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Biotecnologie verdi:
Contributi della genomica e della biologia
molecolare al miglioramento genetico
delle crops.
Eugenio Benvenuto Sezione Genetica e Genomica Vegetale
Workshop ENEA
Cibo ed Energia: un approccio sostenibile
Roma, 11 dicembre 2009
Milestones of Plant Genetics …atB
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early sixties (1960 to date..)
in vitro cell and tissue cultureB
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QuickTime™ e undecompressore Cinepak
sono necessari per visualizzare quest'immagine.
Cultivar migliorata
con interventi
molecolari
Tecniche di
Ingegneria
Genetica
Trasferimento genico
mediato da
Agrobacterium
Propagazione in vitro
Isolamento gene contenente
l’informazione per la sintesi delle
proteine d’interesse
(per es. coinvolte nella resistenza alle
malattie o nell’accumulo di sostanze di
interesse nutraceutico)
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reEarly eighties (1980 to date……)Genetic engineering & GM Crops
Viral vector
PLANT VIRUS INFECTION
TRANSIENT TRANSIENT
TRANSFORMATIONTRANSFORMATION
Plant Cell
Nucleus
Cytoplasm NUCLEAR TRANSFORMATION
MEDIATED BY
A.tumefaciens
or
Particle gun
Chloroplasttransformation
by shot-gun
STABLE STABLE
TRANSFORMATIONTRANSFORMATION
Viral replication
Chloroplast
Plants as cell factoriesPlants as cell factories
“Golden” crops to fight avitaminosis A
Potato Tomato β β β β-carotene
Plant-derived Vaccines
HPV SARS-CoVHIV
�Diagnosis�Immunotherapy�Plant protection
Semi-synthetic antibody
repertoires
2000 to date…..…..‘OMIC’ Technologies
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MODERN MEANING OF GENOMEMODERN MEANING OF GENOME
The The whole whole of the of the GENETIC MATERIALGENETIC MATERIAL
In a In a cellcell, in , in an organisman organism, or in a , or in a speciesspecies
It represents the potential in It represents the potential in
terms of functionality of an terms of functionality of an
organismorganism
Genome Complexity is Genome Complexity is
described as described as the the number number
of of basebase--pairs pairs ((bpbp))
1 1 Megabp Megabp = 1 = 1 million bpmillion bp
Several Overlapping Several Overlapping
RevolutionsRevolutions::
1. The 1. The Original Original OneOne
Nature 409, Nature 409, February February 2001 2001 –– 3000 3000 Mega Mega base base pairspairs
Plant Genomes Publicly ReleasedPlant Genomes Publicly Released
Arabidopsis thalianaArabidopsis thaliana 20012001 125 125 MbpMbp
RiceRice 20022002 370 370 MbpMbp
PoplarPoplar 20062006 530 530 MbpMbp
Vitis Vitis viniferavinifera 20072007 490 490 MbpMbp
PapayaPapaya 20082008 380 380 MbpMbp
PotatoPotato 2009 840 2009 840 MbpMbp
Maize Maize 20092009 2300 2300 MbpMbp
PeachPeach, , AppleApple, , TomatoTomato, 2010 ??, 2010 ??
SorghumSorghum, , CitrusCitrus, , SoyaSoya, Banana, Banana
et et alal
Deciphering the genome ofThe 40 most cultivated crops….
Most Cultivated Crops in 2005 Rank in 2005 Rank in 2000 Area Cultivated in 2005 (HA) Area Cultivated in 2002 (HA)
Wheat 1 1 214666735 215474117
Rice, paddy 2 2 152904158 153942855
Maize 3 3 143798427 139494325
Soybeans 4 4 92443356 74321496
Barley 5 5 55895782 54489630
Sorghum 6 6 43360759 40831541
Seed cotton 7 8 35003800 31924034
Millet 8 7 34815044 37006519
Rapeseed 9 9 27448195 25833433
Beans, dry 10 10 25231390 23531811
Groundnuts, with shell 11 11 23469008 23358847
Sunflower seed 12 12 22372946 21161673
Potatoes 13 13 18872538 20130298
Sugar cane 14 14 18730869 19466758
Cassava 15 15 18320052 17059381
Alfalfa for forage and silage 16 16 15118543 15421302
Oats 17 17 11450461 12676446
Coconuts 18 18 10681368 10596868
Chick peas 19 20 10646032 10100570
Coffee, green 20 19 10007069 10317198
Oil paini fruit 21 21 9384653 9954304
Cow peas, dry 22 25 9170820 7494030
Sweet potatoes 23 23 8810860 9703499
Naturai rubber 24 31 8114849 5640296
Sesame seed 25 27 7596056 7269734
Olives 26 24 7574361 8327400
Grapes 27 26 7470679 7377007
Rye 28 22 6829860 9818138
Peas, dry 29 30 6298663 5769319
Rye grass for forage and silage- 30 5505313
Plantains 31 33 5399072 4976685
Cocoa beans 32 28 5287847 7125153
Sugar beet 33 29 5240711 6011351
Apples 34 32 4774376 5310185
Pigeon peas 35 34 4572039 4244022
Tomatoes 36 38 4570870 3968508
Bananas 37 37 4370342 4030079
Lentils 38 39 4084614 3873738
Tobacco 39 35 3908731 4160103
Triticale5
40 3744001
Plant genomics Plant genomics and and proteomics proteomics --International ConsortiaInternational Consortia--
Sequencing of tomato genome (109 bp)
Grape berry proteomics
Wheat Chromosome Sorting
Toscano
‘omic’ sciences to improvequali-quantitative traits of cultivars
•Identificazione e selezione di geni o assetti genici ideali in genotipi di interesse
•Tipizzazione e origine di varietà tradizionali
S. MarzanoPrincipe Umberto
Corbarino Giallo invernale
5
witheringwitheringpre-veraisonpre-veraison
veraisonveraisonripeningripening
Grapevine System Biology……
Pinot blanc Pinot gris Pinot noir
Sangiovese clones
Identifying Molecular Basis of Clonal Variation
is not a mirage
0
50
100
150
200
250
300
350
45 60 75 90 105 120 135 150 165 180
Days after flowering
Po
lyp
hen
ols
(m
g g
-1 D
W)
CORATINA
TENDELLONE
cv. Tendellonecv. Coratina
135 DAF135 DAF
45 DAF45 DAF
Higher content of polyphenols
Higher content of polyphenols
Lower content of polyphenols
Lower content of polyphenols
DAF: Days After FloweringDAF: Days After Flowering
Plant Material
First olive microarray platform(Genome 1C=1500 Mbp)
CYNARESGenetic Resourses of Cynara spp.
Obiettivi: Recupero caratterizzazione e conservazione delle Recupero caratterizzazione e conservazione delle risorse genetiche tradizionali europee di carciofo e risorse genetiche tradizionali europee di carciofo e cardocardo
Partner: Italia, Spagna, FranciaItalia, Spagna, Francia
Durata del Progetto: 20072007--20112011
Biodiversity
SAFENUT‘Safeguard of hazelnut and almond genetic
resources: from traditional uses to novel agro-industrial opportunities’
Objectives•To increase the knowledge of the European hazelnut and almond germplasm (Corylus avellana and Prunus dulcis) in order to enhance its characterisation, preservation and utilisation.
•To recover and valorise local endangered germplasm in the traditional productive areas of the Mediterranean basin.
• To create a core collection both for hazelnut and almond species.
•To set up a web based inventory linked with the major thematic international databases and to create a strong European network.
• To promote a wider application of traditional knowledge to raise stakeholders awareness on the values of biodiversity in the framework of the sustainable development.
TARGETED ACTION COUNCIL REGULATION (EC) N° 870/2004 Community programme on the
conservation, characterisation, collection and utilisation of genetic resources in agriculture
Duration:April 2007-2010 Partecipants: 6 European Countries (Italy, Spain, France, Portugal, Greece, Slovenia)
Coordinator ENEA
-- Based Based on on Sanger Sequencing Sanger Sequencing
Chemistry Chemistry (1968)(1968)
-- Sequence assebly requiresSequence assebly requires
redundant sequencingredundant sequencing
-- Complex organizationComplex organization
-- Specialized CentersSpecialized Centers
--TimeTime--consumingconsuming::
YearsYears
--CostlyCostly: :
500 500 Megabp Megabp = 1 = 1 Million Million €€
The First The First Genomic Genomic RevolutionRevolution
Phenomenal Improvements Phenomenal Improvements in in several fieldsseveral fields::-- microscopy microscopy and and imagingimaging-- surface chemistrysurface chemistry-- nucleotides biochemistrynucleotides biochemistry-- enzimologyenzimology-- miniaturizationminiaturization-- informatics informatics and and
biobio--informaticsinformatics
““DEMOCRATIZATIONDEMOCRATIZATION”” of of GENOME SEQUENCING GENOME SEQUENCING
Sequencing CentersSequencing Centers Single LabsSingle Labs
ClassicClassic SecondSecond--generationgeneration
High High Throughput Throughput
GenotypingGenotyping
3 micron diameter beads3 micron diameter beads
50,000 features 50,000 features
1,536 SNPs in one run1,536 SNPs in one run
Illumina Illumina
BeadBead--arrayarray
GenotypeGenotype
SeparationSeparation
Alleles Identification isAlleles Identification is NO NO
LONGERLONGER
the the Bottle NeckBottle Neck
Alignement Alignement of of TAGs to TAGs to the the reference sequence reference sequence and and
identification identification of Single of Single Nucleotide PolymorphismsNucleotide Polymorphisms
SecondSecond--Generation SEQUENCING: Generation SEQUENCING:
Comparison among PlatformsComparison among Platforms
Third Third Generation Generation Sequencing Is Already HereSequencing Is Already Here
Single Single Molecule Real Molecule Real Time Time Sequencing performed by Sequencing performed by a single DNA a single DNA
polymerase moleculepolymerase molecule
ZMW is a cylindrical hole ZMW is a cylindrical hole
tens of nanometers in tens of nanometers in
diameter perforating a thin diameter perforating a thin
metal film supported by a metal film supported by a
transparent substrate. transparent substrate.
ZeroZero
ModeMode
WaveguideWaveguide
A DNA pol molecule is attached A DNA pol molecule is attached
at the bottom of the ZMW, which at the bottom of the ZMW, which
allows zeptoliters (10allows zeptoliters (10-- 2121 lt) lt)
reaction in millisecondsreaction in milliseconds
12 Mbp per hour, several thousands nucleotide long12 Mbp per hour, several thousands nucleotide long
Commercialization 2010Commercialization 2010
1 human genome for 100 US dollars by 20131 human genome for 100 US dollars by 2013
First molecular multi-detection test for food and feed analysis:
FoodExpert ID Test ®, bioMerieux -Affimetrix ® Genechip Technology®
•• Based Based on high density DNA on high density DNA
chip chip which supports which supports 80000 80000
oligonucleotide oligonucleotide DNA DNA probes probes
(17 (17 nucleotides nucleotides long)long)
•• Synthesized Synthesized on the on the surface surface
of the chip of the chip by photolithographyby photolithography
•• Probes complementary to Probes complementary to
specificregions specificregions of vertebrate of vertebrate
Cytochrome Cytochrome b geneb gene
•• Can Can identify identify 3 classe of 3 classe of vertebratesvertebrates, , mammals fishes mammals fishes and and birds birds and 33 and 33 species species of of
animalsanimals, , regardless regardless of of whether whether the food the food is raw is raw or or processedprocessed..
•• SensitivitySensitivity: 0.01% of the total : 0.01% of the total weightweight, , validated validated at 5% w/wat 5% w/w
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diagnostics
PerspectivesPerspectives
Sequencing of
large genomes (1010 bp)
Pharmaceuticals
Biofuels
Bioprocesses
Exploitation of plant and
microbial biodiversity
CO2
Solar energySolar energy
BiodieselBiodiesel
HH22
Exploration of microalgae for CO2 mitigation and biofuel production
QuickTime™ e undecompressore
sono necessari per visualizzare quest'immagine.
From Agrigamma (1968)to Agrobiopolis….(2008)
IRRESISTIBLE GENOMIC REVOLUTIONIRRESISTIBLE GENOMIC REVOLUTIONFrom From 2000 2000 toto……. The . The years years of the of the genomesgenomes
GATTACA (1997) “There is no gene for the human spirit…”.