Download GENE - Università degli Studi del Molise

Campobasso, 21 Marzo 2007
La genomica
genomica per
per la
la
La
valorizzazione delle
delle risorse
risorse
valorizzazione
genetiche vegetali
vegetali
genetiche
Luigi Monti
Università degli Studi di Napoli Federico II
CNR Istituto di Genetica Vegetale
PLANT GENETIC RESOURCES
300.000
Plant species
30.000
Useful for humans
3.000
300
Used as food and other
Grown
30
Very important
3
60% of total plant calorie intake
PLANT GENETIC COLLECTIONS
Total
4,038,000
Hammer et al., 2003
GENETIC BOTTLENECKS IMPOSED
ON CROP PLANTS
Wild species
Early domesticates
Modern varieties
Tanksley & McCouch, 1997
Overcoming interspecific barriers:
some examples
Tool
Crop
Traits transferred
Embryo culture
(Tomato)
S. lycopersicum x
S. peruvianum
Resistance to fungi
and bacteria
2n gametes
(Potato)
S. tuberosum (4x) x wild
Solanum spp. (2x)
Resistance to fungi
and frost
Ph gene
(Wheat)
T. durum/aestivum x
Aegilops and other
Triticeae
Resistance to Oidium
and rust
Protein quality
GENOMICS MILESTONES
1953
Watson & Crick:
DNA Structure
1970
Smith &
Wilcox:
Restriction
Enzymes
1975
Southern:
Southern
Hybridizations
Mullis et al:
PCR
PCR
Restriction Enzymes / Ligase
RFLP
AFLP
Linkage mapping
Optical mapping
Library construction
Gene cloning
Tilling
1986
DNA:DNA Hybridization
Southerns / RFLP
Northerns
Microarrays
DArT markers
Taqman Real Time PCR
Chromosome painting
AFLP
RAPD
CAPS
SCAR
STS
Real Time PCR
RT-PCR
DNA sequencing
SNP detection
Genomics
Structural Genomics:
Functional Genomics:
understanding how genomes are
physically organized
understanding how genomes and
genes work
● linkage and gene maps
● physical (BAC) maps
● DNA sequencing
● transcriptomics
● transformation
● gene silencing
The impact of biotechnologies on the use
of genetic resources
Exploitation of genotypes
(Molecular markers)
Exploitation of single traits
(MAS)
Exploitation of single genes
(Forward & reverse genetics)
The impact of biotechnologies on the
conservation of genetic resources
Conservation
Local
varieties
Wild
species
Exploitation of
genotypes (molecular
markers)
+
Exploitation of single
traits (MAS)
+
+
Exploitation of single
gene (Forward &
reverse genetics)
+
+
DNA
+
IDENTIFICATION OF LOCAL VARIETIES
-Pomodoro “Vesuviano”
-Pomodoro “S. Marzano”
-Pomodoro “Sorrento”
-Pomodoro “Corbarino”
-Pomodoro “Africano”
-Pomodoro “Tondo di Sulmona”
-Pomodoro “A pera”
-Pomodoro “Grosso di Maria”
-Fagiolo “di Controne”
-Fagiolo “Occhio nero Oliveto Citra”
-Fagiolo “di Sarcione”
-Fagiolo “Poverello”
-Fagiolo “Marruzzo”
-Fagiolo “Verdolino”
-Fagiolo “Zolfino del Pratomagno”
-Nocciolo “Tonda di Giffoni”
-Nocciolo “Tonda delle Langhe”
-Nocciolo “Napoletanedda”
-Nocciolo “Riccia di Talanico”
-Carciofo “Romanesco”
-Carciofo “Castellamare”
-Carciofo “ Spinoso di Sciacca”
GENETIC TRACEABILITY OF PLANT PRODUCTS
WITH SSR MARKERS
APPLE PRODUCTS
OLIVE OIL
DNA
source
Allelic profile at locus DC4
DNA
source
Allelic profile at 2 loci
no logo!
no logo!
purèe
CH01G12
CH01F02
nectar
128 bp
130 bp
Rao et al., 2006
Molecular assisted selection: resistance genes
pyramided in tomato
Cross
Pyramided
genes
No. F2 analyzed
No. F4 homozygous
at R loci
Momor x Motelle
Momor x Ontario
Frl, Tm2a, I2, Mi, Ve
Frl, Tm2a, Pto
50
30
2
4
Motelle x Ontario
Okitzu x Ontario
Okitzu x Pyrella
I2, Mi, Pto
I2, Tm2a, Pto
I2, Tm2a, py-1
30
30
30
2
6
2
Barone et al., 2004
Use of MAS to transfer useful QTLs from wild species
to the cultivated tomato
Cultivated tomato
chromosomes
1 2 3 4 5 6
Wild species
chromosomes
1
2
3
4
7
8
9 10 11 12
5
6
7
1
7
2
8
3
9
4
9 10 11 12
8
5
6
10 11 12
Inbred line
Grandillo et al. 2000
Plant nuclear genome sequences available
• Arabidopsis thaliana (Nature 408, 769-815)
• Oryza sativa ssp. japonica (Science, 269, 92-100)
• Oryza sativa L. ssp. indica (Science 296, 79-92 )
Sequencing projects under way
9Medicago truncatula
9Lotus japonica
9Solanum lycopersicum
9Solanum tuberosum
9Zea mais
9Brassica oleracea
9Hordeum vulgare
9Glycine max
9Musa spp.
9Populus trichocarpa
The tomato genome sequencing project
Crom. 1
Mb 24
2
3
4
5
6
7
8
9
10
11
12
26
26
19
11
20
27
17
16
10
13
11 T=220
Italy
Usa
Usa
Spain
Japan
GB
China
Korea
Usa
Long arm
France
Usa
Centromere
The Netherlands
Short arm
Heterocromatine
Eucromatine
Satellite DNA
Cytoplasmic DNA sequences are available
The potato (cv. Desiree) plastome
RNA ribosomal genes
Transfer RNA genes
Ribosomal protein genes
Transcription/translation apparatus genes
Photosynthetic apparatus genes
NADH dehydrogenase genes
Other protein genes
Conserved reading frames
Open reading frames
Cardi et al., 2005
The Italian Plant DNA Bank: towards an integrated system for conservation
and utilization of genetic resources
CNR-IGV (project leader Gabriella Sonnante)
A DNA bank is an extension of the concept of gene-bank initially implemented in
seed genebanks and is not meant as a substitute.
Why a DNA bank?
What to store?
¾ Monitoring changes in
populations genetic structure
¾ Landraces of species of interest (e.g.:
Italian tomato landraces)
¾ Assessment of genetic diversity
¾ Typical products (e.g.: Peperone di
Senise PGI)
¾ Disclose taxonomicrelation ships
¾ Search for new gene variants
¾ Development of new markers
¾ Identification and traceability
of typical products
¾ Fraud prevention tools
¾ DNA conservation on request
¾ DNA distribution
¾ Crop wild relatives (e.g.: wild
artichoke)
¾ Wild plants used by man (Eruca,
Borago, etc.)
¾ Model plants (Arabidopsis thaliana,
Medicago truncatula)
¾ Isolated genes, clones, mutants, etc.
¾ Specific DNA markers
Limitazioni nella utilizzazione delle fonti di resistenza
Genomics has expanded from 3 to 5
the number of gene pools
Michelmore, 2003
Resistance genes
isolated and isolated
cloned in tomato whose
is available
Resistance
genes
andsequence
cloned
inintomato
GenBank
whose sequence is available in GenBank
PATHOGEN
GENE
CHROMOSOME
Asc-1
Alternaria alternata f.sp. lycopersici
3
Bs4
Xanthomonas campestris pv vesicatoria
5
Cf2, Cf4, Cf5, Cf9
Cladosporium fulvum
Hero
Globodera rostochiensis
4
I2
Fusarium oxysporum f sp lycopersici
11
Mi *
Meloidogyne spp
6
Pto
Pseudomonas syringae pv tomato
5
Sw5
TSWV
9
Tm2a
TMV
9
Ve1, Ve2**
Verticillium dahliae
9
* confers resistance also to potato aphids
** confer resistance also to Verticillium in potato
1, 6
NEW APPROACHES FOR TRANSGENIC
PLANT PRODUCTION
Use of plant genes and promoters
(e.g.: antisense RNA)
Elimination of marker genes
Direct transformation
(e.g.: gene-gun, electroporation)
In vivo transformation
(no somaclonal variation)
Caratteri presenti in piante transgeniche
in commercio
CARATTERE
GENE UTILIZZATO
Resistenza ad insetti
Cry e Bt (Bacillus thuringensis)
Tolleranza ad erbicidi
EPSPS (pianta)
PAT (batterio)
BXN(batterio
Bar (batterio)
Resistenza a virus
CP (virus)
RNA antisenso
EFFETTO GENE
INSERITO
Proteine tossiche per gli
insetti
Insensibilit allÕ
erbicida
glifosato
Insensibilit allÕ
erbicida
gufosinate ammonio
Resistenza allÕ
erbicida
bromoxynil
Insensibilit allÕ
erbicida
gufosinate ammonio e
fosfinotricina
Resistenza al virus Y
della patata
Interferenza con la
replicazione del virus
Fny (replicasi mutata del virus
CMV)
Maschiosterilit
Barnase (batterio)
Barnase/Barstar (batterio)
Colore del fiore
ACC sintasi antisenso (pianta)
DF reduttasi antisenso (pianta)
Contenuto di acidi grassi
_ ______________
Maschiosterile e
resistente ad erbicidi
Distruzione cellule del
tappeto dellÕ
antera e
ristorazione
maschiosterilit
Blocco della sintesi
dellÕ
etilene
Blocco della sintesi degli
antociani
Aumento del contenuto
di acido oleico
Ca ratt er i pre sent i in p ia nt e transg eniche in s periment az ione
CARAT TER E
GENE UT ILIZZ ATO
EFF ETT O GENE
INSERITO
RIP (pian ta)
Osm otina (pian ta)
Pro teine che inattiva no
la sintesi p rot eica
Resisten za a fu ngh i
Resi ste nz a a c are nza
id ric a
Mann itolo 1P- de idr ogena si
Accumulo di mannito lo
Miglio ram en to qua lit
frut to
Poliga latturona si ant isen so
(piant a)
Bl oc co de lla
de grad azion e d ella
pe ctina
Aumen to de l con ten uto
di frut tan i
Resi ste nz a a fu nghi
Fru ttos ilt ra sfera si (ba tte rio)
Miglio ram en to val ore
nu trizion ale
Fi toene s intasi e Licopen e _
cicl as i (piant a)
Sac car osio fosfato sintas i
Compo st i far mace ut ic i
Or mone de lla crescita (u om o)
Aumen to de lla
produ zione di
prov itami na A
Accumulo di zuc che ro
Si nt esi in piant a d i
com pos ti fa rm ace utici
Eritropo ietina (uomo)
Collagene ( uomo)
Ant ic orpi ricombin anti
IgG ed Ig M
Si nt esi in piant a d i
an ticorp i
Produ zion e di vacc ini
Tossina de l colera , tetan o e
rota viru s
Pro teina Ņenve lopeÓ del virus
de llÕep atite B
Pro teina de l ca pside de l virus
Norw alk
Aut oan tigene pe r il diabe te
Si nt esi in piant a d i
vaccini