Download asexual seed formation for agricultural crop improvement

Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.01
UNRAVELLING APOMIXIS: ASEXUAL SEED FORMATION FOR
AGRICULTURAL CROP IMPROVEMENT
KOLTUNOW A.M.G.
Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization
(CSIRO), Waite Campus, PO Box 350 Glen Osmond, South Australia 5064 (Australia)
apomixis, Hieracium, meiotic avoidance, parthenogenesis, seed
Seeds contribute significantly to world food supply. Flowering plants mainly produce seeds
by sexual reproduction, which is a driver of genetic diversity. The combination of meiosis during
male and female gamete formation, and subsequent gamete fusion at fertilization in the ovule of the
flower to form the embryo compartment of the seed, leads to segregation of parental alleles in
seedling progeny. A second fertilization event generates the endosperm, a nutritive tissue
supporting embryo growth and seedling germination. Remarkably, some flowering plants form
seeds asexually by apomixis. Apomixis comprises an ensemble of developmental processes that
together alter female reproductive functions in the ovule, converting the sexual program to an
asexual one. The result is that the apomictically derived embryo develops solely from cells in
maternal ovule tissues and therefore seedling progeny are genetically identical to the mother.
Apomixis is largely absent in important food crops. Harnessing apomixis as a technology in plant
breeding would increase food yield and security. This talk will focus on the genetic and molecular
control of apomixis in daisy-like Hieracium species which form seeds by avoiding meiosis during
female gamete formation and do not need double fertilization for seed initiation.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.02
DUPONT PIONEER'S PATHS TOWARDS SELF-REPRODUCING HYBRIDS
LAWIT S., CHAMBERLIN M., AGEE A.
Agricultural Biotechnology, DuPont Pioneer, 7300 NW 62nd Ave, PO Box 1004, Johnston, IA,
50131-1004 (USA)
apomixis, megagametophyte, Arabidopsis thaliana, adventitious embryony, embryo sac
Altered plant reproductive biology has been utilized in advancing agricultural productivity for
decades. Recent studies have focused on the gene networks expressed in each of the four specific
cell types within the female gametophyte and have identified cell specific promoters for the egg,
synergids, central cell and antipodals. We have built upon this knowledge by fusing cell type
specific promoters to different fluorescent proteins within a single transcriptional unit and
expressing them in Arabidopsis plants. This created a unique tool set which allows simplified
simultaneous visual tracking of each cell type from gametophyte cellularization
(megagametogenesis) through fertilization and early embryo development. We have used these
constructs for tracking cell fates within Arabidopsis ovules following molecular manipulations. We
have demonstrated egg- and synergid-ablation, and synergids becoming egg-like in the absence of
the egg. We report on the dedifferentiation of somatic ovular cells, which become egg and embryolike, reminiscent of adventitious embryony. The ultimate aim of these manipulations is synthetic
apomixis resulting in seed derived wholly from the maternal plant, and thus fixation of important
agronomic traits such as hybrid vigor, a key benefit to agricultural productivity.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.03
FANCM LIMITS MEIOTIC CROSSOVER
CRISMANI W., MERCIER R.
INRA IJPB, Versailles (France)
meiotic recombination, meiosis, Arabidopsis, FANCM
The number of meiotic crossovers (COs) is tightly regulated within a narrow range, despite a
large excess of molecular precursors. The factors that limit COs remain largely unknown. Here,
using a genetic screen in Arabidopsis thaliana, we identified the highly conserved FANCM
helicase, which is required for genome stability in humans and yeasts, as a major factor limiting
meiotic CO formation. The fancm mutant has a three-fold increased CO frequency compared to
wild-type. These extra COs do not arise from the pathway which accounts for most of the COs in
wild-type, but from an alternate normally minor pathway. Thus, FANCM is a key factor imposing
an upper limit on the number of meiotic crossovers and its manipulation holds significant promise
for plant breeding.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.04
EVOLUTIONARY APPROACHES TO DECIPHERING THE FUNCTIONAL
SWITCH FROM SEXUAL TO ASEXUAL (APOMICTIC) REPRODUCTION
IN NATURAL PLANT POPULATIONS
SHARBEL T.F.
Apomixis Research Group, IPK Gatersleben, 06466 Gatersleben (Germany)
apomixis, genome, polyploid, evolution, gene expression
An organism’s choice to reproduce with or without sex has long puzzled evolutionary
biologists. Apomixis, a natural form of reproduction in plants whereby seeds are produced
asexually, has evolved repeatedly from sexual ancestors in many taxa. Apomixis is of interest on a
number of levels, ranging from population genetics to evolution, but also from an applied
perspective, as it represents a disruptive technology which could significantly change agricultural
practices (e.g. fixing heterosis in hybrid crops). The switch from sex to apomixis is hypothesized to
result from deregulation of developmental pathways leading to sexual seed development, and the
trigger for deregulation involves the global genomic effects of hybridization and polyploidy.
We study apomixis in wild plant populations, and use evolutionary theory to guide our
experimental approaches. High-throughput methods are employed to understand population-level
phenotypic (seed production) and genetic (polyploidy, genetic structure) variability. These data are
then used to design targeted experiments, whereby candidate genes for apomixis are identified using
tissue-specific “omics” methods in particular genotypes. These candidates are then used (1) in
transformation experiments to attempt apomixis induction in sexual plants, and (2) in populationlevel studies to understand the origin and evolution of apomixis with respect to sexuality in natural
populations.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.05
ARF AND Aux/IAA PROTEINS IN THE DEVELOPMENTAL TRANSITION
LEADING TO FRUIT SET IN THE TOMATO
BOUZAYEN M.*,**, WANG H.*,**, ZOUINE M.*,**, YANG Y*,**, MILA I.*,**,
FRASSE P.*,**, VAN DER REST B.*,**
*) University of Toulouse, INPT, Laboratory of Genomics and Biotechnology of Fruit, Avenue de
l'Agrobiopole BP 32607, Castanet-Tolosan F-31326 (France)
**) INRA, UMR990 Génomique et Biotechnologie des Fruits, Chemin de Borde Rouge, CastanetTolosan, F-31326 (France)
tomato, fruit set, parthenocarpy, auxin signaling, transcriptional regulation, Solanum lycopersicum
The onset of ovary development into fruit, the so-called fruit set is naturally triggered by
successful pollination of the flower. Yet, the the signals that drive fruit growth after fertilization are
not clearly understood, even though the involvement of some plant hormones, such auxin and GA,
in the fruit set is becoming well documented. However, the molecular mechanisms underlying the
auxin control of fruit initiation remain largely unknown. We previously showed that downregulation of SlIAA9, a tomato member of the Aux/IAA gene family, leads to fruit development prior
to flower fertilization giving rise to parthenocarpy. Subsequently, the search for putative IAA9
interacting partners identified SlARF8 as a solid candidate. Expression analysis revealed that
SlARF8 transcript levels remain low in unpollinated flowers but increase dramatically after flower
pollination. Interestingly, the over-expression of SlARF8 in transgenic tomato results in the
production of seedless fruit indicating that the altered expression of either of the two genes leads to
pollination-independent fruit set and parthebnocarpy, fully supporting their direct role in the fruit
initiation process. Further investigation revealed that the tomato microRNA, miR167, causes
degradation SlARF8 transcripts and that under-expressing this microRNA in transgenic tomato lines
yields parthenocarpic fruit. All together, the data define a new regulome controlling the flower-tofruit transition involving SlARF8 as a key actor. The expression of SlARF8 is under double control,
by SlIAA9 at the post-translational level and by microRNA at the post-transcriptional level.
Moreover, genome-wide transcriptomic profiling of the fruit set process suggested an active
hormonal interplay involving, mainly auxin, GA and ethylene, that is likely to be the essential
features underlying this developmental transioning. The present study uncovers a control
mechanism of the fruit set process in which the down-regulation of the central player Sl-IAA9
represents the initial event that triggers the cascade of changes in gene expression associated with
the flower-to-fruit transition. Overall,the work indentifies new targets for breeding programs aiming
at improving fruit yield and at producing parthenocarpic fruit.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.06
HISTONE HYPERACETILATION AFFECTS RECOMBINATION IN
ARABIDOPSIS MALE MEIOSIS
CREMONA G., AIESE CIGLIANO R., TERMOLINO P., PAPARO R., CONSIGLIO M.F.,
CONICELLA C.
CNR-IGV, Institute of Plant Genetics, UOS Portici, Via Università 133, 80055 Portici (Italy)
microsporogenesis, chiasma, crossover, histone acetylation
Histone post translational modifications are increasingly recognized as playing important
roles in meiotic events. In particular, histone acetylation was shown to be involved in the meiotic
recombination of budding yeast either by local analysis at hotspots or by genome-wide analysis.
The level of histone acetylation is modulated by the action of histone acetyltransferases (HATs) and
histone deacetylases (HDACs). Furthermore, histone acetylation can be modified by treatments
with inhibitors of HDACs, i.e. trichostatin A (TSA) and nicotinamide.
In Arabidopsis, we studied the effect of histone hyperacetylation on meiotic recombination
using different approaches such as loss-of-function and gain-of-function mutations as well as
regulation of TSA-sensitive HDACs. We obtained evidence that chiasma frequency and distribution
per chromosome pair change in male meiocytes as a consequence of the histone hyperacetylation
that was induced in different ways: (i) over-expression of a GCN5-related HAT; (ii) downregulation of a RPD3 type HDAC; (iii) TSA-treatment. Male meiosis was investigated in Pollen
Mother Cells (PMCs) in the mutants and in TSA-treated material. At metaphase I, univalent
chromosomes were observed indicating a loss of the obligate Crossing Over (CO). As a
consequence of the random segregation of the univalents, uneven chromosome distribution occurred
at later stages. Fluorescence in situ hybridization (FISH) performed with 45S and 5S rDNA probes
showed that different chromosomes were involved in pairing failure in the mutants. Analysis of
bivalent configurations revealed significant variations of ‘rod’ bivalents (chiasmata restricted to a
single arm) and ‘ring’ bivalents (chiasmata present in both arms). Overall, these evidences suggest
that histone acetylation is involved in recombination control in a chromosome specific manner.
However, it is likely that hyperacetylation alters the pattern of other histone modifications (i.e.
histone methylation) demonstrated to interplay with the histone acetylation, especially at level of
specialized subchromosomal domains.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.07
DIFFERENTIAL EXPRESSION IN SEXUAL AND APOMICTIC
GENOTYPES OF THREE MODEL SPECIES STRENGTHENS A CRUCIAL
ROLE OF APOSTART IN THE FORMATION OF SEEDS BY APOMIXIS
ALBERTINI E.*, MARCONI G.*, CONNER J.**, GALLA G.***, MASIERO S.****,
SHARBEL T.F.*****, COLOMBO L.****BARCACCIA G.***, OZIAS-AKINS P.**,
FALCINELLI M.*
*) Dept. of Applied Biology, University of Perugia (Italy)
**) Dept. of Horticulture, University of Georgia Tifton Campus, GA (USA)
***) DAFNAE, University of Padova (Italy)
****) Dept. of Biosciences, University of Milano (Italy)
*****) Apomixis Research Group, IPK (Germany)
apomixis, genomics, Poa pratensis, Hypericum perforatum, Pennisetum squamulatum
Seed is the key factor of crop productivity. The commercial success of a newly selected
cultivar depends not only on its vegetative attributes but also on its ability to produce seeds.
Breeding for seed yield and quality requires new sophisticated technologies, such as apomixis, that
will allow overcoming the conventional breeding limits. Apomixis is a naturally occurring mode of
asexual reproduction in flowering plants, resulting in embryo formation without meiosis or
fertilization of the egg. Seed derived progenies are genetically identical to the maternal parent. In
crop species, apomixis would enable the instantaneous fixation of the complete genome of the best
plants. The introduction of apomixis in crop species is one of the key desirable traits since it would
allow the fixation of heterosis in F1 hybrids leading to efficient and consistent production of highquality seeds, fruits, and vegetables. The development of Apomixis Technology (AT) is expected to
have a revolutionary impact on food and agriculture production by reducing cost and breeding time,
avoiding some complications of sexual reproduction (incompatibility barriers) and of vegetative
propagation (viral transfer). It is estimated that AT in the production of hybrid rice alone could
provide benefits exceeding €1800 million per annum. The development of AT in agriculture require
a deeper knowledge of reproductive development in plants. We have isolated one gene, which was
termed APOSTART because of its START domain and its putative involvement in apomixis
(Albertini et al. 2005, Plant Phys 138:2185-2199). Our previous results demonstrate that some
APOSTART members are expressed exclusively in inflorescences of Poa pratensis. In situ
hybridization analyses revealed that APOSTART is expressed during both male and female meiosis
in all micro- and megaspores. Strong signals were recorded up to the mature embryo sac stage. The
overall data suggest that APOSTART may be related to the programmed cell death that is involved
in the non-functional megaspore and nucellar cell degeneration events that permit enlargement of
maturing embryo sacs. Functional characterization of the Arabidopsis thaliana APOSTART1 gene
(AtAPO1), shows that AtAPO1 is expressed in mature embryo sacs and developing embryos. To
confirm the involvement of APOSTART in apomixis in P. pratensis, we have then created 3
genomic and 3 cDNA libraries using 2 apomictic and 1 sexual genotypes with three aims: i) to
isolate all possible APOSTART members/alleles; ii) to identify DNA regions containing
APOSTART members/alleles; and iii) to identify genes linked to APOSTART. Moreover, to
strengthen the hypothesis of an involvement of APOSTART in apomixis, we have isolated
APOSTART members from two other aposporic species: Pennisetum squamulatum and Hypericum
perforatum. In each species several members/alleles have been identified and characterized. Our
results, demonstrating that APOSTART is expressed differentially in all 3 species, will be reported
and critically discussed.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.08
DE NOVO SEQUENCING AND ANNOTATION OF THE HYPERICUM
PERFORATUM FLOWER TRANSCRIPTOME
GALLA G.*, SHARBEL T.F.**, BARCACCIA G.*
*) Department of Agriculture Food Natural Resources Animals and Environment, University of
Padova, Campus of Agripolis, Viale dell’Università 16, I-35020 Legnaro, Padova (Italy)
**) Apomixis Research Group, Department of Cytogenetics and Genome Analysis, Institut für
Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben (Germany)
apomixis, sexual reproduction, meiosis, gametogenesis, next generation sequencing
St. John’s wort (Hypericum perforatum L.) is a medicinal plant that produces important
metabolites with antidepressant and anticancer activities. Moreover, recently gained information has
shown that it is also an attractive model system for the study of apomixis. The lack of genomic and
transcriptomic data represents a severe complication for understanding the genetic and molecular
mechanisms that control the expression of apomixis in this species. The aim of present research is
the sequencing, annotation and comparative investigation of the Hypericum flower transcriptome,
which is a critical step towards a deep understanding on how aposporic apomeiosis is primed and
parthenogenesis accomplished in this species. We adopted the high-throughput Roche 454
technology to sequence the whole Hypericum flower transcriptome of single flower parts collected
from three apomictic and two sexual genotypes. On the whole, we generated 1,469,947 sequences,
which were globally assembled to originate 33,860 contiguous sequences, with an average length of
1,002 bp. Computational procedures based on BLAST program were used to annotate the sequence
data and evaluate the transcriptome coverage. Protein databases originated starting from all known
sequences involved in key biological processes in both plants and animals were implemented and
queried to better annotate our sequence sets. The analysis of sequence composition of each library
enabled the identification of transcripts that are likely exclusive of single flower parts and
reproductive strategies. As an example, 993 sequences were isolated exclusively from carpels at
different developmental stages, encompassing sporogenesis and apomeiosis, as well as the
formation of embryo sacs and egg cells. Starting from this dataset, we identified 234 transcripts of
genes specifically expressed in apomictic genotypes. In addition, 45 of the sequences uniquely
identified in apomictic genotypes scored high similarity with pre-miRNA precursors targeting
transcription factors. Computational annotation of the remaining sequences, according to the main
vocabularies of Gene Ontology and Plant Ontology, allowed us to select GO terms related to
transcription factor and protein binding activities as well as germ cell differentiation and embryo
sac developmental processes. Visualization of data associated to functional pathways with MapMan
was useful to identify gene products and biological processes in the frame of cell developmental
and molecular networks. Significant over/under contributions of individual libraries in assembling
each contiguous sequence were estimated with the statistical package edgeR implemented in the
software robiNA. Following this method we identified 758 and 347 sequences that were
differentially represented in carpels when compared to stamens and young buds, respectively. The
expression pattern of most promising transcripts was further investigated by quantitative Real-Time
PCR and in situ hybridization assays to demonstrate cell domain identity, and tissue and stage
specificity of genes potentially involved in plant reproduction. Annotation of all identified flower
transcripts as well as their qualitative and quantitative expression patterns will be presented and
critically discussed as they may allow a far better understanding of the molecular bases of gamete
and embryo formation in sexual and apomictic H. perforatum plants.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.09
THE ROLE OF THE CHROMATIN REMODELLING FACTOR nfc102 IN
THE MAIZE FLOWERING PATHWAY
MASCHERETTI I., ROSSI V.
Maize Research Unit, Agricultural Research Council (CRA), Via Stezzano 24, 24126 Bergamo
(Italy)
chromatin modification, Zea mays, flowering, antisense RNA, transcription regulation
Maize nfc102 is a WD-repeat protein that forms a β-propeller structure and that belongs to the
MSI family, originally identified in yeast. nfc102 gene shows high homology with Arabidopsis
FVE, a component of the autonomous flowering pathway and a key regulator of the epigenetic
stability. Previous studies showed that nfc102 interacts with histone deacetylases (HDACs) to
repress transcription. In this study, we have employed different nfc102 down-regulated mutants and
we have observed that all of them exhibit various developmental defects, including a delay of
flowering. However, this effect is not associated with a change of the timing of the flowering
transition phase, but it is related to a more general developmental delay, probably due to a
pleiotropic effect of nfc102.
Since FVE is involved in the regulation of flowering in Arabidopsis, we have analyzed
whether the RNA levels of maize flowering regulators are also affected in nfc102 mutants. Among
the many MADS-box genes identified in maize, only two floral identity genes, ZMM4 and ZMM15,
are down-regulated in nfc102 mutants, but their regulation is indirect. Conversely, the expression in
meristematic area of Indeterminate1 (Id1), a gene involved in the control of floral transition, is
inhibited by a direct binding of nfc102 to Id1 promoter, thus provoking HDAC recruitment and
histone modification changes. Recently, Zea mays centroradialis 8 (ZCN8), a maize gene with
florigenic properties, has been identified. ZCN8, like F T in Arabidopsis, is transcribed and
translated in leaves and its protein moves to the shoot apex meristematic area via the phloem, where
it promotes the floral transition. Interestingly, in meristematic area-enriched tissues (MA) ZCN8
transcript is detected almost exclusively in an unspliced form, while the spliced transcript form is
produced only in leaf blades (LB). Furthermore, ZCN8 expression is indirectly activated by Id1.
Using RNA-blot and strand specific qRT-PCR, we have observed that the unspliced form is mainly
represented by an antisense ZCN8 RNA. Analysis of ZCN8 sense and antisense RNA levels in
nfc102 mutants showed that nfc102 down-regulation affects the levels of both ZCN8 sense and
antisense transcripts, but in an opposite manner. In fact, in these mutants ZCN8 spliced and
unspliced sense RNA increases, while the antisense RNA decreases. Furthermore, we have
observed that, both in MA and LB, nfc102 binds specifically to ZCN8 3’-end, causing histone
modification changes. Anyway this binding is not associated with HDAC recruitment. On the basis
of these observations, we speculate that nfc102 can mediate chromatin modifications during the
ZCN8 sense RNA transcription and processing, by modulating the synthesis of ZCN8 antisense
RNA.
Altogether, our results indicate that nfc102 is involved in the control of maize flowering, by
modulating expression of different flowering regulators, via different mechanisms associated with
chromatin remodeling of nfc102 targets. A molecular model describing nfc102 role in controlling
maize flowering pathway will be discussed.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.10
MATERNAL CONTROL OF FERTILIZATION AND SEED DEVELOPMENT
MIZZOTTI C.*, MENDES M.A.*, CAPORALI E.*, SCHNITTGER A.**, KATER M.M.*,
BATTAGLIA R.*, COLOMBO L.*
*) Dipartimento di BioScienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan
(Italy)
**) Institut de Biologie Moléculaire des Plantes du CNRS, 12, rue du Général Zimmer,
67084 Strasbourg (France)
fertilization, ovule development, seed development, Arabidopsis, endothelium
The life cycle of flowering plants alternates between a diploid sporophytic and a haploid
gametophytic generations that only occur in reproductive organs where the two generations coexist.
After fertilization, the sporophytic seed coat develops in a coordinated manner with the
gametophytic generation and undergoes several changes including accumulation of secondary
metabolites such as proanthocyanidins and mucilage.
In the arabidopsis bsister (abs) mutant, the innermost layer of the seed integuments, called
endothelium, does not accumulate proanthocyanidins and cells have an abnormal morphology. ABS
encodes for a transcription factor belonging to MADS-box transcription factor family. SEEDSTICK
is another MADS-box transcription factor that regulates ovule identity, redundantly with
SHATTERPROOF 1 (SHP1) and SHP2. We have recently found that STK also plays a role during
seed development: stk seeds present defects in seed coat morphology, abnormal proanthocyanidins
accumulation and problems in mucilage release. Interestingly, ABS and STK genes redundantly
control some aspects of seed formation as demonstrated by the phenotype of the abs stk double
mutant plants. In these plants we observed very few seeds due to both a reduced number of
fertilized ovules and to seed abortions. Morphological analysis revealed the complete absence of the
endothelium and massive starch accumulation was observed in the embryo sac. The phenotype of
the abs stk double mutant strongly suggests a maternal role in the regulation of the fertilization
process and it highlights the importance of the endothelium for the development of the next
generation.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Oral Communication Abstract – 1.11
AUXIN AND JASMONIC ACID INTERACTION IN THE CONTROL OF
LATE STAMEN DEVELOPMENTN IN ARABIDOPSIS THALIANA
CECCHETTI V.*, PETROCELLI V.**, FALASCA G.***, ALTAMURA M.***, COSTANTINO
P.*, CARDARELLI M.**
*) Dipartimento di Biologia e Biotecnologie, Sapienza Università di Roma, P.le Aldo Moro 5,
00185 Rome (Italy)
**) Istituto Biologia e Patologia Molecolari, CNR, Sapienza Università di Roma, P.le Aldo Moro 5,
00185 Rome (Italy)
***) Dipartimento di Biologia Ambientale, Sapienza Università di Roma, P.le Aldo Moro 5,
00185 Rome (Italy)
auxin, jasmonic acid, Arabidopsis, stamen, anther dehiscence
Auxin controls developmental processes during late stamen development coordinating anther
dehiscence and pollen maturation and triggering filament elongation. We measured the content of
free IAA in anthers at different developmental stages and we demonstrated that auxin concentration
peaks at the very beginning of late development and subsequently declines when anther dehiscence
begins. We demonstrated that in the auxin-perception mutants afb1 and tir1afb2afb3 endothecium
lignification and anther dehiscence occur earlier than in the wild type. Consistently auxin regulates
the expression of the transcription factor MYB26 required for endothecium lignification.
Anther dehiscence is also controlled by jasmonic acid (JA) as dad1 and opr3 mutants,
defective in JA biosynthesis, show indehiscent anthers. We demonstrated that the transcript levels of
these genes are higher in afb1 and tir1afb2afb3 flower buds at late developmental stages, and the
production of JA in tir1afb2afb3 flower buds is increased compared to controls. We found that afb13 opr3 double mutant anthers exhibit premature endhotecium lignification whereas opr3 mutant
anthers are not altered in the timing of this process. The role of auxin and jasmonic acid in the
control of the anther dehiscence process will be discussed.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.12
RECOMBINATION DIFFERENCE BETWEEN SEXES: IS HISTONE
ACETYLATION A KEY CONTROL FOR HETEROCHIASMY IN
ARABIDOPSIS?
TERMOLINO P., CREMONA G., AIESE CIGLIANO R., PAPARO R., CONSIGLIO M.F.,
CONICELLA C.
CNR-IGV, Institute of Plant Genetics, UOS Portici, Via Università 133, 80055 Portici (Italy)
heterochiasmy, chiasma, crossover, histone acetylation, polymorphism
Heterochiasmy that is the difference for crossover (CO) rate and distribution between male
and female meiosis in the same species, occurs in many organisms including Arabidopsis but its
basis is not well understood. One attractive hypothesis is that it can arise through sex-specific
variations in chromatin modifications. To address this important issue we are investigating the
effect of histone hyperacetylation on the heterochiasmy at a chromosomal level. For this purpose,
we used Meiotic Control of Crossovers1 (Atmcc1) Arabidopsis mutant showing histone
hyperacetylation due to over-expression of AtMCC1, a GCN5-related histone acetyltransferase, that
leads to an overall change in CO distribution in male meiocytes. We created two BC1 reference
populations, derived from F1 plants [Landsberg (Ler) x C24] backcrossed as male or female with
C24 (C24 x F1 and F1 x C24), and two BC1 mutant-derived populations, coming from F1 plants
(Ler x Atmcc1) backcrossed as male or female parent with Ler (Ler x F1 and F1 x Ler). We will
genotype all four populations with 48 SNPs markers homogenously distributed on chromosomes 4
and 5. Genotyping will be performed using the patented KASP SNP genotyping system that uses
Fluorescent Resonance Energy Transfer (FRET) coupled with the power of competitive allele
specific PCR, allowing array-scale detection of SNPs without the need for a separation step.
Any significant variation in meiotic recombination due to chromatin organization (histone
hyperacetylation) will be observed on the basis of differences in CO rates between the reference and
the mutant-derived populations. In this way, we could answer to the question whether histone
acetylation plays a role in heterochiasmy in Arabidopsis.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.13
LOSS OF DNA METHYLATION AFFECTS THE RECOMBINATION
LANDSCAPE IN ARABIDOPSIS
MIROUZE M.*,**, LIEBERMAN-LAZAROVICH M.*, AVERSANO R.***, BUCHER E.****,
NICOLET J.*, REINDERS J.*****, PASZKOWSKI J.*
*) Department of Plant Biology, Sciences III, University of Geneva, CH-1211 Geneva 4,
(Switzerland)
**) Institut de Recherche pour le Développement, Unité Mixte de Recherche UMR232 Diversité
Adaptation et Développement des Plantes, Université Montpellier 2, 34394 Montpellier (France)
***) Department of Soil, Plant, Environmental and Animal Production Sciences, University of
Naples Federico II, 80055 Portici (Italy)
****) Botanical Institute, University of Basel, 4056 Basel (Switzerland)
*****) DuPont/Pioneer Crop Genetics Research, DuPont Experimental Station E353, Wilmington,
DE 19880 (United States)
epigenetic, chromatin, epigenetic recombinant inbred lines, met1-3
Regulation of meiotic recombination depends on both DNA sequence and chromatin
properties. Indeed, it is known that cross-overs (COs) are not evenly distributed along chromosomes
and are suppressed in chromosomal regions encompassing compact, hypermethylated centromeric
and pericentromeric DNA. Among the epigenetic marks playing an essential role in stabilizing
heterochromatin, DNA methylation is the best-characterized one. The objective of this work was to
determine whether specific variations in DNA methylation would affect recombination and whether
these effects would differ in euchromatic vs. heterochromatic chromosomal regions. To address this
question we used hypomethylated mutant of Arabidopsis thaliana (met1) andanalyzed its meiotic
recombination in comparison to a wild-type plant. We observed unexpected and counterintuitive
effects of DNA methylation losses on CO distribution. Moreover, our data revealed that
recombination was further promoted in the hypomethylated chromosome arms while it was
inhibited in heterochromatic regions encompassing pericentromeric DNA. Notably, the total
number of COs was not affected, implying that loss of DNA methylation led to a global
redistribution of COs along chromosomes. To determine whether altered levels of DNA
methylation influence recombination directly in cis or indirectly in trans by changing expression of
genes encoding recombination components, we analyzed CO distribution in mapping populations
derived from two different epigenetic recombinant inbred lines (epiRILs) characterized by
randomly distributed and well-mapped hypomethylated chromosomal segments. The results of
these experiments, supported by expression profiling data, suggest that DNA methylation affects
meiotic recombination in cis. Because DNA methylation exhibits significant variation even within a
single species, our results imply that it may influence the evolution of plant genomes through the
control of meiotic recombination.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.14
THE TEN MOST CITED ARTICLES PUBLISHED ON APOMIXIS IN THE
LAST FIVE-YEAR PERIOD
BARCACCIA G., GALLA G.
Laboratory of Plant Genetics and Genomic Analysis, DAFNAE, University of Padova, Campus of
Agripolis, Viale dell’Università 16, 35020 Legnaro (Italy)
apomixis, model species, recent advances
Apomixis is highly desirable in agriculture as a reproductive strategy for cloning plants by
seeds. Because embryos derive from the parthenogenic development of apomeiotic egg cells,
apomixis results in offspring that are exact genetic replicas of the parent. Introgression of apomixis
from wild relatives to crop species and transformation of sexual genotypes into apomictically
reproducing ones are long-held goals of plant breeding. In fact, it is generally accepted that the
introduction of apomixis into agronomically important crops will have revolutionary implications
for agriculture. During the last two decades, many scientists have speculated on the isolation of
gene/s controlling key steps of the apomictic pathway and many papers have postulated the
production of engineered plants exhibiting apomictic-like phenotypes. As a matter of fact, none of
the major crop plants has been bred for apomixis and only some features of apomixis have been
genetically engineered in model species. Consequently, even in the era of genomics, understanding
the genetic control and molecular regulation of apomixis appears much more complicated than
expected. This contribution deals with a critical review of the 10 most cited articles published on
apomixis in the last indexed quinquennial (2006-2010). In particular, original articles dealing with
apomixis in plants were retrieved from the major databases of scientific journals and ordered
according to the number of citations using the appropriate tools of Web of Knowledge [v. 5.6], a
premier research platform for information in life sciences. Reviews on apomixis were not taken into
account. A massive set of cytological and ecological information, along with genetic and molecular
data have been collected mainly in model species (i.e. Boechera holboellii, Hieracium spp.,
Hypericum perforatum, Paspalum spp., Ranunculus spp., Taraxacum officinale) and often tested in
Arabidopsis to elucidate the mechanisms of apomeiosis, parthenogenesis and apomixis. Several
genes involved in the formation of unreduced embryo sacs and egg cells, or responsible for the
autonomous development of the embryo and endosperm have been cloned and characterized, but
none of them mimes the apomictic pathway as a whole in crop plants. Hence, after two decades of
substantial studies conducted in several laboratories and model plants, the asexual reproductive
strategy called “gametophytic apomixis” by Nogher (1984) still seems an unsolved puzzle. This
situation resulted in a loss of confidence by the major seed companies, making it difficult to acquire
funds for conducting research on apomixis. Nowadays, novel views and original concepts are
however emerging from the fog, including the parallel between Y-chromosome and apomixisbearing chromosome (e.g., comparative genomic analyses revealed common features as repression
of recombination events, accumulation of transposable elements and degeneration of genes) from
the most primitive to the most advanced in evolutionary terms, and the link between apomixis and
gene-specific silencing mechanisms (i.e., likely based on chromatin remodelling factors or
transacting and heterochromatic interfering RNAs involved in both transcriptional and post-
transcriptional gene regulation). More recently, merging lines of evidence regarding the role of
auxin in cell fate specification of embryo sac and egg cell development have been reported in
Arabidopsis. Last but not least, under the evolutionary point of view, apomixis may now be
regarded as a consequence of sexual failure rather than as a recipe for clonal success.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.15
DEFECTIVE ENDOSPERM 18-ENCODED YUCCA1 PROTEIN IS
ESSENTIAL FOR NORMAL AUXIN-DEPENDENT ENDOSPERM
DEVELOPMENT IN MAIZE
BERNARDI J.*, LANUBILE A. *, LI Q-B.**, KUMAR D.***, CHOUREY P.**,
MAROCCO A.*
*) Institute of Agronomy, Genetics and Crop Science, Università Cattolica del Sacro Cuore,
Via Emilia Parmense 84, 29122 Piacenza (Italy)
**) United States Department of Agriculture, Agricultural Research Service, CMAVE, 32608
Gainesville, FL (USA) and Departments of Plant Pathology and Agronomy, University of Florida,
32611 Gainesville, FL (USA)
***) Interdisciplinary Center for Biotechnology Research, University of Florida, 32610 Gainesville,
FL (USA)
auxin, endosperm development, ZmYucca1, Zea mays
The phytohormone auxin (IAA) plays a fundamental role in vegetative and reproductive plant
development. Developing seeds accumulate the highest level of IAA of all tissues in a plant, but
poor is known on genes/enzymes critical to endosperm development. We analyzed a seed-specific
viable maize mutant, defective endosperm 18 (de18), that accumulates 10- to 15- fold less IAA in
endosperm and ~40% less dry matter compared to De18. Gene expression analyses of selected
putative IAA-biosynthesis genes, showed an high expression of Trp-aminotransferase co-orthologs
in both De18 and de18, while only the ZmYucca1 correlated with the reduced IAA levels in the
mutant throughout endosperm development. Further, sequence analyses of ZmYuc1 cDNA and
genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that
generated a premature stop codon and a truncated YUCCA1 protein of 212 amino acids, compared
to the 400 amino acids in the De18. Consistent with these results are the recombinant protein data
from E. coli where full length cDNA clones of ZmYuc1 of De18 and de18 endosperm showed a
normal and greatly reduced sizes of fusion proteins, ~70 kD and ~50 kD, respectively (each
included a GST tag of 26.0 kD). The putative ~1.5 kb ZmYuc1 promoter region also showed many
rearrangements, including a 151 bp deletion in the mutant. Our concurrent high density mapping
and annotation studies of chromosome 10, contig 395, showed that the de18 locus was tightly
linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1
gene that encoded the YUCCA1 protein is the causal basis of impairment in a critical step in IAA
biosynthesis, essential to endosperm development in maize. The lack of compensation of the
ZmYuc1 function despite the high transcript abundance of the two ZmTar genes suggests that
YUCCA1 constituted a rate-limiting step in a single pathway of multiple steps of IAA biosynthesis,
consistent with the recent evidences in Arabidopsis.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.16
GENES INTERACTION IN MAIZE ENDOSPERM DEVELOPMENT
SANGIORGIO S., GABOTTI D., MANZOTTI P., CARABELLI L., CONSONNI G.,
GAVAZZI G.
Dipartimento di Scienze Agrarie e Ambientali- Produzione, Territorio, Agroenergia,
Università degli Studi di Milano, Via Celoria 2, 20133 Milano
empty pericarp, cryptic variability, modifier, interaction, second site non complementation
In this report we present the results of a complementation test involving nine emp (empty
pericarp) mutants of maize representing single gene mutants, isolated as independent events.
These mutants are embryo lethal and drastically reduced in their endosperm size. As to the
endosperm, they can be subdivided into two major subgroups: one including those with a flat
appearance of the kernel and smooth pericarp and another one with a wrinkled pericarp.
In all mutants, the analysis of longitudinal sections of mature seeds reveals absence of
morphogenesis in the embryo proper, an observation the correlates with the failure of these mutants
to germinate.
By crossing inter-se plants heterozygous for the nine emp mutants we identified those non
complementing (i.e. allelic) and those complementing (i.e. not allelic) in the F1 generation. Most
results in the F1 were concordant to those obtained in the F2 generation with the exception of three
cases where the F1 results suggest allelism (i.e. one gene) whereas those in the F2 suggest
segregation of two genes.
These intriguing results seem to suggest an interaction between different emp mutants,
attributable to a phenomenon that is often referred to as second site non-complementation (SSNC).
SSNC can be explained by assuming interaction between two different mutant proteins leading to a
toxic product or that the mutant form of one protein sequesters the wild-type form of the other
protein into an inactive complex.
In some cases of 9 to 7 segregation in the F2 generation we recovered the two phenotypes
(emp flat and emp wrinkled) with values fitting the 9:4:3 ratio suggesting the presence of an
epistatic interactions between the two mutants.
For example, in the case of the cross between emp4 and emp*-9475 the scoring of F2 ears
revealed a segregation ratio of 9:4:3 (wt: emp wrinkled: emp flat) in which we can assume that
emp*-9475, that has a wrinkled phenotype, is epistatic to emp4, that has a flat phenotype. However
the data need to be increased to demonstrate that the hypothesis postulated is correct.
Another important point was the finding that in some ears segregating for a single emp mutant
in different genetic backgrounds (A636, W23 and Mo17) some mutant seeds were identified
exhibiting a more abundant endosperm tissue and occasionally an embryonic axis. A low
percentage of these seeds germinate.
This observation could be explained in two ways. The first implies that emp mutants uncover
a cryptic variability and the second possibility is that the improved endosperm of the mutant is the
result of an interaction between the mutant and a second factor originally present in the inbred line.
The results of crosses made to test for a segregation of a modifier have allowed us to identify a
dominant and a recessive modifier.
If these results are confirmed, emp mutants could be used as a tool for the detection of genetic
factors enhancing the amount of endosperm in the maize kernel to be exploited in breeding
programs.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.17
THE INTERACTION BETWEEN parthenocarpic fruit AND Curl INDICATES
A ROLE OF CLASS I KNOX GENES IN TOMATO FRUIT SET
MAZZUCATO A., PICARELLA M.E., RUIU F.
Dept. of Science and Technologies for Agriculture, Forestry, Nature and Energy (DAFNE),
University of Tuscia, Via S.C. de Lellis snc, 01100 Viterbo (Italy)
Solanum lycopersicum L., flower development, fruit set, parthenocarpy, class I KNOX genes
Knotted1-like homeobox (KNOX) transcription factors (TFs), which have been described as
negative regulators of gibberellin 20-oxidase (GA20ox) genes, are highly expressed at anthesis in
the ovary of wild-type (WT) tomato plants and transcript levels decrease after pollination. In the
parthenocarpic fruit (pat) mutant, where the ovary develops into seedless fruits independently of
pollination and fertilization, the expression levels of Tomato Knotted 2 (TKn2), ortholog of the
Arabidopsis SHOOTMERISTEMLESS (STM) TF, decrease prior to anthesis. These finding indicate
that members of the KNOX gene family might act as negative regulators of fruit growth, possibly
repressing gibberellin biosynthesis in unpollinated WT ovaries. Following these observation, a
phenotypic and molecular characterization of the interaction between the mutations pat and Curl
(Cu, overexpressing TKn2 and showing abnormal leaf development) was carried out by studying
segregating progenies. All phenotypes typical of the pat syndrome were evaluated in F3 and F4
individuals representing all the genotypic combinations (Pat cu, pat cu, Pat Cu and pat Cu). For all
the reproductive traits examined, including the frequency of stamen and ovule aberrations and yield
traits such as fruit and seed set, a reduction of the pat expressivity was observed in the pat Cu
double mutant background. When specifically testing for parthenocarpy, emasculated not-pollinated
flowers of pat Cu plants did not set fruit or developed fruitlets significantly smaller compared to the
pat single mutant. Thus, the GA-overdose phenotypes showed by the pat mutation are likely
mediated by a deregulation of Tkn2 in the mutant ovary. Expression analysis in parthenocarpic
systems other than pat indicated that the developmental regulation of Tkn2 in the ovary is conserved
in different wild-type backgrounds, but is not paralleled in all the parthenocarpic mutants. In
conclusion, the phenotypic and molecular characterization of the pat Cu double mutant strongly
suggests an interaction between the gene underlying the pat mutation and Tkn2 in the regulation of
tomato fruit set.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.18
A TRANSCRIPTOMIC APPROACH TO IDENTIFY REGULATORY GENES
INVOLVED IN FRUIT SET OF WILD TYPE AND PARTHENOCARPIC
TOMATO GENOTYPES
RUIU F.*, PICARELLA M.E.*, IMANISHI S.**, MAZZUCATO A.*
*) Dept. of Science and Technologies for Agriculture, Forestry, Nature and Energy (DAFNE),
University of Tuscia, Via S.C. de Lellis snc, 01100 Viterbo (Italy)
**) NARO Institute of Vegetable and Tea Science (NIVTS), Tsu (Japan)
Solanum lycopersicum L., fruit set, transcript profiling, parthenocarpy
The tomato (Solanum lycopersicum L.) parthenocarpic fruit (pat) mutation associates a strong
competence for parthenocarpy (seedless fruit production) with homeotic transformation of anthers
and aberrancy of ovules. To dissect this complex floral phenotype and to detect genes involved in
the pollination-independent fruit set of the pat mutant, a transcriptomic approach was used. Ovaries
from two pre- and one post-anthesis stages were collected to monitor and compare the expression
profile of genes in the wild type (WT) and in its near-isogenic pat line. A microarray platform
consisting of over 41,000 probes was used in this study. Normalized expression data were subjected
to one-way ANOVA and 3,627 genes showed significant expression differences (p<0.01). Among
them, 1,706 genes displayed a greater than 3-fold change in at least one of the pair-wise
comparisons analyzed. Gene sequence distribution following by Gene Ontology annotation showed
“catalytic activity” (43.9%) and “binding” (38.9%) as the most represented categories at the
molecular function level. A clustering analysis allowed the selection of 12 clusters containing genes
developmentally regulated in the WT ovary during fruit set and de-regulated in the mutant. These
genes are putatively involved in the determination or in the expression of the parthenocarpic
phenotype. These clusters, that were enriched of sequences encoding transcription factors (TFs),
included orthologs of Arabidopsis genes involved in the regulation of flower organs development,
such as BIG PETALp, APETALA3 and CRABS CLAW. Interestingly, de-regulation in the pat mutant
of such genes represents the first direct evidence of the association of these TFs with parthenocarpic
fruit growth. Their expression profile was confirmed by qRT-PCR, which was also extended to
other genes already known to be involved in controlling tomato fruit set, such as A U X I N
RESPONSE FACTOR7 (ARF7), ARF8 and INDOLE-3-ACETIC ACID9 (IAA9). Finally, selected
genes showing a de-regulated expression pattern in pat compared to the WT were also studied in
other tomato parthenocarpic systems (pat-2, pat3/4, aux/iaa9, RNAi-ARF7). This comparative
approach raised interesting cues for developing the present model of the molecular network
regulating parthenocarpy in tomato with stamen-ovule-ovary interaction as a driving feature of this
trait.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.19
CHARACTERIZATION OF GRAPEVINE YABBY GENE FAMILY
INVOLVED IN SEEDLESSNESS
DI RIENZO V., BLANCO A., MONTEMURRO C.
Department of Plant Biology and Chemestry, Section of Genetic and Plant Breeding, University of
Bari “Aldo Moro”, Via Amendola 165/A, 70126 Bari (Italy)
seedlessness, grapevine, yabby gene family, ino gene, high resolution melting PCR
Seedlessness is an highly relevant trait in many fruit crop species, and it is a primary target of
breeding programs for table grape. Two main mechanisms are responsible for the formation of
seedless fruits: parthenocarpy, where the fruits develop in absence of fertilization, as in cultivated
pineapples, some Citrus species, and bananas; and stenospermocarpy, in which occur both
pollination and fertilization, but the embryo does not form or it abort before completion of seed
formation, as in seedless watermelon and grape. Mutation leading to failure in seed formation have
been studied in the model system Arabidopsis thaliana, where complete absence of seeds has been
associated to defects in ovule development. The mutation of A. thaliana inner no outer gene
(known as ino or yabby4 gene) exhibits a partial failure in seed formation due to the absence of the
outer integuments of the ovule, and ino orthologs showed a good conservation of the spatial
expression pattern in other plant genera, such as Annona, Impatiens, and Nymphaea.
The aim of our work was to investigate the putative role in seedlessness of the ino gene in
grapevine. Its genomic sequence was analysed in two table grape cultivars: Italia (seeded) and Big
Perlon (seedless). The ino gene was isolated, cloned and sequenced in both varieties. Several SNP
(Single Nucleotide Polymorphism) between Italia and Big Perlon were evidenced and one of them
was used for High Resolution Melting analyses in a population of 192 F1 plants derived from a
pseudo-test cross, in order to map the gene.
A further approach to better understand the role of ino gene in grape was the set up of its
expression pattern analysed in different tissues (small, medium and large leaves, stems, tendrils and
flowers) and at different development stage. The gene expression experiments will allow to
elucidate the spatial gene pattern and the outer integument specificity that is an exclusive
characteristic of this gene among the other members of the Yabby family.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.20
OLIVE FRUIT SIZE IS REGULATED EARLY IN THE FLOWER:
MORPHOLOGICAL AND MOLECULAR EVIDENCE
CIRILLI M.*, CAPORALI S.**, FRIONI E.*, PAOLETTI A.**, RAPOPORT H.***,
SILVESTRI C.*, PERROTTA G.****, ROSATI A.**, RUGINI E.*, MULEO R.*
*) Dipartimento di Scienze e Tecnologie per l’Agricoltura, le Foreste, la Natura e l’Energia,
Via S. Camillo DeLellis s.n.c, Università della Tuscia, Viterbo 01100 (Italy)
**) CRA OLI, Via Nursina 2, 06049 Spoleto (Italy)
***) Instituto de Agricultura Sostenible, CSIC, PO Box 4084, 14080 Cordoba (Spain)
****) ENEA CR Trisaia, S.S. 106 Jonica 75026 Rotondella (Italy)
fruit size, ovary, cell division, Olea europeae, qPCR
Differences in fruit size among olive cultivars arise mostly at the ovary level at bloom, where
larger-fruited cultivars display larger ovaries and flowers. Larger ovaries have more cells of similar
size. Hence, fruit size differences appear to be related to cell division before bloom. In the present
work, we isolated a group of genes involved in cell number regulation, organ size, and, ultimately,
crop yield. Subsequently we tested whether their expression levels are different in the two cultivars
with diverse ovary and fruit size (i.e. cv Canino, small, and cv Ascolana Tenera, large
fruits/ovaries). OeCNR1 and OeCNR2 genes, identified in an SSH cDNA flower library of O.
europaea cv Leccino (confirmed by large scale 454 pyrosiquencing of cDNA), are putatively
orthologs of S. lycopersicum fruit weight gene fw2.2, which governs a quantitative trait locus that
accounts for 30% of tomato fruit size variation. To expand investigation of how related genes may
affect organ size, we have also isolated other genes involved in cell division and organ size
regulation, such as ERECTA (OeERE) and ERECTA-like (OeERE-like) both of which interact with
auxin AINTEGUMENTA (OeANT), which might play a pivot role between signal delivered by the
former genes and signal carried out by OeCNR1, on the cell proliferation. Organ growth is also
dependent on cell enlargement and the OeBEE1gene, carrying the brassinosteroids-signals, which is
one of the most involved genes in this phenomena. Cell cycling genes and genes involved in
polyamine synthesis have been also isolated. Gene expression analyses were carried out using
quantitative real-time PCR from the initial phase of flower development until the phases of fruit set
and drupe development, along with histological analyses of ovary and fruit dimension.
The data show an increased level of transcripts of OeCNR1 in cv Canino compared to cv A.
Tenera, during the phase of ovary development and in initial fruit development, whereas OeCNR2
levels of transcript were found to be higher in Canino than A. Tenera in initial fruit development
phase. These data agree with that found in tomato, where the fw2.2 gene negatively regulate cell
division and it is suspected to be the major player in the reduction of ovary cell number.
The expression of genes will be discussed in a framework that involves the role of hormones
and growth regulators in determining the destiny of organ size, and whether this destiny could
depend on the number of cells vs their size.
Acknowledgement: This research was partially supported by the “Fondazione Anna Maria
Catalano – ONLUS” and by Progetto Strategico MIPAF “OLEA - Genomica e Miglioramento
genetico dell’olivo”, D.M. 27011/7643/10. We thank the Roche Diagnostic Spa, Applied Science to
support the OLEA Italian Project.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.21
MONITORING OF CULTIVAR FIDELITY OF GLOBE ARTICHOKE
GERMPLASM IN A MEDIUM-TERM CONSERVATION UNDER MINIMAL
GROWTH CONDITIONS USING SSR AND ISSR MARKERS
TAVAZZA R.*, REY N.A.**, PAPACCHIOLI V.*, PAGNOTTA M.A.**
*) ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Rome (Italy)
**) Department of Science and Technologies for Agriculture, Forestry, Nature and Energy
(DAFNE), Tuscia University, Via S. C. de Lellis, 01100 Viterbo (Italy)
vitro, micropropagation, genetic fidelity, molecular markers
In vitro cultivation for conservation purposes is recognized as efficient technique
complementary to in situ practices for preservation of valuable plant species. Since maintenance of
globe artichoke (Cynara cardunculus var. scolymus L) germplasm in seed form is restricted due to
their heterozygosity and segregation, in vitro technologies provide a complementary approach to
field genebanks which still act as both active and base collections. In vitro storage is excellent for
medium-term storage and is used as the active collection in some genebanks. Moreover, dual mode
of conservation of the accessions in vitro provides protection from pests and climatic hazards
increasing their availability for distribution.
In the frame of an Italian National project (CARVARVI) evaluation of selected clones of
globe artichoke under different slow-growth conditions (using osmotic stress) is ongoing, aiming to
establish the best condition for in vitro storage.
Slow growth storage, refers to the techniques enabling the in vitro conservation of shoot
cultures, in aseptic conditions, by reducing markedly the frequency of periodic subculturing through
the reduction of cellular metabolism, without affecting the viability and regrowth of shoot cultures.
This approach has been used, in our study, to successfully preserve, for 6 months, 7 clones
representative of 3 different Romanesco typologies (early, medium, late). During the 6-months
preservation, the explants reduced greatly plant growth without lousing the explants survival.
Since one of the major objectives of germplasm conservation is to maintain the genetic
diversity of one species in a stable condition, the storage techniques used should not endanger plant
genetic stability. For this reason DNA markers technologies were used to monitor, at 6 months, the
effect of the in vitro storage conditions used, on the genetic stability of the preserved clones.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.22
ANALYSIS OF POSSIBLE GENETIC VARIATION INDUCED BY
MICROPROPAGATION IN GLOBE ARTICHOKE PLANTLETS
PAGNOTTA M.A.*, TAVAZZA R.**, REY N.A.*
*) Department of Science and Technologies for Agriculture, Forestry, Nature and Energy
(DAFNE), Tuscia University, Via S. C. de Lellis, 01100 Viterbo (Italy)
**) ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Rome (Italy)
vitro, micropropagation, genetic fidelity, molecular markers
Propagation of plant material by in vitro culture could face with genetic variants occurring
during in vitro phases. Genetic fidelity of micropropagated plantlets of Globe Artichoke is a key
issue for germplasm conservation and commercialization; objective of the present study is the
assess of genetic variation across subcultures. Leaves from both in-vivo (mother plants in the field)
and from in-vitro material (multiplicated shoots) were analyzed over two years to determine
intraclonal variation and to evaluate the influence of the number of subcultures on the generation of
somaclonal variants. Two different DNA-based techniques, Inter Simple Sequence Repeats (ISSR)
and simple sequence repeat (SSR) markers was used and evaluated, in assessing the genetic stability
of micropropagated plants of six ecotypes. Eight primers were successfully used to amplify the
DNA demonstrating a different ability to detect genetic variation.
ISSR amplicons detected by automatic sequencer resulted as an efficient marker typology in
showing the occurrence of the genetic changes that occur during the micropropagation process of
Globe Artichoke. We conclude that when the tissue culture technique is used, the analysis of
somaclonal variability could require more than one DNA-based technique. In fact as our results
showed different markers detect different genome regions and have different polymorphic ability.
The pathway of variation is not constant across the different subcultures and this give useful
information to be utilized during breeding activities and variety registration.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.23
EFFECT OF COCONUT WATER AND GROWTH REGULATOR
SUPPLEMENT ON THE IN VITRO PROPAGATION OF CORYLUS
AVELLANA L.
SANDOVAL PRANDO M.A.*, CHIAVAZZA P.**, CONTESSA C.*, BOTTA R.*
*) Dipartimento di Colture Arboree, Università degli Studi di Torino, Via Leonardo da Vinci 44,
10095 Grugliasco (Italy)
**) Dipartimento di Agronomia, Selvicoltura, Gestione del territorio, Università degli Studi di
Torino, Via Leonardo da Vinci 44, 10095 Grugliasco (Italy)
Gibberellic acid, N6-benzyladenine, Indole-3-acetic acid, DKW
Corylus avellana L. represents an economically important crop in the European Community.
The availability of efficient and reliable in vitro propagation methods can contribute to exploit the
local genetic resources and the breeding obtentions and provide tools for biotechnological
objectives. Several protocols of micropropagation have been realised but the methods have not yet
been developed to allow large–scale mass propagation, as requested for commercial purposes, due
to poor proliferation yield. The aim of this study was to contribute to improve proliferation and
growth of hazelnut using different supplements to the basal medium. Single axillary buds from
mature trees of Corylus avellana L. were cultured on the modified DKW (Driver & Kuniyuki
Walnut) basal medium enriched with gibberellins, cytokinins, auxins and coconut water in several
combinations for the growth of hazelnut shoot explants, cultivar ‘Tonda Gentile delle Lange’. The
addition of 20% coconut water to the modified DKW medium increased the number of adventitious
shoots per explant. None of the used doses of cytokinin provided similar results. The results also
showed that gibberellins improved the elongation and the coconut water improved the quality of
plant growth in term of plant vigour. The supplement of 3 µM gibberellic acid (GA3), 2.2 µM N6benzyladenine (BAP), 0.02 µM Indole-3-acetic acid (IAA), was effective for increasing the growth
of the shoot explants. Shoot multiplication differed with the position on the medium (horizontal or
vertical). The highest rates occurred with propagules in a horizontal position. Preliminary rooting
tests are being carried out both in vitro and using a minicutting system under greenhouse.
Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress
Perugia, Italy – 17/20 September, 2012
ISBN 978-88-904570-1-2
Poster Communication Abstract – 1.24
IN VITRO PROPAGATION STRATEGIES FOR INDUCTION AND
REGENERATION OF POLYPLOID PLANTS IN DENDROBIUM
PHALENOPSIS HYBR.
GROSSO V.*, NARDI L.**, FARINA A.*, GIORGI D.*, ARACRI B.***, PASHKOULOV
D.***, FATTA DEL BOSCO S.****, CATALANO C.****, MOTISI A.****, ABBATE L.****,
LUCRETTI S.*
*) ENEA Centro Ricerche Casaccia, Unità Tecnica AGRI, Laboratorio GEN, Via Anguillarese 301,
00123 Roma (Italia)
**) ENEA Centro Ricerche Casaccia, Unità Tecnica RAD, Laboratorio FARM, Roma (Italia)
***) FLORAMIATA, Loc. Casa del Corto, Pian del Castagnaio (Italia)
****) Istituto di Genetica Vegetale CNR, Corso Calatafimi 414, Palermo (Italia)
Dendrobium hybrid, flow cytometry, polyploidization, Protocorm Like Body
Orchid plants are monocots belonging to the Orchidaceae family, which is one of the most
numerous families listing more than 25,000 species and more than 150,000 commercial hybrids.
They hold a high economical value on the international flower markets and are sold both as pot
plants and cut flowers. Breeding in orchids is mostly directed to generate new varieties and hybrids
to correspond market needs; the introduction of novel genetic variability it’s crucial to fullfil this
target. Manipulating ploidy levels in commercial and wild species is one of the possible way to
create new genetic variability. Polyploidy may allow breeders to overcome sexual incompatibility
barriers and to recover hybrid fertility by the generation of allopolyploids, which, in turn, may have
an improved pest and disease tolerance. Polyploids may also show an enhanced vigor and large
general size. Actually, known polyploid orchids have larger flowers and exhibit recurrent and
longer blooming time. Our work has been focused on protocorm like bodies (PLBs) of a
Dendrobium interspecific hybrid (Dendrobium phalaenopsis x Dendrobium loddigesi), which
exhibit an high regeneration capability. PLBs were in cultivated in liquid and solid media,
generating fast growing PLBs or callus tissue. Both types of explants were used for a series of
experiments devoted to polyploidy induction using antimitotic drugs at different concentrations and
incubation time. We applied colchicine (COL) and amiprophos-methyl (APM) treatments to inhibit
cell cycle and mitotic spindle organization into PLB and callus cells, both in liquid and solid media.
To investigate ploidy levels, immediately after treatments, explants were analyzed by flow
cytometry, which is a method for a rapid and early screening of DNA content on isolated nuclei in
suspension. Nuclei were isolated from 20-30mg chopped tissues in a MgCl2 TRIS lysis buffer. The
effectiveness of the treatments for polyploidy induction were assessed on regenerated plantlets after
six months from the incubation. The 3 days treatment showed to be effective in polyploidization for
all concentrations. Also, regenerated PLB polyploid plants showed a greater vigor and a higher
proliferative capacity than untreated ones, probably due to the fact that the polyploidization was
able to balance their hybrid genetic constitution. Experiments performed using callus tissue
presented a similar behavior than PLB ones, with a slower production of plantlets which showed an
incrased percentage of mixoploids at the higher drug concentrations together with a lower growth
vigor. The flow cytometry approach was able to indicate which treatments were the most active and
less toxic to orchid cells, which in turn avoided an excessive strength of mutagenic treatments and
an useless in vitro propagation of true-to-type materials, thus reducing costs and experiment
working time.
This work is supported by MIPAAF, Project NOVAORCHID (D.M.11074/7643/09)