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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Take a step back……

Take a step back…..

First results on durum wheat….

“omic” sciences

In a continous development

From mutagenesis…..to……

Cell and Tissue culture….

early sixties (1960 to date..)

in vitro cell and tissue cultureB

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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

Developing biotechnological tools to controlTomato yellow leaf curl disease

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

High throughput flow cytometer cell sorter forbiotech manipulations

(Wheat genome = 16000 Mbp)

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

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(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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re‘Omic’ Technologies in molecular food

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

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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…”.

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reThank you for your attention!