politecnica delle Marche,
Via Brecce Bianche, I-60131 Ancona, Italy. [email protected] 2
Department of Plant Sciences, North Dakota State University, Fargo, ND, 58105 USA
Genomics and Bioinformatics Program, North Dakota State University, Fargo, ND, 58105 USA
Identifying regions of the genome that have been the targets of selection will provide important
insights into the evolutionary history and facilitate the identification of important agronomic genes.
The structure of genetic diversity of modern crops is deeply influenced by the processes of
domestication and plant breeding. Both domestication and plant breeding reduced genetic diversity
due to random genetic drift (bottlenecks) and because of selection for target genes. World-wide
common bean (Phaseolus vulgaris L.) is the most important source of proteins for direct human
consumption, and the identification of genes of agronomic importance may facilitate improved
productivity and quality of this important crop. We sequenced gene fragments spanning 6 cM of a
genomic region of the linkage group B8 where several major QTLs related to the domestication
syndrome have been previously located. We compared three sets of accessions representing the
various stage of common bean improvement (wild, landraces and improved cultivars). Gene
fragments representing other genomic regions were sequenced for comparison. Overall, we
analysed 52 genotypes chosen on the basis of SSR data in order to represent the largest diversity
within each set of accessions. We performed several statistical tests to identify the signature of
selection due to domestication and crop improvement. This research is very promising because we
may be able to identify genes of potential agronomic importance and to determine the effect of
domestication and breeding on the structure of genetic diversity in the common bean genome.
10
From genomics to breeding (and vice versa): a new strategy to associate resistance gene
analogs to the root-knot nematode resistance in Phaseolus vulgaris L.
Francesca Del Bianco, Paolo Ranalli and Andrea Carboni
CRA - Istituto Sperimentale per le Colture Industriali, Bologna
The first SCAR markers linked to the root-knot nematodes resistance were selected using an
innovative strategy called Genomic-Pedigree. With this approach we were able to associate an
evolutionary study of the NBS-LRR gene family and the heritability of resistance genes through a
long and complex breeding program. The evolutionary genomics study followed the dynamics of
this resistance gene family in 7 different genotypes (3 Mesoamerican resistant accessions and one
Andean derivated cultivar, one Mesoamerican wild plus Bat 93 and Jalo EEP558). The 176
Resistance Gene Analog sequences were validated using a very conservative scientific and statistic
plan of action. Some RGA sequences were found unexpectedly equal in Mesoamerican and Andean
materials and used to design specific PCR primers; these markers were extraordinary effective in
the selection of new breeding lines resistant to Meloidogyne spp. but these sequences are also under
investigation for expression genetics and functional genomics studies.
11
A new phylogenetic analysis of Phaseolus species: Patterns of diversification and
biogeography
Servín L.E.1,2
and Márquez-Ortíz Y. 1,2
1
Instituto de Biotecnologia, UNAM, Cuernavaca, Morelos, Mexico; 2
Licenciatura en Ciencias
Genomicas, UNAM, Cuernavaca, Morelos, Mexico.
The phylogeny of genus Phaseolus has been resolved with increasing detail and support (Delgado-
Salinas et. al. 1999, Freytag and Debouck 2002, Delgado-Salinas et al. 2006). Uncertainties about
ancestral biogeography patterns remain unsolved and nowadays there are only a few attempts about
the Phaseolus phylogenetic dating.
The tribe Phaseolae has three economically important genus: Phaseolus, Glycine (soybean) and
Vigna (Asiatic beans); this tribe is part of the Papilionoideae subfamily of the family Fabaceae
(Leguminosae) (Mercado-Ruaro, 2000). The cultivated species of the genus Phaseolus: P.
acutifolius A. Gray, P. coccineus L., P. dumosus Greenman, P. lunatus L. and P. vulgaris L. are the
most important grain legumes for direct human consumption in the world (CIAT, 1993, Delgado
Salinas et al. 2006). Phaseolus consist of about 70 strictly New World species (Delgado et al. 2006,
Freytag and Debouck 2002) that thrive in a broader range of altitudes (730 – 2 000 m. above sea
level) in lowland dry and wet forest up to oak, pine-oak, pine forest and humid forest from
southeastern Canada to northern Argentina (Toro et al. 1990, Delgado-Salinas et. al. 1999,
Delgado-Salinas et. al 2006). Some species (P. lignosus, P. mollis, P. lunatus, P. filiformis and P.
acutifolius) can inhabit American islands and show a broader range of endemism. There is some
evidence that some species that inhabit continental land show specific biogeography patterns of
distribution (Beebe et al. 2000, 2001, Freytag and Debouck 2002, Delgado-Salinas et al. 2006).
Delgado-Salinas et al. 1999 performed a study based on a combined phylogenetic analysis of
nrDNA ITS/5.8S sequences and morphological characters. Resulting from this classification,
approximately 50 species of Phaseolus into nine groups with little clade support were obtained. The
group composed by P. microcarpus was resolved as the earliest branch within the Phaseolus genus.
Several domesticated species belong to the P. vulgaris and P. lunatus groups. Freytag and Debouck
(2002) studied 76 species of Phaseolus and classify them into 14 groups. Four groups are
containing the five domesticated species. These two studies reflect that molecular phylogenetic
studies using nrDNA sequences are not totally congruent with traditional morphology based
taxonomy in the case of Phaseolus genus. The Delgado-Salinas et al. (2006) phylogenetic study
based on a combined parsimony analysis of nrDNA ITS/5.8S and plastid trnK loci, resulted in a
classification of two major clades of the genus. Clade A comprises the pauciflorus, pedicellatus and
tuerckheimii groups and clade B comprises the filiformis, lunatus, vulgaris, leptostachyus and
polystachios groups. Also, eight secondary clades are recognized as previously reported in
Delgado-Salinas et al. (1999). The major clade phylogenetic relationships show inconsistencies
with the present study. Besides the high parsimony bootstrap support for the groups reported in
Delgado-Salinas et al. (2006), five enigmatic species (P. glabellus, P. macrolepis, P. microcarpus,
P. oaxacanus and P. talamancensis) are weakly resolved as a sister clade to the tuerckheimii group.
This work presents a Phaseolus phylogenetic analysis where only wild accessions are considered.
It includes new wild accessions of Phaseolus from North, Middle and South America together with
the wild Phaseolus sequences reported on GeneBank. First, we give an exhaustive revision of our
phylogenies to asses the congruence with the previously published analysis and the present
taxonomic system (Delgado-Salinas et. al. 1999, Freytag and Debouck 2002, Delgado-Salinas
2006). Second, we assign the new and the enigmatic species to their possible clade in our
phylogeny as well, we increase or maintain the bootstrap support of the Phaseolus groups. Third,
we are currently determining divergence times for all major clades and we will try to compare them
with the previously reported by Delgado-Salinas et al. (2006). Finally we wish to reconstruct
ancestral areas of distributions for all major clades and generate a hypothesis for a possible centre
of Phaseolus genus origin.
12
Perspectives into factors affecting the nutritional quality of common bean (Phaseolus vulgaris
L.)
M. Carbonaro
Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN) - Roma
Plant seeds represent a major source of dietary proteins, the amount of proteins varying from about
20% to 40% (d. wt.) in certain legumes and oilseeds. Phaseolus spp. L. are the most important
legumes for human consumption in the world. Common bean (Phaseolus vulgaris L.) contains 20-
25% protein, 40% starch, 20% dietary fiber, several micronutrients (minerals, vitamins) and
bioactive compounds (phenolics, soluble fiber). There is a general consensus that antinutritional
factors of protein nature (protease inhibitors, lectins) are inactivated by proper heat-treatment.
However, data on the effects of heating on non-protein antinutritional compounds, such as phytic
acid and polyphenols, are conflicting.
Evidence from in vivo and in vitro studies have previously indicated that digestion of native legume
seed storage proteins is limited because of conformational constraints. However, this statement has
recently been questioned by the results of in vivo ileal digestibility studies. Despite loss of solubility
of proteins has generally been observed to take place after cooking of legumes, it has to be
established how far the thermal aggregation phenomenon may impair digestibility. It is also
possible that essential amino acids, minerals, trace elements and even phenolics remain trapped
inside undigested protein aggregates, with significant decrease in their availability.
This dissertation will focus on recent acquirements on the nutritional quality of common bean, in
comparison with that of other legume species, coming out by the application of novel, specific
methodologies to whole legume seed flour or extracted fractions.
13
Identification and characterisation of a low phytic acid (lpa ) mutant in bean
Francesca Sparvoli
1, Erik Nielsen
2, Bruno Campion
3, Enrico Doria
2, Incoronata Galasso
1, Marzia
Fileppi3, Giovanni Tagliabue
1, Maria Gloria Daminati
1, Søren Rasmussen
4 and Roberto Bollini
1
1Istituto di Biologia e Biotecnologia Agraria-CNR, Milan, Italy;
2Dipartimento di Genetica e
Microbiologia, Università di Pavia, Italy; 3
CRA – Istituto Sperimentale per l’OrticolturaMontanaso
Lombardo, Lodi, Italy;4 Royal Veterinary and Agricultural University, Department of Agricultural
Sciences, Frederiksberg, Denmark
Phytic acid is the major form of P storage in the seeds and acts as an antinutrient for humans and
monogastric animals. Applied interest in seed phytic acid is due to its role in human health and
animal nutrition. In fact, this compound binds mineral cations, such as Fe, Zn and Ca, forming
mixed salts (phytin) that are largely excreted by humans and other non ruminant animals, since they
have no or limited phytase activity in their digestive tract. Phytin excretion contributes to
micronutrient deficiencies in developing world populations and to water pollution (eutrophication).
The development of low phytic acid (lpa) grains might represent a useful tool to obtain nutritionally
improved food and feed as well as environment friendly and sustainable productions.
Many data are accumulating in the literature indicating that phytic acid and inositol polyphosphates
also play key roles in the regulation of several plant functions, like root architecture, nutrient
uptake, stomata opening, etc. Therefore studies on this metabolic pathway, besides being important
for the nutritional aspects are also important to understand its role in plant-environment
interactions.
Our group has started a study with the aim of to identify lpa mutants in bean by both forward and
reverse genetic (TILIING) approaches and succeeded in isolating a lpa mutant (lpa-280-10)
showing a 90% reduction of phytic acid and a 25 % reduction in raffinose content in the seed. Data
will be presented on the phenotypic and molecular characterisation of this mutant.
Research partially supported by Ministry of Agricultural Alimentary and Forest Politics with funds
released by C.I.P.E. (Resolution 17/2003).
14
Bowman-Birk inhibitors in common bean
Incoronata Galasso
1, Angela R. Piergiovanni
2, Lucia Lioi
2, Bruno Campion
3, Roberto Bollini
1 and
Francesca Sparvoli1
1
Istituto di Biologia e Biotecnologia Agraria, IBBA-CNR, Milano (Italy) 2 Istituto di Genetica Vegetale, IGV-CNR, Bari (Italy)
3 Istituto Sperimentale per l’Orticoltura, CRA, Montanaso Lombardo, Lodi (Italy)
The Bowman-Birk inhibitors (BBIs) are serine proteinase inhibitors with a low molecular mass (8–
9 kDa) and high cysteine content. In legume seeds, proteinase inhibitors are considered anti-
nutritional compounds due to their ability to irreversibly inhibit the action of digestive enzymes.
Hence, their removal may be desirable. However, there are also indications that proteinase
inhibitors may need to be retained in plants, since they have an active role against pest and diseases.
Recent work demonstrated that soybean BBI, besides being a storage protein and a recognised
agent for seed protection, is also effective in preventing or suppressing carcinogenic processes in
both in vitro and in vivo models (Kennedy 1998, Armstrong et al., 2003).
In common bean (Phaseolus vulgaris L.) the presence of BBIs has been demonstrated and it has
also been evaluated the seed trypsin inhibitor activity in several bean genotypes (Piergiovanni and
Galasso, 2004). Analysis carried out on the primary structure of the BBI genes has evidenced
several polymorphisms in the two binding loops. This result suggests that the genome of common
bean contains several types of BBI genes. In order to confirm this hypothesis and also to understand
their genomic organization we analysed a BAC library from P. vulgaris accession G12949, (CIAT,
Cali, Colombia) which covers 6 times the bean genome. After filter hybridisation, using as probe a
BBI nucleotide sequence previously isolated (Piergiovanni and Galasso, 2004), three positive BAC
clones were identified. All these BAC clones contained an identical genome insert since they
showed a very similar profile after digestion with the restriction enzyme HindIII and hybridization
with the BBI probe. The BAC clone P2B8 was chosen and partially sequenced. Our results
confirmed that a small multigene family codes for BBIs and showed that three double-headed
inhibitors are present in a single BAC clone of about 130 Kbp. Two of them, one containing the
binding loop trypsin/chymotrypsin and the other one the elastase/trypsin, showed to be very close
to each other and to be arranged in opposite orientation (head to head) in a PstI fragment of 5.351
bp (EMBL acc. number AM492522).
Research partially supported by Ministry of Agricultural Alimentary and Forest Politics with funds
released by C.I.P.E. (Resolution 17/2003).
15
Role of rhizobial carbohydrates in nodulation of beans
Patricia Lariguet, Dale Noel*, William J. Broughton, and William J. Deakin
LBMPS, Université de Genève, 30 quai Ernest-Ansermet, 1211 Genève, Switzerland. *Department of Biological Sciences, Marquette University,
PO Box 1881, Milwaukee, WI 53201-1881, USA.
Although the common bean, Phaseolus vulgaris L., has a very broad host-range, large variations in
the abilities of nodulating Rhizobium species to fix nitrogen on various plant genotypes have been
observed. Our studies on the interaction of the broad host-range Rhizobium sp. NGR234 with many
Phaseolus species, cultivars and accessions has shown that specific bacterial carbohydrates are
essential for the development of efficient, nitrogen-fixing nodules. Included amongst these are
flavonoid-inducible polysaccharides containing rhamnose. These rhamnose-containing O-antigens
are attached to a modified core-lipid A carrier (lipopolysaccharide, LPS). The primary sequence of
the O-antigen is [-3)-α-L-Rhap-(1,3)-α-L-Rhap-(1,2)-α-L-Rhap-(1-]n, and the LPS core lacks the
acidic sugars commonly associated with the antigenic outer core of LPS from non-induced cells.
This rhamnan, which is absent from non-induced cells, has the same primary sequence as the A-
band O-antigen of Pseudomonas aeruginosa, except that it is composed of L-rhamnose rather than
the D-rhamnose characteristic of the latter.
16
EMS mutagenesis of BAT 93 for TILLING
T. Porch*, P. Lariguet**, M. Blair***, W. Broughton**
*) USDA/ARS/TARS, Mayaguez, Puerto Rico, USA
**) University of Geneva, Geneva, Switzerland
***) CIAT, Palmira, Colombia
TILLING (targeted induced local lesions in genomes) offers a unique opportunity for performing
reverse genetics in common bean due to the absence of an effective transformation protocol and
thus the inability to develop transposon-based or T DNA-based mutagenesis systems. TILLING is
complementary to traditional plant breeding programs as it provides a source of characterized genes
for gene-based marker development for use in molecular selection of important traits (e.g. for
nutritional quality, yield, or disease resistance). EMS (ethyl methane sulfonate) induced
mutagenesis creates numerous point mutations in the genome, with the mutagenesis efficiency
based on the EMS concentration, the length of the treatment, and the ambient temperature. This
study evaluated EMS concentrations of 20 to 60mM in order to confirm approriate levels of EMS
for TILLING of the genotype BAT 93. Germination, plant survival, plant height, and seed yield
were evaluated based on protocols developed previously (C. Pankhurst, P. Lariguet and W.
Broughton, unpublished) using a 15 hour EMS treatment at 21°C. Development of the mutagenesis
population is being carried out collaboratively at the University of Geneva in Geneva, Switzerland,
at the International Center for Tropical Agriculture (CIAT) in Palmira, Colombia, and at USDA-
ARS-TARS in Mayaguez, Puerto Rico.
17
FLUOTILL: a high-throughput platform for TILLING and ECOTILLING analyses
Pietro Piffanelli(1), Fabio Celestini(1) and Ilaria Losini(1) and Silvio Salvi(2)
(1) PTP Genomics Platform, CERSA - AgBiotech Research Centre, Parco Tecnologico Padano –
PTP, Via Einstein, 26900, Lodi – Italy
(2) (2) Department of Agroenviromental Science and Technologies-DiSTA, University of Bologna,
Viale Fanin 44, 40127 Bologna - Italy
The Parco Tecnologico Padano (PTP) and University of Bologna (DiSTA) have joined forces to
increase the automation and throughput of the TILLING screening process. The entire workflow -
DNA extraction, pooling, amplification, digestion and purification - has been automated using
Tecan liquid handlers. The automated FLUO-TILL Platform coupled with the use of the PGP-
LIMS (Laboratory Information Management System) and of a barcode system ensures thorough
quality controls and full traceability of each processed DNA sample. The final capillary
electrophoresis step is performed using an Applied Biosystems’ 3730 DNA Analyzer. The two
primers used during the amplification step are both labelled using a different fluorophore, allowing
to unambiguously distinguishing the two digestion fragments. In addition, a Perl-CGI script was
developed, enabling mutant detection automatically. The ECOTILLING protocol was developed
using both plant and animal DNA collections and enables detection of multiple polymorphic sites in
the same analysis.
18
Functional Genomics Analysis of a Highly Expressed Nod Gene Family in Phaseolus
vulgaris.
Olivares JE, Estrada G, Campos F, Gabriel G, Alvarado X, Díaz-Camino C, Santana O, and
Sanchez F.
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional
Autónoma de México. Av. Universidad 2001, Chamilpa. Apdo. Postal 510-A. Cuernavaca,
Morelos, México. Email: [email protected]
A functional genomics approach was used to unravel the biological role in root nodules of a highly
expressed nodulin gene family of Phaseolus vulgaris. Genomic analysis indicated that one of the
most abundant transcripts in the bean nodule EST data base reported by Ramirez et al, 2005 is
encoded by gene members of the npv30 gene family. Highly expressed ESTs from nodule were also
found expressed in the leaf cDNA library. Although Npv30 transcripts are very abundant, they
encode 30-32 kDa proteins that are hardly detected by biochemical and immunological methods,
suggesting that these proteins have a short half-life and/or mRNAs are strongly regulated at the
translational level (Campos et al, 1995). Although, npv30 transcripts are mostly localized in the
infected cells from the bean nodule central zone, they are also accumulated in vascular bundles and
the nodule parenchyma. Surprisingly, Npv30 proteins were mostly immunolocalized in the
neighboring uninfected cells and in the intercellular spaces around the infected cells. Npv30
proteins have N-terminal signal peptides suggesting that they are membrane-associated or secreted
proteins. In addition, they have two regions with zinc finger-like binding motifs (Cys-X7-Cys).
Finally, at the C-terminal end, all members have different tracks of prolines with variable lengths.
Some members with large proline tracks are able to tightly bind profilin, an actin binding protein
with a key role in the endocytic membrane pathway. We recently described that composite plants
can be efficiently nodulated by rhizobia (Estrada et al., MPMI 19:1385-93, 2006). The phenotype
of nodules with knocked down levels of NPV30 (antisense constructs) showed that infected and
uninfected cells have entered a programmed cell death program, suggesting that the npv30 gene
family has a role in repressing a default death pathway or has an anti-death function in root nodules.
CONACYT 42562-Q and PAPIIT IN2084075 are acknowledged.
19
RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common
bean (Phaseolus vulgaris)
K. Bonfim* J. C. Faria**, E. O. P. L. Nogueira*, E. A. Mendes*, F. J. L. Aragão*
*Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 Norte, 70770-900, Brasília, DF, Brazil.
**Embrapa Arroz e Feijão, Rodovia GO-462, km 12 Zona Rural C.P. 179, 75375-000 Santo
Antônio de Goiás, GO, Brazil.
Corresponding author: F. Aragão ([email protected])
BGMV is transmitted by the whitefly Bemisia tabaci (Gennadius) in a persistent, circulative
manner, and causes the golden mosaic of common bean (Phaseolus vulgaris L.). The characteristic
symptoms are yellow-green mosaic of leaves, stunted growth or distorted pods. Control
measurements have been primarily focused on controlling the vector by contact or systemic
insecticides, with the concomitant problems of development of insecticide resistance, low cost-
benefit ratio, and environmental concerns. The disease is the largest constraint to bean production
in Latin America and causes significant yield losses (40−100%) in South and Central America,
Mexico, and the United States. In Latin America, BGMV causes severe yield losses, particularly
during the warmer months when B. tabaci population is higher, leading to a great reduction of
summer plantings of common bean. Extensive screening of common bean germplasm for resistance
to BGMV has revealed no genotypes with high levels of resistance. The resistance often is
unsatisfactory and commercial cultivars are susceptible under early, moderate, or severe infection.
Several strategies have been employed for genetically engineering resistance to viruses in
transgenic plants. For begomoviruses, most of them have involved the expression of truncated
defective genes and antisense RNA. Here, we explored the concept of using RNAi [e] construct to
silence the AC1 viral gene and generate highly resistant transgenic common bean plants. AC1
encodes a complex, multifunctional protein (Rep) that acts as a rolling-circle replication initiation
factor, which is the only protein strictly essential for viral genome replication and is capable of
regulating its own expression. Twenty transgenic common bean lines were obtained using the
biolistic process with an intron-hairpin construction to induce post-transcriptional gene silencing
against the AC1 gene. Two lines (named 5.1 and ahas 3.2) presented high resistance (approximately
93% of the plants were free of symptoms) upon inoculation at high pressure (about 500 viruliferous
whiteflies per plant during the whole plant life cycle) and at a very early stage of plant
development. Transgene-specific siRNA were detected in both inoculated and non-inoculated
transgenic plants. A semiquantitative PCR analysis revealed the presence of viral DNA in
transgenic plants exposed to viruliferous whiteflies for a period of 6 days. However, when insects
were removed, no virus DNA could be detected after an additional period of 6 days. Geminiviruses
cause severe disease problems on several other crops throughout the world, such as cassava,
cowpea, mungbean, pepper, melon, tomato, cotton blackgram, lima bean, soybean, potato,
eggplant, pepper, chili pepper, watermelon, squash, and papaya and have been considered the “pest
of the century”. The strategy presented here could be extended to achieve resistance against
geminiviruses from other plant species.
20
Phaseomics: Generation and Analysis of Expressed Sequence Tags (ESTs) From Early and
Late Pod Stage Tissue of Phaseolus vulgaris L. Variety BAT93
Bhore Subhash J.*, Amelia K., Nurhuda J., and Shah Farida H.
Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade
Center City, 75450 Ayer Keroh, Melaka, Malaysia. *To whom correspondence should be addressed
(Email: [email protected]).
Beans are important source of proteins in human diet. Thousands of legume species exist but more
common beans (Phaseolus vulgaris L.) are cultivated on commercial scale. These beans are also
vital in agricultures as they form associations with bacteria that ‘fix-nitrogen’ from the air. This
amounts to internal fertilization which contributes to the high protein content in the legumes than
any other plants. However, the yields of common beans are low and the quality of their seed
proteins is sub-optimal. Therefore, exploitation of modern genetic techniques could help to solve
the problems. An international consortium called ‘Phaseomics’ has been formed to establish the
necessary framework of knowledge and materials that will result in disease-resistant, stress-
tolerant, and high-yielding beans with high-quality proteins. This project is a part of the
‘Phaseomics’ consortium loop. The objectives of this project are to construct cDNA libraries from
the bean [(Phaseolus vulgaris L.) (BAT93)] pod tissue at different developmental stages (early and
late pod stage), and to generate Expressed Sequence Tags (ESTs). The generated ESTs will then be
used to fabricate the micro-array slides, and genes that are stage-specific will be identified through
the micro-array analysis. In later stage, the Phaseolus vulgaris L. (BAT93) could be challenged
with pathogens such as the white mould and genes which are expressed only during the pathogen
attack will be identified. Early and late pod stage tissue cDNA libraries have been constructed
successfully using CloneMinerTM
cDNA library construction kit (Invitrogen), and 4100 ESTs has
been generated. Research work of ESTs generation and analysis of the generated ESTs is in
progress. This paper reports the functional annotation of the analyzed 4100 ESTs. This work is
fully supported by a grant from the Ministry of Science, Technology, and Innovation (MOSTI) of
Malaysian Government [Grant Code: BSP (M) / BTK / 004 (3)].
21
What we know and what we do not know about European beans
A. M. De Ron, M. De la Fuente, A. P. Rodiño, A. M. González, M. Pérez-Barbeito, M. Santalla
Misión Biológica de Galicia – CSIC. Pontevedra. Spain
Characterization of crop germplasm from specific regions helps understand the patterns of genetic
variation that facilitate further germplasm collection, characterization, management and their
efficient utilization in genetics and breeding. Although there is a lack of knowledge about
Phaseolus vulgaris germplasm dissemination, Gepts & Bliss (1988) and Gepts & Debouck (1991)
suggested that the European germplasm was primarily from the Andes. However, based on historic
evidence it is probable that the initial common bean accessions introduced in Europe were from
Mesoamerica, arriving the Andean germplasm later. Thus, subsequently new cultivars may have
evolved within and between the two gene pools in Europe. There was likely a quick distribution of
bean seeds as curiosities, being the seed exchange very frequent in many areas of Europe. No
records of common bean earlier than 1543 have been found in European herbariums, suggesting
that the common bean was distributed in this area after 1540, and in 1669 it was already cultivated
on a large scale, as reported by Zeven (1997).
The milestones in the study of European beans were: i) to define the relevant European market
classes (PHASELIEU Project), ii) to set up a wide germplasm working collection (currently 1403
European accessions together with 198 from other Mediterranean countries), iii) to assess the
phenotypic diversity for morpho-agronomic traits, iv) to analyze the phaseolin and other seed
proteins as evolutive markers proteins and isozyme markers, and v) to analyze the microsatellites or
SSRs – Simple Sequence Repeats polymorphisms.
Up to now the secondary diversification of common bean in Europe was documented based on the
genetic variation of bean populations for phenotypic and biochemical markers. During this process,
selection by man, genetic drift and outcrossing played a relevant role that allowed the emergence of
new bean forms being some of them recombinant types between the Mesoamerican and Andean
genetic pools. The DNA polymorphism based on analysis of SSRs is contributing to the
understanding of the structure of this new genetic variation through Europe. Phaseolin and seed
protein pattern can be used as a marker to resolve phylogenetic problems in Phaseolus, and they
can also be useful descriptors for cultivars.
22
Bridging science and society dynamics in crop research: common bean as a model
R. Remans and J. Vanderleyden
Centre of Microbial and Plant Genetics (CMPG), K.U.Leuven, Belgium
A research framework for common bean under low input systems was discussed on the BEAN
workshop held in CIAT, November 22-24, 2006.
The aim of the discussion was to approach research on common bean in a demand driven way: how
can we bridge common needs and expectations in bean production with research accomplishments
and research project objectives? Here we will present a summary and highlights of the 3-day
workshop.
Increase in global dynamics (markets, environments, urbanization, trading agreements,..) urges on
new dynamics and solutions in agriculture. Scientific research can contribute to farmers’ adaptation
to this dynamic situation by creating more options for farmers, adapted to their and global market
conditions and thus increasing their flexibility. Common bean as a highly nutritional and genetic
diverse crop possesses opportunities to offer small farmers more food security and a more stable
income generation. However, problems in common bean cultivation are numerous. Basic needs as
increase in yield potential and yield stability, optimization of nutritional value and minimization of
input requirements were highlighted as common needs in bean cultivation during the workshop.
Related to these common needs, some common denominators to increase plant efficiency were
identified including the root system development, biological nitrogen fixation and grain quality.
Further it was discussed how bean research tools can contribute to tackle these common needs.
Advances in molecular research technologies and increasing availability of new breeding material
offer an exciting combination to exploit the potential of common bean as a high nutritional and low
input crop. As our knowledge on common bean is increasing, so should the yield on the farmers’
field.
This presentation aims to stimulate the discussion in the Phaseomics V session on ‘Bridging science
to agronomy’.
23
Nodular diagnosis for integrated improvement of symbiotic nitrogen fixation in cropping
systems.
1Drevon J.J.,
2Gugliemni S.,
4Boyer G.,
5Hernandez G.,
3Lafosse-Bernard E.,
1Pernot C.,
1Vailhe H
1INRA-Montpellier-Supagro, France;
2CT Montpellier-Supagro, France;
3CIVAM-Bio,
Montpellier, France 4Chambre d’Agriculture, Catelnaudary, France ;
5IS-La Renée, Quivican,
Cuba.
The nodular diagnosis consists into measuring the nodulation of a legume in an area of production,
and relate it to growth and subsequent yield of the legume. Its objective is to assess the spatial
variation in the expression of the legume potential in rizobial symbiosis, and to search for local
factors that limit this symbiosis. The sampling of the field sites where measurements are performed
is based on the participation of farmers. Results will be shown for the nodulation of bean and the
variation of native rhizobia into wheat-rotation systems of Lauragais with the Syndicat des
Producteurs de Haricot à Cassoulet for high-value canned-bean. The relation of the nodular
diagnosis with the selection of new variety will be illustrated with the participatory assessment of
recombinant inbred lines contrasting for their efficiency in use for symbiotic nitrogen fixation in
horticultural systems of Languedoc, with organic farmers of the region, as part of a contribution to
the grainlegume project of the EU FP6. It will be concluded about the contribution of the nodular
diagnosis to an integrated approach to improve symbiotic nitrogen fixation under osmotic
constrains in mediterranean areas, like in the previous Fysame and on-going Aquarhiz project of
the EU-INCOMED program. As a prospect, will be presented a pre-project for adapting the legume
rhizobial symbiosis to low P soils for mediterranean areas of Africa and Europe.
24
Development of bean breeding lines for food and feed uses
Bruno Campion1, Marzia Fileppi
1, Enrico Doria
2, Erik Nielsen
2, Giovanni Tagliabue
3, Incoronata
Galasso3, Francesca Sparvoli
3, Gloria M. Daminati
3, Roberto Bollini
3
1CRA – Istituto Sperimentale per l’OrticolturaMontanaso Lombardo, Lodi, Italy;
2Dipartimento di
Genetica e Microbiologia, Università di Pavia, Italy; 3Istituto di Biologia e Biotecnologia Agraria,
CNR,Milan , Italy. [email protected]
In many countries of Central and South America and Africa, common bean seeds are consumed in
large quantities. In Europe and North America, vegetarian diets and consumption of vegetables in
general (common bean included) are increasing. However, when the primary source of protein are
vegetables, and especially leguminous seeds, many nutritional problems arise, above all, those due
to the presence of antinutritional factors that hamper protein digestibility (by 47-55%) and cation
absorption (Fe, Zn, Ca, Mg) at intestinal level. It is known that two main groups of anti-nutritional
factors, usually very abundant in common bean seeds, are responsible for the undesirable effects
mentioned above: a) a group of proteins called lectins, b) a group of non-protein compounds such
as tannins, polyphenols, phytate and some oligo-saccharides. While lectins are heat labile factors
and can be largely denatured by cooking, the second group is heat-stable and maintains a high
chelating ability towards proteins, causing a reduction of their digestibility, and towards cations,
leading to a drastic decrease of their absorption in the intestine. On the other hand, it has been
demonstrated that these compounds are responsible for good health, not only at intestine level, but
also for the entire body, since they exert a direct or indirect protective effect against a few
important human diseases (nutraceutical activity). Thus, the presence of such compounds in bean
seeds may be important, in particular where human diet is very poor of crop food (Europe and
North America).
The balance of different foods to include in many different optimised diets suitable to the different
geographical, ethical, cultural situations and personal necessities, can be possible or much easier if
adequate bean genetic materials (having particular genetic traits) are developed. Therefore, we
started a research focused on the development of new bean genetic materials containing higher,
lower or null amounts of nutraceutical compounds in the seeds.
The objective of the first breeding program was the complete removal at genetic level of the major
lectins usually accumulated in the seeds. Plant materials used in this breeding work were a few
accessions provided by CIAT (Centro Internacional de Agricultura Tropical, Cali, Colombia) and
some Italian commercial cultivars showing good agronomic traits. After evaluation and selection of
numerous breeding lines, two advanced lines were chosen. The first, called “P501”, shows
determinate growth habit and red-vine mottled seeds of around 500 mg, while the second, called
“938”, shows determinate growth habit and brown seeds of about 200 mg. The seeds produced by
these lines are devoid of major lectins, it means without arcelin, phytohemagglutinin and a-amylase
inhibitor, which are usually present in wild-types. A new group of lectin-free breeding lines have
been produced later on from a few crosses made between three commercial climbing cultivars and
“P 501”. The aim of the selection was the introduction of the lectin-free character into the best
climbing varieties, showing good agronomic traits for pod and seed. Although these materials are
only at the F3 generation, their agronomic traits are almost in accordance with what the market
requires.
The goal of the second breeding program was the introgression of the character “lectin-free” into
cultivated materials producing seeds with lower contents in tannins and polyphenols. It is well
known that these two compounds are almost exclusively accumulated in the integument of the seed
and that genetic traits controlling their amount are positively correlated to the seed coat colour. The
trait “white seed coat” is associated with lower levels of these two compounds and can be
considered as the main marker in our breeding programs. Genetic materials “lectin-free + white
25
seed coat” are now available as breeding lines of two types: a) F3 climbing large seeded (seed >
400 mg) and, b) F5 bush small seeded (seed < 250 mg).
With a new research started on 2004, we tried to reduce the high amount of phytic acid
accumulated in the seeds of common bean. A mutagenic treatment with ethyl-methane-sulphonate
solution (EMS) was applied to an F3 population of 7,200 lectin-free seeds. A number of
biochemical analyses performed on later generations allowed to identify and isolate a genetic “low-
phytic acid (lpa)” (see Sparvoli et al., this meeting). At present we have M3 and M4 “lectin-free -
low-phytic” progenies producing small black seeds (around 200 mg) that have already been used in
a new cycle of crosses in order to obtain new breeding lines useful for food and feed carrying all
the three main traits “lectin-free (lf) + low polyphenols and tannins (lpt) + low phytic acid (lpa)”,
responsible for most anti-nutritional problems in common bean. Five main advantages are expected
from the use of the new bean seeds (lf+lpt+lpa) for food and feed: more digestible proteins, higher
levels of free phosphorous (thus more bio-available), more bio-available bivalent cations (Fe++
,
Zn++
, Ca++
, Mg++
), less flatulence, possible use of these new beans as direct and raw protein source
also for mono-gastric animals. Next year, when an adequate amount of these new beans will be
available, we will perform nutritional tests in order to verify how much of the expected advantages
are real.
At present, the development of new genetic materials producing phaseolin-free and low/high
trypsin-inhibitor seeds is also under way.
Research partially supported by Ministry of Agricultural Alimentary and Forest Politics with funds
released by C.I.P.E. (Resolution 17/2003).
26
The effects of Plant Growth Promoting Rhizobacteria on bean nodulation
Roseline Remans, Anja Croonenborghs, Roldan Torres Gutierrez, Jan Michiels and
Jos Vanderleyden
Centre of microbial and plant genetics (CMPG), K.U.Leuven, Belgium
Several plant growth promoting rhizobacteria (PGPR) have shown potential to enhance nodulation
of legumes when coinoculated with Rhizobium. To optimize the efficiency of these Rhizobium-
PGPR-host plant interactions, unraveling the underlying mechanisms and analyzing the influence of
specific environmental conditions is crucial. In this work the effect of four PGPR strains on the
symbiotic interaction between Rhizobium and common bean (Phaseolus vulgaris L.) was studied
under deficient versus sufficient phosphorus supply. It was observed that the effect on nodulation of
three out of four PGPR tested was strongly dependent on P nutrition. Further, the use of specific
PGPR mutant strains indicated that bacterial indole-3-acetic-acid production (IAA) and 1-
aminocyclopropane-1-carboxylate (ACC) deaminase activity play an important role in the host
nodulation response, particularly under low P conditions. Moreover, it was shown that the
differential response to PGPR under low versus high P conditions was associated with changes in
the host hormone sensitivity for nodulation induced under P deficiency.
These findings contribute to the understanding of the interplay between Rhizobium, PGPR and the
plant host under different environmental settings.
27
Studies on the nodulation preference in Phaseolus vulgaris-rhizobium coevolution
O. Mario Aguilar
1, Maria Pia Beker
1, Daniela Bruzesse
1, Eitel Peltzer Meschini
1, Flavio Blanco
1,
María Eugenia Zanetti1, Helge Küster
2, Alfred Pühler
2, and Gabriel Favelukes
1
1Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas, UNLP, 1900-La
Plata, Argentina 2Institute for Genome Research and Systems Biology, CeBiTec, Bielefeld
University, D-33594 Bielefeld, Germany. [email protected]
Phaseolus vulgaris is preferentially nodulated by R. etli lineages which are geographically related
to the centre of host diversification. This is shown by polymorphism of the bacterial gene nodC,
being nodCα and nodCδ the most common alleles in nodules of Mesoamerican and Andean beans,
respectively. To gain insight into the basis of this coevolution, kinetics of infection threads
formation and nodulation were performed using four strains of each lineage. The number of
infection threads and nodules formed on the Mesoamerican bean cultivar Negro Xamapa by nodCα
strains was significantly higher during the first days after inoculation. R. etli strains carrying the
alleles nodCα and nodCδ were differentially tagged with DsRed and GFP and used in competition
assays. Two days after inoculation, only infection threads produced by nodCα strain were detected,
whereas nodCδ strains initiated the formation of this structure later, about four days after
inoculation. These results provide evidence that the mechanisms underlying nodulation preference
begins very early in the symbiotic association. In order to identify genes responsible for the
selectivity in the symbiotic association, we constructed a cDNA macroarray containing 2108 non-
redundant sequences corresponding to TCs of root hair from Negro Xamapa inoculated with R. etli
SC15 (nodCα) or 55N1 (nodCδ). Macroarrays were hybridized to cDNA of control (mock treated),
SC15 or 55N1 inoculated beans. Differential clones were selected and confirmed by qRT-PCR.
They showed homology to a diversity of genes involved in primary and secondary metabolism,
signal transduction pathways, protein transport and folding, transcription factors, pathogen defence,
etc. Currently, the role of these genes in the interaction is being assessed by RNAi silencing and
over-expression using composite plants of common beans.
28
Bean responses to the Type Three Secretion System of Rhizobium sp. NGR234
Patricia Lariguet, Silvia Ardisonne, William Broughton and William Deakin
Laboratoire de biologie moléculaire des plantes supérieures (LBMPS), Sciences III, Université de
Genève, 30 quai Ernest Ansermet, 1211 Genève 4, Switzerland. [email protected]
Rhizobia are nitrogen-fixing Gram-negative bacteria that form a mutually beneficial symbiotic
interaction with legumes. Some of them, including Rhizobium etli and the broad range host
Rhizobium sp. strain NGR234 possess an active type III secretion system (T3SS). T3SS are found
in many Gram-negative pathogenic bacteria and they deliver effector proteins into eukaryotic host
cells. Effector proteins perturb signal transduction pathways of their hosts, thereby facilitating a
disease. Some signalling modifications, or perhaps the effector proteins themselves are detected by
resistant hosts and trigger a strong defence reaction against the pathogen. Effector proteins
recognized in this way are called avirulence (Avr) proteins.
Inoculation of common bean varieties (Phaseolus vulgaris) with NGR234 results in mainly small,
white, non-nitrogen fixing nodules. We found that in all the commercial varieties tested so far, the
activity of the T3SS is detrimental for efficient nodulation. NGR234 secretes at least nine proteins
in a T3SS dependent fashion: NopA, NopB, NopC, NopL, NopM, NopP, NopX, NopT and NopJ
(Nop stands for Nodulation outer protein). The negative effect of the NGR234 T3SS for the
symbiosis is caused by one or more of 3 effectors depending on the bean variety: NopJ, NopL and
NopT. NopL is a rhizobial protein with no homology to any known motifs. In contrast NopJ and
NopT exhibit partial homologies to Avr proteins of pathogenic bacteria. The fact that the symbiotic
bacterium NGR234 produces homologues of Avr proteins that might be recognized by the plant as
negative effectors prompted us to test whether similar mechanisms of effector recognition operate
for symbiotic bacteria and for pathogenic bacteria. Genetic data using bean varieties known to be
resistant or susceptible to the Avr homologues of NopJ and NopT suggested that NopJ and NopT
are not recognized by the same processes as their Avr counterparts. We are currently searching for
bean proteins targeted by the rhizobial T3SS effectors.
29
Dual role of quorum sensing molecules in swarming motility of Rhizobium etli
K. Braeken and J. Michiels
Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics,
K.U.Leuven, Leuven, Belgium.
Swarming is considered as a bacterial social behavior associated with migration on semi-solid
surfaces. In the presence of extracellular slime, bacteria exhibit a flagella-driven movement on top
of the agar (0.4-1.2 %) enabling them to spread in group over the surface. As for other social
phenomena, quorum sensing (QS) by means of small signal molecules was demonstrated to be
involved in the regulation of swarming. For many Gram-negative bacteria, QS is mainly depended
on N-acylhomoserine lactones (AHLs)-based LuxIR-type systems. Here, we describe a dual role for
the signal molecules in controlling swarming behavior of the bacterium Rhizobium etli, the
symbiotic partner of the common bean plant. Previously, two AHL-based QS systems, cin and rai,
were identified and we demonstrated that mutants in the R. etli cin system are no longer able to
move over a semi-solid surface. By studying expression levels of cin- and rai-gusA fusions induced
by different AHLs and by evaluating the effect of exogenous complementation of swarming
behavior with AHLs, we concluded that the cin system is the major swarming regulator in R. etli.
This system was induced by its cognate AHL as well as by other long chain AHLs. Hence, long
chain AHLs play an important signaling role, as they are required for high-level expression of the
rai and cin QS systems. In contrast, no effect of a cin mutation on swarmer cell differentiation was
observed. By measuring surface activities of AHLs, a second role for the long chain AHLs was
disclosed: as they possess significant surface activity and induce liquid flows, known as Marangoni
flows, as a result of gradients in surface tension at biologically relevant concentrations, these
molecules also fulfill a direct role during swarming as biosurfactants.
30
Dissecting the impaired signalling pathway in a non-nodulating Phaseolus vulgaris mutant
A. L. Ramos, N. Nava, E. Alemán, O. Santana, L. Cárdenas, C. Quinto.
Dept. de Biología Molecular de Plantas, Instituto de Biotecnología, UNAM. Apdo Postal 510-3,
62250. Cuernavaca, Morelos, México
The development of nitrogen-fixing nodules in legume plants involves a subtle, two-way
interaction between a bacterium and its host plant. Legume roots exude flavonoids that induce the
expression of the bacterial nodulation genes, which encode proteins implicated in the synthesis and
secretion of signals called Nod factors (NFs). NFs signal back to the plant root and induce several
responses, leading to bacterial invasion and nodule formation. Herein, the cellular and molecular
characterization of a Phaseolus vulgaris non-nodulating mutant (NN-mutant) is described. Root
hair cells of the NN-mutant plant respond with swelling and branching when inoculated with
Rhizobium etli, even though without curling induction. Furthermore, initiation of cell division in the
outer cortex, or entrapment of bacteria and infection thread formation were not found. Both the
bean wild-type and the NN-mutant showed elevated intracellular calcium changes in the root hairs,
after NFs treatment. Whereas the NN-mutant is deficient in early nodulin gene expression when
inoculated with R. etli, it can be effectively colonized by arbuscular mycorrhizal fungi. Together,
our results indicate that the P. vulgaris NN-mutant is not blocked at the NFs perception level, but
rather at later downstream stages between calcium signaling and early nodulin gene induction. Our
data suggests that this mutation is in a gene homologous to nsp-1 described in Medicago truncatula
that encodes a presumed transcription factor belonging to the GRAS family.
Funded by DGAPA IN204305 and CONACyT U42560-.
31
Molecular diversification of pgip gene family members in Phaseolus vulgaris
Giulia De Lorenzo1, Manuela Casasoli
1, Luca Federici
2, Nicoletta Vella
1, Renato D’ovidio
3, Juan
Fernandez-Recio4, Adele Di Matteo
5, Felice Cervone
1
1Dip. Biologia Vegetale, Università di Roma “La Sapienza”, Italy;
2Ce.S.I. Centro Studi
sull’Invecchiamento and Dip Scienze Biomediche, Università di Chieti “G. D’Annunzio”, Italy; 3Dip. Agrobiologia e Agrochimica, Università della Tuscia, Viterbo, Italy;
4Molecular Modeling
and Bioinformatics Unit, Parc Cientific de Barcelona, Spain; 5Dipartimento di Scienze
Biochimiche, Università di Roma “La Sapienza”, Italy.
Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal
endopolygalacturonases (PGs) that belong to the superfamily of leucine-rich repeat (LRR) proteins
and play a role in defence against necrotrophic fungi. In many species, PGIP is encoded by small
gene families. Pgip families provide an interesting case of adaptive molecular evolution, because
their sequence and structure is the result of co-evolution with PGs of pathogenic organisms.
Moreover, PGIPs, like PGs, are subject to both functional constraints and selection pressure for
diversification. The Phaseolus vulgaris pgip gene family comprises four members that are clustered
in a 50-kb region and, based on their similarity, form two pairs of paralogous genes
(Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4); these genes show both partial redundancy and sub-
functionalization against fungal and insect PGs. We have studied intra-specific nucleotide
variability in pgip genes of wild P. vulgaris accessions from the three centres of origin and
diversification of this species in Central and South America. A different evolutionary dynamics is
apparent in the four family members: Pvpgip1 is likely evolving to a pseudogene, Pvpgip2 e
Pvpgip3 are very conserved, while Pvpgip4 is the most variable. Models of codon evolution
showed evidence of positive selection in the 3rd
LRR of Pvpgip3. Analysis of 50 dicot pgip genes
revealed that legume pgips cluster separately from the other dicots genes. More than 20 positively
selected sites were identified, mainly located at the concave face of the LRR solenoid. A different
and unrelated approach, i.e. the Optimal Docking Area (ODA) method, was used to predict the
propensity of surface residues to be involved in protein-protein interactions. A significant
correlation was observed between the ratio ω of non-synonymous (dN) and synonymous (dS) substi-
tution rates and ODA values leading to a cross-validation of both approaches and to the prediction
of sites involved in the recognition of fungal PGs. These analyses provide useful information for
site-directed mutagenesis studies aimed at defining structure-function relationship of these proteins
and developing PGIPs with novel recognition capabilities.
32
Sequence variation and functional analysis of PvPGIP2 in common bean (Phaseolus vulgaris
L.) and related species
Michela Janni
1, Valentina Rocchi
1, Anna Farina
1, Stefano Benedettelli
2, Giulia De Lorenzo
3,
Renato D'Ovidio1
1Dept Agrobiologia e Agrochimica, Univ. della Tuscia, Viterbo, Italy;
2Dept Scienze Agronomiche
e Gestione del Territorio, Univ. di Firenze, Firenze, Italy; 3Dept di Biologia Vegetale, Univ. La
Sapienza, Roma, Italy
PGIPs are extracellular plant protein inhibitors of endopolygalacturonases (PGs). These proteins
belong to the leucine-rich repeat (LRR) family and are encoded by small gene families. In bean, the
Pgip family is composed by four expressed members among which Pvpgip2 transcript is the more
abundant and its encoded product is the most wide-spectrum and efficient inhibitor. In order to gain
information on the structure-function relationship of this protein, we have analysed a number of
wild bean (P. vulgaris) accessions and cultivated genotypes. Our analyses indicate that the protein
encoded by Pvpgip2 is well conserved. Most variants contains synonymous substitutions and only
two accessions show non synonymous replacements. A slightly larger variation has been found by
analysing the pgip2 gene in the related bean species P. coccineous, P. acutifolius and P. lunatus.
The most pronounced variability was observed between PvPGIP2 and PlPGIP2, with 9 non
synonymous substitutions, while the lower polymorphism was between PvPGIP2 and PcPGIP2,
with 5 non synonymous substitutions.
All polymorphic pgip2 genes were expressed in Nicotiana benthamiana using PVX as vector and
the inhibitory activity of the transiently expressed proteins were compared. Inhibitory assays
demonstrated that the PGIP2 variants possess slightly different efficiencies. These results will be
discussed taking into account the position of amino acid substitutions.
33
Functional genomics of beans under abiotic stress conditions
G Hernandez*, M Ramírez*, O Valdés-López*, M Lara*, MA Graham**, M Tesfaye***, M
Wandrey†, T Czechowski†, A. Schlereth†, F Cheung‡, HC Wu‡, CD Town‡, PM Reddy*, L
Girard*, JL Reyes&
, F Sánchez&
, M Udvardi†&&
, CP Vance***&&&
* Centro de Ciencias Genómicas-Universidad Nacional Autónoma de México, Cuernavaca, México
** USDA-ARS, Corn Insects and Crop Genetics Research Unit, Ames, IA, USA
*** University of Minnesota, St. Paul, MN, USA
† Max Planck Institute for Molecular Plant Physiology, Golm, Germany
‡The Institute for Genomic Research (TIGR), Rockville, MD, USA
& Instituto de Biotecnología- Universidad Nacional Autónoma de México, Cuernavaca, México
&& Samuel Robert Noble Foundation, Ardmore, OK, USA
&&& USDA-ARS, Plant Research Unit, St. Paul, MN, USA USDA-ARS, Plant Research Unit, St.
Paul, MN, USA
Our research in collaboration with groups from different institutions/countries, has contributed to
develop resources for common bean functional genomic research, such as: EST sequences
(Ramírez et al. 2005) and the common bean gene index (www.tigr.org), transcriptomics,
transcription factors profiling platform and reverse genetics to modulate specific gene expression.
We are using functional genomic approaches to investigate the responses of bean to abiotic stress
such as phosphorus (P) deficiency and metal toxicity. Macroarray hybridization analysis has been
performed to define relevant genes differentially expressed in roots and nodules of bean plants
grown under phosphorus deficiency (-P) as compared to those grown under normal P conditions
(+P). We performed a complementary in silico study by combining bioinformatics analysis with
available micro/macroarray data, and have identified 52 P. vulgaris candidate genes as induced by
P-stress response (Graham et al. 2006). We have developed a common bean transcription factors
(TF) profiling platform based in quantitative real-time RT-PCR, consisting of 372 TF TCs/ESTs
identified from TIGR common bean Gene Index. Using this platform we will perform TF transcript
profiling of different bean plants grown under various stress conditions, beginning with roots and
nodules from P deficient bean plants. We are using the system of composite bean plants with
transgenic root system induced by Agrobacterium rhizogenes to determine – via overexpression or
RNAi technologies- the functions/relevance of selected candidate genes in nodulation, abiotic stress
and gene regulation networks (TF genes).
34
Molecular Characterization of Common Bacterial Blight Resistance Genes in Phaseolus
vulgaris
Perry, GE, Reinprecht, Y, Chan, J and Pauls, KP
Department of Plant Agriculture, University of Guelph, Guelph Ontario, Canada N1G 2W1
Common bacterial blight (CBB) is endemic to all regions of the world where dry beans are
cultivated. The disease is caused by the bacterium, Xanthomonas axonopodis pv. Phaseoli, and
results in reduced seed yield and the contamination of future seed. CBB resistance has been studied
for a number of years, and has led to the development of several lines that have resistance to X.
axonopodis pv. Phaseoli. Recently, a CBB-resistant cultivar, OAC-Rex was developed.from a
cross between HR20-728 and MBE 7 made in 1988. MBE 7 was a selection from the cross of ICA
Pijao/PI 440795//Ex Rico 23 and was used to provide the CBB resistance. Another CBB-resistant
line, HR67, was produced by a series of crosses between Centralia, HR13-621, OAC Rico and
XAN159. A number of molecular markers have been identified for various lines for the resistance
genes in these lines In HR67, the marker BC420 on linkage group B7 has been associated with
CBB-resistance and in OAC Rex identified the markers Bng21, Bng71 and pvCTT001 on linkage
groups B3, B4 and B5, respectively, are associated with resistance. The objectives of the current
project are to 1) produce BAC libraries with OAC Rex and HR67, 2) screen the libraries with the
markers to identify clones containing the genes for resistance and 3) test the effectiveness of the
clones to reduce CBB symptom expression when transiently expressed by Agroinfiltration. We
expect that the studies will identify clones for sequencing to ultimately allow identify CBB
resistance genes in Phaseolus.
35
Communicating grain legume research
N. Terryn, M. Van Montagu and Anne Schneider*
Institute for Plant Biotechnology for Developing Countries, UGent, Belgium *) European
Association for Grain Legumes (AEP), Paris, France
Authors also represent AEP and the GLIP consortium
Within all kind of research the need to disseminate your results to both other scientists and the
community at large is always considered as important, but often neglected. Science communicators
play the crucial role to act as facilitators between scientists and the broader public, linking and
transforming scientific information into a format understandable to the media and a general
audience. In addition, disseminating the information is the first step to make the results being
exploited into various applications, as well as to get feedback or questioning for further
investigations.
For research in Phaseolus and grain legumes in general, researchers need to communicate on their
work not only with fellow scientists, but also to the stakeholders like breeders and farmers, as well
as to the press and decision makers, as to make sure they understand the importance of the research
that has been performed in the light of sustainable development in agriculture. Dissemination of
information and communication adapted to each audience are therefore also important to secure
further funds for your project.
The Grain Legumes Integrated Project (GLIP) is a large multi-national project co-funded by the 6th
RTD Framework Programme of the European Union (FP6), striving to develop new strategies to
enhance the use of grain legumes crops in food for human consumption and animal feed in Europe
and beyond. A final dissemination event will be held in Lisbon, 12-16 Nov 2007, where other
groups working on legume are also invited to disseminate their results.
Specialised organisations like AEP, the European association for grain legumes research, but also
its members and any legume scientist all over the world can help to broker between scientists and
end-users from economic sectors. To facilitate this, a specific platform has been established, named
the Technology Transfer Platform, with a priority for the plant breeding area.
For more information check following websites:
www.grainlegumes.com
www.eugrainlegumes.org
36
Phaseolus and Internet
M. Nenno, Vimodrone (MI), Italy, web site: www.nenno.it
The internet came into beeing as the “network of networks” more than 40 years ago but only when
Tim Berners-Lee and Robert Cailliau “invented the Web” in 1992, it was ready to build up what
today is becoming the most important resource of information and communication. In 2006, the
number of websites has reached 100 millions, hosting any kind of data and images including a
substantial amount of information relevant for scientific research. Applications like web browers,
email, and mailing list are used daily and presention of workgroups and online database searches on
institutional websites are common tasks. The new opportunities that the internet offers are blogs,
Wikis, social bookmarking, communities, and portals enabling everybody to publish in less time
and with less effort (Web 2.0).
How did the Phaseolus research community profit from these possibilities? Most of the specific
information about Phaseolus can be found on genebanks sites like e.g. CIAT, GRIN, ECP/GR
Phaseolus, Phaseolinae Collection, IPK, and ILCB as well as in sequence and mapping databases
like e.g. EMBL, NCBI, Phaseolus coccineus EST Project, and BeanGenes. The number of
Phaseolus specific websites and link directories (BeanRef, Beangenes site) is relatively small.
BeanRef, a collection of links to sites with different aspects of research on beans, was opened in
1995 and is a helpful resource until today. The Bean Seed Images site (online since 2005) is the
only collection of Phaseolus seeds images with a systematic overview of detailed seed pictures for
all Phaseolus species available today, although, some genebank databases have also images as
supplementary information such as CIAT and GRIN.
In order to find potential grant partners the web sites of workgroups like the Gepts Lab, the
Phaseolus Seed Protein Group (PSPG), the Phaseomics global initiative or the repository of the
Annual reports of the Bean Improvement Cooperative (BIC) can be an interesting starting point.
The latest online resource for Phaseolus is the Legume Message Board site which can be used as an
online platform to discuss the different research topics.
For those who are interested in related species or comparisons with related genomes some valuable
resources are the ILDIS, Medicago and LIS site.
Links on: http://www.nenno.it/Beanref/
37
POSTERS
1. When gene flow counteracts domestication: the case of common bean (Phaseolus vulgaris L.) Maria I. Chacón S., Rosa I. González and Daniel G. Debouck
2. Evolutionary history of wild Lima bean (Phaseolus lunatus L.) and the history of its domestication in the Americas Jenny R. Motta, Martha L. Serrano, Genis Castillo, Daniel G.
Debouck & Maria I. Chacón S.
3. Phylogeographic analysis of the chloroplast DNA variation in wild common bean (Phaseolus vulgaris L.) in the Americas Maria I. Chacón S., Barbara Pickersgill, Daniel G.
Debouck & J. Salvador Arias
4. Genetic diversity in a common bean (Phaseolus vulgaris L.) ex situ collection of Italian landraces. B. Tiranti, L. Macaluso, P.L. Spagnoletti Zeuli and V. Negri
5. Genetic variation and structure of a Phaseolus coccineus L. collection. B. Tiranti, G.
Spataro, P. Arcaleni, G. Attene, R. Papa, P. Spagnoletti Zeuli, V. Negri
6. What can a landrace case study tell us about adaptation traits? B. Tiranti, V. Negri
7. Functional genomics in beans (Phaseolus vulgaris L.): the Phaseomics consortium. Emmanuel Jayko Jaiyeola
8. Genetic diversity in common bean core collection of INIFAP-México. Homar R. Gill-
Langarica, M. L. Patricia Vargas-Vázquez, José S. Muruaga-Martínez, Patricia Pérez-Herrera,
Rigoberto Rosales-Serna & Netzahualcoyotl Mayek-Perez
9. Management of some Italian common bean landraces maintained on-farm. A.R.
Piergiovanni, L. Lioi
10. Genetic diversity of common bean (Phaseolus vulgaris L.) ecotype “fagiolo di Controne” revealed by SSR and ISSR molecular markers: a preliminary study. L. del Piano, C.
Sorrentino, C. Capone, M. Abet, A. Cuciniello
11. Variation for AFLP in a European ‘core collection’ of common bean (Phaseolus vulgaris l.). Logozzo Giuseppina, Donnoli Rosa, Spagnoletti Zeuli Pierluigi
12. Molecular analysis of the Phytic acid pathway in common bean Fileppi M, Galasso I,
Campion B, Daminati MG, Bollini R, Sparvoli F
13. PROM – National research project for the improvement of vegetable crops for Southern-Italy. Campion B, Fileppi M, Lioi L, Piergiovanni AR, Carbonaro M, Lo Scalzo R, Galasso I,
Tagliabue G, Daminati MG, Sparvoli F, Bollini R
38
When gene flow counteracts domestication: the case of common bean (Phaseolus vulgaris L.)
Maria I. Chacón S.1, Rosa I. González
2 and Daniel G. Debouck
2
1Universidad Nacional de Colombia, Facultad de Agronomía, Bogotá, [email protected];
2CIAT Genetic Resources Unit, Cali, Colombia.
Domestication is the human-driven process by which our crop plants originated. The three main
evolutionary forces that act during domestication are: unconscious and/or conscious selection,
founder events in time and space, and gene flow between crop and wild relatives. Domestication
may occur once or on multiple occasions, at one or several locations, and given enough time and
geographic isolation, domesticated races may evolve in a pre- or post-domestication context. In the
first one, races evolve from multiple domestications of wild populations already genetically
divergent and locally adapted, and in the second one, races evolve from a unique domestication
event followed by geographic diffusion and “secondary domestications”. Secondary domestications
are conversions of local wild relatives to cultigens through hybridization with introduced
domesticated types. Here we report recent evidence for multiple and secondary domestications in
wild common bean, gene flow between the bean crop and its wild relative and document the
opposite effects of these two forces on the genetic diversity in this crop. This evidence is based on
analyses of chloroplast DNA polymorphisms and microsatellite (SSR) alleles. In the Andes, wild
beans seem to have been domesticated once in Southern Peru followed by post-domestication
differentiation into races (races Peru, Nueva Granada and Chile) (the post-domestication context).
On the other hand, in Mesoamerica, multiple domestications of different wild populations and
secondary domestications may have taken place, giving rise to the Mesoamerican races (races
Mesoamerica, Durango, Jalisco and Guatemala) (the pre-domestication context). In general,
domestication events introduced a strong founder effect in Mesoamerica and in the Andes.
Opposing to this, wild and domesticated beans may experience gene flow resulting in what is called
wild-weed-crop complexes. These complexes have been observed along the range of distribution of
wild beans. We have analyzed chloroplast haplotypes and SSR alleles typical of wild populations
and also the presence of these molecular polymorphisms in the different elements of the complexes.
The results suggest that chloroplast capture and flow of SSR alleles have taken place in these
complexes and in both directions (wild to domesticated and vice versa, the former direction being
predominant), thus enriching the genetic diversity of these complexes over time.
39
Evolutionary history of wild Lima bean (Phaseolus lunatus L.) and the history of its
domestication in the Americas
Jenny R. Motta1, Martha L. Serrano
1, Genis Castillo
2, Daniel G. Debouck
3 &
Maria I. Chacón S.4
1 Escuela de Biología, Universidad Industrial de Santander, Carrera 27 Calle 9, Bucaramanga,
Colombia; 2 Laboratorio CINBIN, Escuela de Biología, Universidad Industrial de Santander,
Carrera 27 Calle 9, Bucaramanga, Colombia; 3 Unidad de Recursos Genéticos, CIAT, Km 13 via
Cali-Palmira, Colombia; 4 Facultad de Agronomía, Universidad Nacional de Colombia, Bogotá,
D.C., Colombia.
Lima bean (Phaseolus lunatus L.) is one of the five cultivated Phaseolus bean species, which
originated in the Neotropics. Lima beans have two recognized varieties, var. silvester and var.
lunatus that refer to the wild and domesticated Lima beans, respectively. Within the silvester
variety there are two gene pools, the Mesoamerican and the Andean pool. These two gene pools are
characterized by contrasting geographical distributions, morphological, biochemical and molecular
traits. The patterns of genetic differentiation between these two gene pools may have arisen partly
as a result of the geological history of the Americas (e.g. the relatively recent closure of the Isthmus
of Panama (7 mya) and the relatively recent uplift of the Andes) and partly as a result of the
historical and current patterns of gene flow. It has been suggested that the species has an Andean
origin and very little is known on how it reached its current geographical distribution in South
America, Central America (including the Caribbean) and Mesoamerica. On the other hand, very
recently in the history of the species (about 8,000 years ago), wild Lima beans populations were
brought into cultivation and eventually were domesticated giving rise to three cultigroups: two
small-seeded ones called Sieva and Potato (their place of origin is still controversial) and one large-
seeded called Big Lima of Andean origin (Ecuador-northern Peru). It is widely known that the
domestication process has important consequences on genetic diversity and phenotypic traits (e.g.
domestication syndrome), therefore the importance to determinate where and how many
domestication events took place in a given species. In this research, we propose: (1) to assess the
hypothesis of an Andean origin of wild Lima beans, (2) to study the history of migrations of wild
Lima beans among Mesoamerica and South America, and (3) to study domestication patterns in
Lima beans and the effect of domestication on genetic diversity. To approach these questions, we
are using a variety of statistical and analytical tools in the fields of phylogeography, population
genetics and phylogenetics, to (a) uncover relationships among wild Lima beans and related species
from Mesoamerica and South America, (b) date divergence times among wild Lima beans and
closely related species, (c) define relationships among lineages within wild Lima beans and among
wild and domesticated Lima beans, and (d) study population structure of a representative sample of
wild and domesticated Lima beans. We will show preliminary results of a pilot study on
polymorphisms of 11 non-coding chloroplast regions and the internal transcribed spacer of the
ribosomal DNA (ITS). So far, two cpDNA regions (the atpB – rbcL and the trnL – trnF intergenic
spacers) and the ITS region have shown informative intra- and interspecific variation.
Note: Jenny R. Motta and Martha L. Serrano have contributed equally to this study.
40
Phylogeographic analysis of the chloroplast DNA variation in wild common bean (Phaseolus
vulgaris L.) in the Americas
Maria I. Chacón S.1, Barbara Pickersgill
2, Daniel G. Debouck
3 & J. Salvador Arias
4
1Facultad de Agronomía, Universidad Nacional de Colombia, Colombia, South America. E-mail:
Department of Agricultural Botany, School of Plant Sciences, The
University of Reading, Whiteknights, PO Box 221, RG6 6AS, UK; 3 Genetic Resources Unit,
Centro Internacional de Agricultura Tropical, CIAT, A. A. 6713, Cali, Colombia; 4 Grupo de
Biodiversidad, Escuela de Biología, Universidad Industrial de Santander, Carrera 27 Calle 9,
Bucaramanga, Colombia.
The wild common bean (Phaseolus vulgaris L.) being an original floristic component of montane
subhumid forests is widely but discontinuously distributed from northern Mexico to northern
Argentina on both sides of the Isthmus of Panama. Little is known on how the species has reached
its current disjunct distribution. In this research, chloroplast DNA polymorphisms in seven non-
coding regions were used to study the history of migration of wild P. vulgaris between
Mesoamerica and South America. A penalized likelihood analysis was applied to previously
published Leguminosae ITS data to estimate divergence times between P. vulgaris and its sister
taxa from Mesoamerica, and divergence times of populations within P. vulgaris. Fourteen
chloroplast haplotypes were identified by PCR-RFLP and their geographical associations were
studied by means of a Nested Clade Analysis and Mantel Tests. The results suggest that the
haplotypes are not randomly distributed but occupy discrete parts of the geographic range of the
species. The current distribution of haplotypes may be explained by isolation by distance, and by at
least two migration events between Mesoamerica and South America: one from Mesoamerica to
South America, and another one from northern South America to Mesoamerica. Age estimates
place the divergence of P. vulgaris from its sister taxa of the Phaseoli section from Mesoamerica at
or before 1.3 Ma, and divergence of populations from Ecuador-northern Peru at or before 0.6 Ma.
As these ages are taken as minimum divergence times, the influence of past events, such as the
closure of the Isthmus of Panama and the final uplift of the Andes, on the migration history and
population structure of this species cannot be disregarded.
41
Genetic diversity in a common bean (Phaseolus vulgaris L.) ex situ collection of Italian
landraces.
B. Tiranti*, L. Macaluso**, P.L. Spagnoletti Zeuli** and V. Negri*
* Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali e Zootecniche (DBVBAZ),
Università di Perugia, Borgo XX giugno 74, 060121 Perugia, Italy - [email protected]
** Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli Studi della
Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy.
Landraces (LRs) are vital genetic resources for breeding purposes, diversification of production,
developing new farming systems and new quality products. The extent and distribution of the
genetic diversity in a crop depends on its breeding system, geographical, ecological and human
factors. Conservation of genetic variability is essential for present and future human well-being.
To date, the in situ or ex situ conservation strategies have been applied with little information on the
genetic diversity that is being conserved. In order to improve use and management of conserved
germplasm, it is necessary to understand the genetic diversity that is present in collections.
The DBVBAZ presently maintains a relative large collection of germplasm (over 5000 accessions)
under long term storage conditions (-18°C, vacuum sealed seed packets). The amount and the
regional distribution of genetic diversity relative to 19 Italian LRs were assessed using different
approaches that included morphological (international descriptors), biochemical (phaseolin seed
proteins) and molecular analysis (Simple Sequence Repeats markers). Results obtained showed a
wide variation overall morphological traits, especially seed characters. The three major phaseolin
types were found, ‘C’ (38.9%), ‘S’ (33.1%) and ‘T’ (28.0%) types. Nine of ten loci analysed were
polymorphic and 82 different alleles were detected overall SSR loci (average per locus: 8.2 alleles).
The wide variation observed shows how rich and interesting the Italian germplasm is. Our findings
offer the opportunity to rationalize the collection, to develop a core collection and to exploit these
resources for valuable traits.
42
Genetic variation and structure of a Phaseolus coccineus L. collection.
B. Tiranti*, G. Spataro*, P. Arcaleni*, G. Attene**, R. Papa***, P. Spagnoletti Zeuli****, V.
Negri*
* Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali e Zootecniche, Università di
Perugia, Italy- [email protected]; ** Dipartimento di Scienze Agronomiche e Genetica Vegetale
Agraria Università of Sassari, Italy; *** Dipartimento di Scienze degli Alimenti, Università
Politecnica delle Marche, Ancona, Italy; **** Dipartimento di Biologia, Difesa e Biotcnologie
Agro-Forestali, Università degli Studi della Basilicata, Potenza, Italy.
P. coccineus L. (2n = 2x = 22) originated in Mexico and Central America. It was introduced in
Europe after the discovery of America and, nowadays, is widely cultivated because of its suitability
to cool environments.
At the present, few information about the level of genetic diversity of the whole European
germplasm of P. coccineus are available, even thought investigations on genetic variation of some
populations from Spain, Central Italy and Poland were carried out. All these studies detected a high
level of polymorphism.
Aims of this study were to :
a) obtain a core collection representative of the world wide variation held in germplasm banks,
b) investigate its genetic variation by using molecular markers (SSR),
c) compare the level of genetic diversity in groups of accessions coming from European and
Meso-America,
d) improve the knowledge of the evolutionary process caused by introduction in Europe,
e) obtain an overall picture of diversity which can be useful for future plant breeding activities.
The SSRs used were efficient markers in detecting P. coccineus diversity. The obtained core
collection appears to be representative of the species diversity and can be useful in future studies.
Data based on it are in agreement with Meso-America being the domestication centre of the species
and with its later introduction in Europe. In the area of original adaptation and domestication
germplasm shows a wide diversity and appears to be continuously enriched by introgression from
wild forms. Introduction in Europe, which was presumably realised with a few seeds and LRs,
caused a consistent reduction of diversity. Drift, selection and lack of relevant migration among
European populations triggered a differentiation process from original gene pool which appears to
be in progress. Genetic diversity of European and Italian germplasm deserves to be preserved
through in situ ed ex situ conservation programs in order to allow future plant breeding activities
aimed at developing varieties suitable to the European environment.
43
What can a landrace case study tell us about adaptation traits?
B. Tiranti, V. Negri
Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali e Zootecniche, Università di
Perugia, Borgo XX giugno 74, 060121 Perugia, Italy- [email protected]
Awareness of the need for biodiversity conservation is now universally accepted. To date,
conservation activities have mainly focused on ex situ and in situ conservation of wild species.
However, the diversity between and within crop species also has a significant value. In
domesticated crops, landraces have been, and still are, the primary source of genetic diversity for
plant breeding. As such, landraces are vital genetic resources which should be maintained on farm
and ex situ for future use. Few it is known about the organization of landrace diversity and about
the forces acting in shaping it, although this knowledge is fundamental for breeding and
conservation activities. A primary aim of this study was to obtain an insight in how variation has
been built on under a cultivated environment and to identify loci that potentially underlie selective
effects by using a landrace case study whose natural and human environment is known in details.
Another aim of this study was to define an appropriate on farm conservation strategy for this
threatened bean landrace, which can serve as a model for other threatened populations. Farmer seed
lots of this landrace were examined for 18 morpho-physiological traits and 28 SSR molecular
markers. Significant differences were found among them for both the morpho-physiological and
molecular traits. A high level of genetic diversity and a significant genetic structure was detected
among the farmer’s populations (Fst = 0.367). The landrace appears to be structured as a
metapopulation in which a substantial differentiation is maintained at the subpopulation level.
Evidence of locus-specific selective effects was obtained for four of the thirteen loci-differentiating
subpopulations by either one of the statistical tests used (DetSel, Fdist2). Both the statistical tests
showed one of these loci to be under selective pressure due to altitudinal gradient. Our data suggest
that differential micro-environmental selection pressures and drift explain the observed pattern of
LR diversity. An appropriate on farm conservation of a structured LR requires that subpopulations
be maintained on the farms from which they come.
44
Functional genomics in beans (Phaseolus vulgaris L.): the Phaseomics consortium
Emmanuel Jayko Jaiyeola
Rose Kali International Senior Secondary School, Banjul, Gambia
Fifty percent of the grain legume consumed worldwide are beans ( Phaseolus sp.). Common bean
(P. vulgaris ) is economically and nutritionally important as a primary source of protein in the
human diet in several countries. That makes common bean an important candidate for genomic
studies. An international consortium called Phaseomics ( Phaseolus genomics) has been formed to
establish the necessary framework of knowledge and materials for the advancement of bean
genomics. A major goal of Phaseomics is to generate new common bean varieties that are not only
suitable for but also desired by the local farmer and consumer communities. As part of the global
project of Phaseomics, the research efforts from our group are oriented towards: i) establishing an
efficient genetic transformation system for P. vulgaris and ii) sequencing ESTs from different bean
cDNA libraries and performing expression analyses. In collaboration with C.P. Vance,
approximately 3000 EST’s were sequenced from a common bean root nodules cDNA library. The
source of the RNA for library preparation was 15 day-old nodules of P. vulgaris cv Negro Jamapa
81, induced by Rhizobium tropici CIAT899. Clones were sequenced from the 5’-terminus and
analyzed by BLASTX. The ESTs were grouped into four main functional categories: metabolism
(24.7%), development (23.9%), interaction with the environment (21.6%) and unknown function
(29.7%). Expression will be measured via macro and micro-arrays. Other cDNA libraries, from
young pods and from P-limited roots of P. vulgaris cv. Negro Jamapa plants, have been prepared.
45
Genetic diversity in common bean core collection of INIFAP-México
Homar R. Gill-Langarica
1, M. L. Patricia Vargas-Vázquez
2, José S. Muruaga-Martínez
2, Patricia
Pérez-Herrera2, Rigoberto Rosales-Serna
3 & Netzahualcoyotl Mayek-Perez
1*
1Centro de Biotecnología Genómica-Instituto Politécnico Nacional. Reynosa, México. C. P. 88710.
e-mail: [email protected]; 2 INIFAP-Campo Experimental Valle de México. Chapingo, México. C.
P. 56230; 3INIFAP-Campo Experimental Valle del Guadiana. Durango, Dgo. México. C. P. 34170.
** Financial support by CONACYT and SIP-IPN.
In México common bean (Phaseolus vulgaris L.) is a popular food cultivated in 2.2 million hectares
annually. Limited use of common bean genetic diversity has caused low yield increments in genetic
breeding programs. There are 7,846 accessions at INIFAP’s (National Institute for Forestry,
Agriculture and Livestock Research) common bean gene bank. A subset consisting in 200
accessions were selected to represent the gene bank’s entire common bean collection. Selection was
made considering seed color and germplasm origin. Objective was to evaluate genetic diversity
included in a subset of 200 common bean accessions selected as INIFAP´s core collection.
INIFAP´s core collection was morpho-agronomically characterized during 2003 using 45 traits
including passport data, phenology, leaf characters, plant architecture, yield components, seed
quality and disease reaction (anthracnose, rust, common bacterial blight, halo blight, angular leaf
spot and white mold). Four AFLP (Amplified Fragment Length Polymorphisms) oligonucleotide
combinations were also used to evaluate core collection genetic diversity. Ten common bean bred
cultivars released by INIFAP and 30 landraces from Oaxaca, Veracruz and Chiapas were included
as outgroups. Morpho-agronomical data were used to perform a principal component analysis
(PCA). AFLP data were used to obtain the number of amplified and polymorphic AFLP products,
genetic diversity index (DI) and Neighbor-Joining cluster analysis. Geographical origin (State and
altitude) was considered for the cluster analysis. The PCA showed low values for PC1 (12.7%) and
PC2 (12.5%) due the high variation observed in the core collection. Three main accession groups
were found: Group I included Nueva Granada race cultivars with determinate bush (Type I) growth
habit; Mesoamerican black seeded cultivars formed group II and group III included Jalisco and
Durango races cultivars. AFLP analysis detected 530 amplified products, from which 469 (89.3%)
resulted polymorphic. Highest DI values were found in accessions from Jalisco (35%) and
Aguascalientes (33%). Lowest DI values were observed in landraces from Oaxaca (20%) and bred
cultivars (22%). Cluster analysis based on States of origin showed two major groups. Group A with
two subgroups: A1 included accessions from Central México and related plant introductions from
Tamaulipas and A2 formed with accessions from Michoacán, landraces from Oaxaca, Veracruz and
Chiapas and bred cultivars. Group B included accessions from Central and Northern México
(subgroup B1) and Central and Southern México (subgroup B2). A third, more diverse, group for
accessions from Zacatecas was observed. Cluster analysis based on collecting site altitude showed
clear separation among accessions from core collection, landraces and bred cultivars. Accessions
collected in altitudes from 0-500 masl and 501-1000 masl showed highest values for genetic
similarity. Increasing values were observed for genetic distance among accessions according to
increments in collecting site altitude. Genetic Diversity Complex observed in Central México
influenced common bean diversity in Northern and Southern México. Genetic similarities detected
between accessions collected in contrasting regions resulted from an intensive common bean seed
exchange and gene flow. Collecting site altitude and State origin influenced accessions clustering.
Results showed that INIFAP’s common bean core collection is truly representative of the many
diverse environments in which beans evolved and are grown in México. Results in core collection
molecular analysis and morpho-agronomical characterization will be used in broadening common
bean genetic base in México and elsewhere.
46
Management of some Italian common bean landraces maintained on-farm
A.R. Piergiovanni, L. Lioi
Istituto di Genetica Vegetale, IGV, CNR, Bari, Italy
Common bean (Phaseolus vulgaris L.) is a traditional crop in Italy, where farmers grow landraces
selected and maintained by themselves. One of the strategies applied to the safeguard of the crop
genetic resources is the on-farm conservation, defined as the continued cultivation and management
of landraces by farmers in the agro-ecosystem where the crop evolved. Possible on-farm
conservation should rely on an accurate study of level of landrace diversity in order to define the
most appropriate strategy of safeguard. In respect of this, examples from different cases identified
during Italian common bean germplasm study are briefly discussed.
In Italy, common bean cultivation started in the Belluno province (northern Italy) in the XVI
century, where still today four ‘borlotto’ types named Fagioli di Lamon are cultivated. About fifty
years ago, a seed sample from Lamon, was probably introduced in the Cuneo province (Piedmont
region, northern Italy) giving rise to the ecotype named Billò. A multidisciplinary approach
comparing the Billò and the Lamon common beans was undertaken to evaluate either the rightness
of the Billò provenance or the genetic differentiation attributable to farmer and environment
selective pressures. At Gradoli and Acquapendente (Viterbo province, central Italy) a white small-
seeded common bean named Fagiolo del Purgatorio is cultivated since XVIII century. The
screening of 23 populations belonging to this ecotype showed that two distinct nuclei constituted its
genetic structure. Consequently, safeguard initiatives correctly designed should be finalized to
avoid the loss of one of them. An appreciable genetic differentiation was detected among
populations of Poverello bianco, a landrace cultivated in both Pollino massif slopes (Basilicata and
Calabria regions, southern Italy) by using molecular markers. The landraces named Cioncone and
Fagiolo pane aquilano cultivated in two close valleys located in central Italy (Valle Aniene and
Valle Peligna, respectively) represent an example of different names used by local farmers to
designate the same genetic material. Finally, how the attribution of PGI, one of the quality marks
introduced by EU, to a group of ecotypes cultivated in the same area can affect their survival is
discussed for more than 10 ecotypes named Fagioli di Sarconi (Basilicata region, southern Italy).
These examples evidenced that the interaction among rural communities, national and local
research institutions is fundamental to a correct management of the maintained on-farm ecotypes.
Research partially supported by Ministry of Agricultural Alimentary and Forest Politics with funds
released by C.I.P.E. (Resolution 17/2003).
47
Genetic diversity of common bean (Phaseolus vulgaris L.) ecotype “fagiolo di Controne”
revealed by SSR and ISSR molecular markers: a preliminary study
L. del Piano, C.Sorrentino,.C. Capone, M. Abet, A. Cuciniello
Istituto Sperimentale per il Tabacco, CRA, Scafati (SA) Italy
Analysis of genetic diversity within populations could be a great benefit to plant genetic resources
conservation. The French bean Phaseolus vulgaris L. ecotype “fagiolo di Controne” is a valuable
crop grown near the Calore river, in the province of Salerno (Campania, Italy). In order to reveal
genetic diversity both within and among populations of domesticated Italian common bean, seven
different populations of “fagiolo di Controne” and some commercial and typical italian varieties,
were investigated by mean of Simple Sequence Repeats (SSR) and Inter Simple Sequence Repeats
(ISSR) molecular markers.At present four SSR primers, obtained by current literature, specific to a
given place in the genome, were tested. The three primers designed for related but different
phytoemagglutinin gene sequence, produced a single amplification product which characterizes
only “Marconi”, a variety of green bean. The fourth primer, designed on cellulase gene sequence,
generated bands which characterize two typical varieties of Campania.
ISSR analysis was performed with five different primers, previously selected on the basis of clarity
and reproducibility of bands. A very low polymorphism was revealed within and among the seven
populations of “fagiolo di Controne”. The cluster analysis, based on ISSR polymorphic fragments,
grouped the populations of “fagiolo di Controne” together with “Coco” and “Canari” ecotype
common beans. The collected data are going to be improved with some other SSR and ISSR
primers in order to have more information useful for domesticated landraces “fagiolo di Controne”
germoplasm characterization.
48
Variation for AFLP in a European ‘core collection’ of common bean (Phaseolus vulgaris l.)
Logozzo Giuseppina, Donnoli Rosa, Spagnoletti Zeuli Pierluigi
Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli Studi della
Basilicata, viale dell’Ateneo Lucano, 10 – 85100 Potenza, Italy - [email protected]
Common bean (Phaseolus vulgaris L.) was introduced in Europe from both Mesoamerican and
Andean centres of origin. In a previous study a European collection (n=544) was characterized by
phaseolin patterns and morphological traits to investigate the contribution of the two gene pools in
European germplasm. A European bean core collection (23 countries – accessions ‘C’ type = 33%,
‘T’ type = 35.7% and ‘S’ type = 31.3%) was developed and validated using random-stratified
sampling by logarithm of frequency of phaseolin pattern based on phaseolin patterns and on the
information available in the genebank databases to assess the contribution of the two American
gene pools to the European germplasm and their relative importance for breeding purposes. At the
DNA level, the core collection (n=300) and a set of American accession (n=82) have been
fingerprinted using 4 AFLP primer combination markers: E-AGT/M-GAC, E-AGT/M-GTA, E-
ACC/M-AGA and E-ACC/M-ATG. They gives access to a very large range of polymorphisms,
because of access to the complete genome. Across all primer combinations, fragment size ranged
from 79 to 422 bp. The profiles yielded a total of 221 polymorphic scorable bands, with an average
of 55.25 bands per primer combination. 209 fragments were polymorphic in European gene pool,.
129 in Meso-American gene pool and 187 in Andean gene pool: 40 bands were common among all
Phaseolus germplasm. Nei’s gene diversity and Shannon’s index were higher in European
accessions (He=1.17 and SDI=1.29) than American gene pools. European and Andean gene pools
were minor genetic distance (0.004). Multivariate analysis (UPGMA) using the AFLP data
clustered 71% of accessions in 2 distinct groups and comparison with phaseolin types and passport
data showed for each cluster the clear contribution to Meso-American and Andean centres of origin
to European gene pool.
49
Molecular analysis of the Phytic acid pathway in common bean (Phaseolus vulgaris L.)
M. Fileppi*, I. Galasso**, B. Campion*, M.G. Daminati,** R. Bollini**, F. Sparvoli**
* Istituto Sperimentale per l’Orticoltura, Montanaso Lombardo, Lodi, Italy
** Istituto di Biologia e Biotecnologia Agraria, CNR, Milano, Italy
Phytic acid is the major form of P storage in the seeds and acts as an antinutrient for humans and
monogastric animals since it is able to chelate cations such as Fe, Ca, Zn. Therefore, there is a
strong interest to develop Phaseolus vugaris seeds with a reduced content in phytic acid. However,
the manipulation of phytic acid in the seed requires knowledge on the key enzymes involved in its
biosynthetic pathway.The phytic acid biosynthesis is believed to follow three types of pathways:
pathways (I) and (II) are inositol lipid-dependent, whereas pathway (III) does not require lipid
synthesis. The lipid-independent biosynthesis (III) starts from a glucose 6P, that is converted in
myo-inositol 3P (InsP1), then several kinases, many of which are yet to be identified, catalyse the
sequential addition of phosphate units to the InsP1, to produce InsP2, InsP3, InsP4, InsP5 and InsP6.
The lipid-dependent phytate biosynthesis (II and III) initiates through phospholipase C and
subsequent sequential phosphorylation of inositol 1,4,5-trisphosphate or inositol 1,3,4-trisphosphate
by several kineses leads to InsP4, InsP5 and InsP6 .
In common bean, we have isolated and characterized the first enzyme myo-inositol-phosphate
syntase (MIPS) which is involved in the conversion of glucose 6P to myo-inositol 3P (InsP1) and
the three major kinases involved in the phytic acid pathway: Inositol-(1,3,4,5,6)-pentakisphosphate
2-kinase (PvIpk1), Inositol-(1,4,5) trisphosphate 3/6-kinase (PvIpk2) and Inositol (1,3,4)
trisphosphate 5/6-kinase (PvIpk3). All these genes showed a high similarity with the orthologous
kinases genes isolated from barley, maize and Arabidoposis and are present in 1 or 2 copies in the
bean genome.
To investigate on the expression pathways of these genes we used a quantitative RT-PCR approach
to follow their mRNA synthesis in the developing cotyledons and in different plant tissues. While
MIPS expression is very high at the very early stages of seed development (from 4 to 10 DAF) and
then decreaes up to 100 times, the expression levels of the three kinases is much more constant
during seed develeopment, even though there is a slightly decrease in gene expression after the
mid-maturation stage, in accordance with the InsP metabolites synthesis.
Research partially supported by Ministry of Agricultural Alimentary and Forest Politics with funds
released by C.I.P.E. (Resolution 17/2003).
50
PROM – National research project for the improvement of vegetable crops for Southern-Italy
Group: “Bean for open field cultivation”
Research: Evaluation and improvement of old bean landraces cultivated in Southern-Italy
Bruno Campion
1, Marzia Fileppi
1, Lucia Lioi
2, Angela Rosa Piergiovanni
2, Marina Carbonaro
3,
Roberto Lo Scalzo4, Incoronata Galasso
5, Giovanni Tagliabue
5, M. Gloria Daminati
5, Francesca
Sparvoli5, Roberto Bollini
5
1CRA - Istituto Sperimentale per l'Orticoltura, Sez. di Montanaso Lombardo, Lodi;
2Istituto di
Genetica Vegetale, CNR, Bari; 3
Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione,
Roma;4CRA - Istituto Sperimentale per la Valorizzazione Tecnologica dei Prodotti Agricoli,
Milano; 5Istituto di Biologia e Biotecnologia Agraria, CNR, Milano
The general objective of this research, is the improvement of old local bean landraces cultivated in
traditional areas of Southern-Italy. Their very high nutritional and culinary quality caused a
significant raise of market demand and prices in the last three years. On the other hand, these old
cultivars often present undesirable characters like a high genetic variability together with a high
susceptibility to various diseases. Germplasm characterization and preservation, plant selection
followed by the recovery of BCMV- (Bean Common Mosaic Virus) or bacterial-free individuals
are very important tools to improve general condition and value of these materials.
The project involves different groups with different expertises in order to: a) characterise the local
bean landraces for their genetic variability, seed protein composition, nutritional and agronomic
traits; b) assure germplasm collection and conservation; c) select superior materials and, where
required, recover virus -free lines.
Research supported by Ministry of Agricultural Alimentary and Forest Politics with funds released
by C.I.P.E. (Resolution 17/2003).
Participants
First Name Last Name Institution Department address City Country e mail
Mario Aguilar Universidad Nacional de La Plata Instituto de Bioquimica y Biologia Molecular, Facultad de Ciencias Exactas
1900 La Plata La Plata Argentina [email protected]
Francisco Aragão Embrapa Recursos Genéticos e Biotecnologia PqEB W5 Norte, 70770900 Brasília Brazil [email protected]
Subhash Bhore Melaka Institute Of Biotechnology
Molecular Biology Division Lot 7, MITC city, 75450 Ayer Keroh, Melaka
Malaysia [email protected]
Matthew Blair CIAT Biotechnlogy Reseacrh Unit Apartado Aéreo 6713 Cali Colombia [email protected]
Roberto Bollini CNR Istituto di Biologia e Biotecnologia Agraria
Via Bassini 15, 20133 Milan Italy [email protected]
William Broughton Université De Genève Laboratoire de Biologie Moléculaire des Plantes Supérieures
30 Quai Ernest-Anserment, 1211
Genève Switzerland [email protected]
Bruno Campion CRA Istituto Sperimentale per l'Orticoltura Via Paullese 28, 26836 Motanaso Lombardo (LO)
Italy [email protected]
Caterina Capone CRA Istituto Sperimentale per il Tabacco Via P. Vitiello 108, 84018 Scafati (Sa) Italy
Marina Carbonaro INRAN Food Chemistry Via Ardeatina 546, 00178 Roma Italy [email protected]
Andrea Carboni CRA Istituto Sperimentale per le Colture Industriali
Via di Corticella 133, 40128 Bologna Italy [email protected]
Maria Chacon Universidad Nacional De Colombia
Agronomia campus universitario, edificio 500, of. 228
Bogotà Colombia [email protected]
Maria Gloria Daminati CNR Istituto di Biologia e Biotecnologia Agraria
Via Bassini 15, 20133 Milan Italy [email protected]
Giulia De Lorenzo Università La Sapienza Dipartimento Di Biologia Vegetale P.le Aldo Moro 5, 00185 Rome Italy [email protected]
Antonio M. De Ron MBG-CSIC Plant Genetic Resources p. o. box 28, 36080 Pontevedra Spain [email protected]
Luisa Del Piano CRA Istituto Sperimentale per il Tabacco Via P. Vitiello 108, 84018 Scafati (Sa) Italy [email protected]
Claudia Díaz-Camino Instituto De Biotecnología/Unam Biología Molecular De Plantas av. universidad 2001, chamilpa
Cuernavaca, Morelos
Mexico [email protected]
Francesca Del Bianco CRA Istituto Sperimentale per le Colture Industriali
Via di Corticella 133, 40128 Bologna Italy
Renato D'ovidio Università della Tuscia Dipartimento di Agrobiologia e Agrochimica
Va San Camillo de Lellis, 01100
Viterbo Italy [email protected]
52
Jean Jacques
Drevon INRA-SupAgro Rhyzosphere and Synìmbiosis Unit Place Pierre Viala 2, 34060 Montpellier France [email protected]
Marzia Fileppi CRA Istituto Sperimentale per l'Orticoltura Via Paullese 28, 26836 Motanaso Lombardo (LO)
Italy [email protected]
Incoronata Galasso CNR Istituto di Biologia e Biotecnologia Agraria
Via Bassini 15, 20133 Milan Italy [email protected]
Homar Gill Langarica Centro De Biotecnologia Genomica-Ipn
Vegetal Blvd. del Maestro Eesq. Eelias Piña s/n, 88730
Reynosa Mexico [email protected]
Georgina Hernandez UNAM Centro De Ciencias Genómicas ap. postal 565-a Cuernavaca, Morelos
Mexico [email protected]
Emmanuel Jayko
Jaiyeola Rose Kali International Senior Secondary School
Asst Head Of Biochemisrty Department
p.o.box 1051 banjul the gambia, 00220
Banjul Gambia [email protected]
Patricia Lariguet University Of Geneva Plant Bioloy 30 Quai Ernest-Anserment, 1211
Geneva Switzerland [email protected]
Lucia Lioi CNR Istituto di Genetica Vegetale Via Amendola 165/a, 70126 Bari Italy [email protected]
Giuseppina Logozzo Università degli Studi della Basilicata
Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali
Viale dell'Ateneo Lucano 10, 85100
Potenza Italy [email protected]
Yamile Márquez Ortiz UNAM, Instituto De Biotecnología
Biologia Molecular De Plantas Av. Universidad #2001, Col. Chamilpa, 62251
Cuernavaca, Morelos
Mexico [email protected]
Francis Mendy Rose Kali International Senior Secondary School
Head Of Bio/Chemistry Department p.o.box 1051 banjul the gambia, 00220
Banjul Gambia [email protected]
Jan Michiels K.U.Leuven Centre Of Microbial And Plant Genetics
Kasteelpark Arenberg 20, b-3001
Heverlee Belgium [email protected]
Jennifer Motta Universidad Industrial De Santander
Escuela De Biología Carrera 27 Calle 9 Bucaramanga Colombia
Valeria Negri Universita di Perugia Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali e Zootecniche
Borgo XX Giugno 74, 06121, Perugia Italy [email protected]
Mario Nenno - - Viale Martesana 31, 20090 Vimodrone (MI) Italy [email protected]
Donal O'sullivan NIAB Centre for Plant Genetics, Breeding and Evaluation
Huntingdon Road, CB3 0LE Cambridge UK [email protected]
Roberto Papa Università Politecnica delle Marche
Facoltà di Agraria via Brecce Bianche 1, 60131 Ancona Italy [email protected]
K. Peter Pauls University Of Guelph Department Of Plant Agriculture 50 Stone Road W., N1G 2W1 Guelph, Ontario Canada [email protected]
Pietro Piffanelli AgBiotech Research Centre, Parco Tecnologico Padano
PTP Genomics Platform, CERSA Via Einstein, 26900 Lodi Italy [email protected]
Tim Porch USDA-ARS-TARS Tropical Agriculture Research Station 2200 P. A. CAMPOS AVE., SUITE 201, 00680-5470
Mayaguez, Puerto Rico
53
Carmen Quinto Instituto De Biotecnología, Unam.
Biología Molecular De Plantas Av. Chamilpa 2001, 62210 Cuernavaca, Morelos
Mexico [email protected]
Roseline Remans K.U.Leuven Centre Of Microbial And Plant Genetics
Kasteelpark Arenberg 20, b-3001
Heverlee Belgium [email protected]
Monica Rossi Università Politecnica delle Marche
Facoltà di Agraria via Brecce Bianche 1, 60131 Ancona Italy [email protected]
Federico Sanchez UNAM, Instituto De Biotecnología
Plant Molecular Biology Av. Universidad #2001, Col. Chamilpa, 62210
Cuernavaca Morelos
Mexico [email protected]
Luis Eduardo Servín Garcidueñas,
UNAM, Instituto De Biotecnología
Biologia Molecular De Plantas Av. Universidad #2001, Col. Chamilpa, 62251
Cuernavaca, Morelos
Mexico [email protected]
Pierluigi Spagnoletti-Zeuli
Università degli Studi della Basilicata
Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali
Viale dell'Ateneo Lucano 10, 85100
Potenza Italy [email protected].
Francesca Sparvoli CNR Istituto di Biologia e Biotecnologia Agraria
Via Bassini 15, 20133 Milan Italy [email protected]
Giovanni Tagliabue CNR Istituto di Biologia e Biotecnologia Agraria
Via Bassini 15, 20133 Milan Italy [email protected]
Nancy Terryn University of Gent Institute for Plant Biotechnology for Developing Countries,
K.L. Ledeganckstraat 35 Gent Belgium [email protected]
Barbara Tiranti Sezione di Genetica e Miglioramento Genetico
Biol. Vegetale Biotec. Agroambientali e Zootecniche
Via Borgo XX giugno 74, 06121
Perugia Italy [email protected]
Jos Vanderleyden K.U.Leuven Centre Of Microbial And Plant Genetics
Kasteelpark Arenberg 20, b-3001
Heverlee Belgium [email protected]
Roseli Wassem University Of Geneva Plant Biology 30 Quai Ernest-Anserment, 1211
Geneva Switzerland [email protected]
Martha Serrano Universidad Industrial De Santander
Escuela De Biología Carrera 27 Calle 9 Bucaramanga Colombia