Davey, MW No 2010
1
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ANTIOXIDANTS:Biofortification and Resistance Breeding
Mark W. DAVEY
Lab Fruit Breeding & Biotechnology
Division Crop Biotechnics
[email protected]
Leuven
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Lab Fruit Breeding & Biotechnol
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Tree architecture•production efficiencies,
•yelds and yield predication
•correlative effects
Fruit quality•antioxidants
•texture
•quality traits (taste,
acidity..)
Development•flowering
•pollination
•fruit set
Resistance•abiotic stress
•biotic stress (Venturia
inaequalis, storage..)
LFBB Apple Breeding & Development
HIGH QUALITY, SUSTAINABLE FRUIT PRODUCTION
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Genetics•Genetic control (breeding)
•Application of molecular markers
•Intra- and inter-species
(bio)diversity
•Inter-species Breeding barriers
Molecular Genetics•Gene expression
•Cloning and transformation
•Differential gene expression
•DNA sequencing (3 x 72)
•Genetic maps, QTL’s,
•Genetic fingerprinting
Biochemistry•(A)biotic stress
•Antioxidant Metabolism
•Proteomics
•Metabolite profiling
•…
Whole Plant Physiology•Fruit quality measurements
(NIR, etc.)
•Plant growth and development
•Fruit storage
•…
Expertisesmadav 1011 madav
2 – OVERVIEW of RESEARCH
O
OH
HO CH2OH
HO
O
L-AA L-DHA
O
O
HO CH2OH
O
O
Davey, MW No 2010
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On-going Projects
1. Proteomics of Malus - Venturia interactions
2. Pear-scab – Venturia pirina
3. Fruit quality traits: molecular marker development
4. Carotenoid (vitamin A) metabolism and diversity in Musa
5. Genetic control of tree architecture
6. Induced resistance and cross-tolerance to Botrytis in Malus
7. Polyploidisation as a means to manipulate crops stress tolerance
THEMES
1. The plant stress adaptive responses
1. role of antioxidant metabolism
2. Molecular breeding; Tools and mapping
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PROTEOMICS:
•Malus – Venturia interaction
•Vit A accumulation, Musa
Bruno DANIELSFrancis AMAOKO-ANDOH
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2D-gel PAGE
SCAB PROTEOMICS: 2D-PAGE
Remains the method of choice for separating complex protein mixtures from cell lysates, tissues etc.
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Scab Phenotyping
days after infection
susceptible
polygenic
resistance
monogenic
resistance
% in
fect
ion
•10 plants, per condition, per time point
•3 leaves per plant
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Proteomics, 2D-PAGE
3 plants
3 leaves/plant
Image processing
Scanning
Image processing
Scanning
Image processing
Scanning
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RESULTS: Image analysis
masters
differential spots
Comparing treatments
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Differential protein expression
days after infection days after infection
days after infectiondays after infection
Scab-specific protein induction
Development and mapping of molecular
markers for candidate genes involved in
apple fruit quality traits
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Markers & Fruit Quality Improvement, KUL
Need for development of new apple cv’s to ensure renewal,
changing consumer preferences etc..
Which Quality Traits?
texture is particularly important and cv’s with crisp and juicy fruit will encourage
consumers to purchase again.
studies suggest that flesh firmness is the main edible quality factor determining
consumer acceptance
Bottle Necks to Breeding
• Long juvenile period, High heterozygosity in apple, traits are multigenic
Molecular Markers for MAB/MAS
• Pyramiding, stability, speed..
quantify expression during fruit ripening and development
expression profiles grouped into 12 clusters of ~appr. 500 diff. expressed tags
>400 tags have been sequenced (appr. 80%, encircled clusters have been
sequenced completely
Identify sequence tags associated with particular phases of development
cDNA-AFLP approach
Gene Discovery
June Sept
Dreesen & Keulemans (2005), ActaHort 738, p293.
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Candidate Gene Approach
genes involved in ethylene-biosynthesis and signaling:
MdACS3, MdERS2, MdETR2, MdCTR1, MdERF2
novel genes with regulatory functions and with expression profiles
peaking at climacterium:
NDPKII-related gene, AP2-related transcription factor,…
genes encoding enzymes involved in cell wall modification:
expansins, XETs, pectate lyases, β-galactosidases, α-furanosidase
cDNA-AFLP and ripening literature search:
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LG 9 – AP2-related TF
MdExpA5
EAATMCCG170EAATMCAT62
AP2-related
CH01h02EAATMCAC77
ECAAMCGA265EAATMCCT131
EAGCMCCT8904h11EAACMCGG106EAATMCCT331
EAGAMCCG331
EAGAMCCG248EACCMCCA195MdExpA5
EAGAMCCA77EAGAMCGG43
CH01f03b
EAGAMCCA183
EAATMCAC77
EAATMCCT17004h11
EAAGMCGA52
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
Telamon-9 Braeburn-9
AP2-related TF might be a regulator of fruit growth
2
3 4
1
2
3
4
Md
Ex
pA
5
EA
AT
MC
CG
17
0
EA
AT
MC
AT
62
AP
2-r
ela
ted
CH
01
h0
2
EA
AT
MC
AC
77
EC
AA
MC
GA
26
5
EA
AT
MC
CT
13
1
EA
GC
MC
CT
89
04
h1
1
EA
AC
MC
GG
10
6
EA
AT
MC
CT
33
1
1
2
3
4
Md
Ex
pA
5
EA
AT
MC
CG
17
0
EA
AT
MC
AT
62
AP
2-r
ela
ted
CH
01
h0
2
EA
AT
MC
AC
77
EC
AA
MC
GA
26
5
EA
AT
MC
CT
13
1
EA
GC
MC
CT
89
04
h1
1
EA
AC
MC
GG
10
6
EA
AT
MC
CT
33
1
1
2
3
4
siz
ew
eig
ht
aft
er
pic
kin
g
QT
L a
na
lysi
s T
ela
mo
n-
20
04
1
2
3
4
Md
Ex
pA
5
EA
AT
MC
CG
17
0
EA
AT
MC
AT
62
AP
2-r
ela
ted
CH
01
h0
2
EA
AT
MC
AC
77
EC
AA
MC
GA
26
5
EA
AT
MC
CT
13
1
EA
GC
MC
CT
89
04
h1
1
EA
AC
MC
GG
10
6
EA
AT
MC
CT
33
1
1
2
3
4
Md
Ex
pA
5
EA
AT
MC
CG
17
0
EA
AT
MC
AT
62
AP
2-r
ela
ted
CH
01
h0
2
EA
AT
MC
AC
77
EC
AA
MC
GA
26
5
EA
AT
MC
CT
13
1
EA
GC
MC
CT
89
04
h1
1
EA
AC
MC
GG
10
6
EA
AT
MC
CT
33
1
1
2
3
4
1
2
3
4
siz
ew
eig
ht
aft
er
pic
kin
g
QT
L a
na
lysi
s T
ela
mo
n-
20
04
size
Weight
at harvestL
OD Scores
1
2
3
4
AP2-like
Davey, MW No 2010
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0
1
2
3
4
Md
Bga
lEC
ACM
CCT
61
02b1
EA
CAM
CCA
280
EACA
MC
CT39
1E
AA
CMC
AT1
35
EAG
AM
CCA
98
Md
AC
S3a
EAA
GM
CGG
384
Md
XET
03EA
AT
MCG
T225
EA
ATM
CCT
82
ECA
TMC
AC1
48EA
GCM
CA
T185
EA
CAM
CCA
272
ECA
AM
CCG
275
EAA
TMC
CG1
23
EAA
GM
CG
G57
EAG
AM
CCT
116
EAA
TMC
GT2
63
EA
CAM
CCA
240
ECA
CM
CCT1
05
EA
AG
MC
CG70
EAA
TMC
CT1
38
ECA
AM
CG
G1
49
EAA
TMC
AT1
64EA
GA
MC
CA1
71
CH
02c
09
EAA
GM
CA
T132
ECA
TMC
AT1
20
Md
ACS
1
firm
ne
ss
pick
ing
QT
L a
na
lysi
s T
ela
mo
n-
20
05
0
1
2
3
4
Md
Bga
lEC
ACM
CCT
61
02b1
EA
CAM
CCA
280
EACA
MC
CT39
1E
AA
CMC
AT1
35
EAG
AM
CCA
98
Md
AC
S3a
EAA
GM
CGG
384
Md
XET
03EA
AT
MCG
T225
EA
ATM
CCT
82
ECA
TMC
AC1
48EA
GCM
CA
T185
EA
CAM
CCA
272
ECA
AM
CCG
275
EAA
TMC
CG1
23
EAA
GM
CG
G57
EAG
AM
CCT
116
EAA
TMC
GT2
63
EA
CAM
CCA
240
ECA
CM
CCT1
05
EA
AG
MC
CG70
EAA
TMC
CT1
38
ECA
AM
CG
G1
49
EAA
TMC
AT1
64EA
GA
MC
CA1
71
CH
02c
09
EAA
GM
CA
T132
ECA
TMC
AT1
20
Md
ACS
1
firm
ne
ss
pick
ing
QT
L a
na
lysi
s T
ela
mo
n-
20
05
LOD Scores
0
1
2
3
4
Firmness
Harvest
LG 15 – MdACS3a, MdBgal and MdXET03
MdBgalECACMCCT6102b1EACAMCCA280EACAMCCT391EAACMCAT135EAGAMCCA98MdACS3aEAAGMCGG384MdXET03EAATMCGT225MdACS1EACCMCAA50EAGAMCGC115ECAAMCGA48EAATMCCT82ECATMCAC148EAGCMCAT185EACAMCCA272ECAAMCCG275EAATMCCG123EAAGMCGG57EAGAMCCT116EAATMCGT263EACAMCCA240ECACMCCT105EAAGMCCG70EAATMCCT138ECAAMCGG149EAATMCAT164EAGAMCCA171CH02c09EAAGMCAT132ECATMCAT120
ECATMCAC148MdXET03MdACS1ECAAMCCT193ECAAMCGA48EAATMCGT225
ECATMCCT142
CH02c09EAATMCCT447EAATMCCT548
02b1ECACMCCT61
EAATMCCT54
ECACMCCT290ECATMCGA439
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
Telamon-15 Braeburn-15
MdACS3a is relevant for fruit firmness at
harvest~ expression profile
qPCR MdACSa - cv Elstar
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
3-A
ug
5-A
ug
7-A
ug
9-A
ug
11
-Au
g
13
-Au
g
15
-Au
g
17
-Au
g
19
-Au
g
21
-Au
g
23
-Au
g
25
-Au
g
27
-Au
g
29
-Au
g
31
-Au
g
2-S
ep
4-S
ep
6-S
ep
8-S
ep
climacterium
MdACS1
MdXET03
MdACS3a
MdBgal
Telamon-15
qPCR, MdACS3
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“Cross-Tolerance and Antioxidant Metabolism as Determinants of Resistance of Apple Fruit to
Postharvest Botrytis Decay”
L-AA
MDHA
DHA 2GSH
GSSG NADPH
NADP+
NADP
NADPH
H2O2
2H2O
1 2
3 4
2 O2.-
2 O2
5
6
H2
O
½ O2
7
O3
O2.-
OH .
OOH.
1
32
Ascorbate peroxidase
Monodehydroascorbate reductaseDehydroascorbate reductase
4 Glutathione reductase
5 Superoxide Dismutase
6 Mehler reaction7
Catalase
L-AA
MDHA
DHA 2GSH
GSSG NADPH
NADP+
NADP
NADPH
H2O2
2H2O
1 2
3 4
2 O2.-
2 O2
5
6
H2
O
½ O2
7
O3
O2.-
OH .
OOH.
1
32
Ascorbate peroxidase
Monodehydroascorbate reductaseDehydroascorbate reductase
4 Glutathione reductase
5 Superoxide Dismutase
6 Mehler reaction7
Catalase
1
32
Ascorbate peroxidase
Monodehydroascorbate reductaseDehydroascorbate reductase
4 Glutathione reductase4 Glutathione reductase
5 Superoxide Dismutase5 Superoxide Dismutase
6 Mehler reaction6 Mehler reaction7
Catalase7
Catalase
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Harvest date, vit C & Botrytis susceptibility
Davey et al 2007, J Agr Food Chem
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0
20
40
60
80
100
120
140
160
nm
ol to
t L
-AA
/gfw
Infe
cti
on
Sc
ore
Harvest date as days post July 27th, 2004
Botrytris 6 weeks
Gloeosporium 9 weeks
tot L-AA SL
tot AOX SL corrected
H A R V E S T D A T E
S E N S I T I V I T Y
Correlations between apple cv harvest date, mean fruit AsA content and
susceptibility to post-harvest phytopathogen infection
V I T C C
O N T E N T
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RESULTS: disease development
Botrytis Susceptibility: Disease symptoms were quantified by measuring the %
incidence (I) and size (S/mm) of lesions around the infection site.
Results expressed as infection severity (IS) = (I x S)/100 at 14 DPI
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
0 5 14
Infe
ctio
n S
ev
eri
ty
DPI
GD red
GD green
Br Red
Br Green
Br
GD
GD
Br
Br > GD
Green > Red
Braeburn, t=14 DPI
0.000.050.100.150.200.250.300.350.400.450.50
Control Mock Healthy
Infected
Diseased
Infected
Br, t=14 protein Red
Green
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Control Mock Healthy
Infected
Diseased
Infected
Br, APX, t=14 Red
Green
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Control Mock Healthy
Infected
Diseased
Infected
Br, t=14 CAT Red
Green
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Control Mock Healthy
Infected
Diseased
Infected
Br. T=14, POX Red
Green
Red vs Green Samples Relative changes in H2O2 Detoxification
STRESS
WOUNDINGINFECTION
O2.- H2O2
H2O + ½O2
AsA
MDHA
+H2O
DHA
GSH
GSSG
Flavonoid
Flavonoid.
+ H2O
APx MDHAR DHAR GR
POX
CAT
SOD
43
2
1
-2-3
-4
Diseased Infected Br, t=14
STRESS
WOUNDINGINFECTION
O2.- H2O2
H2O + ½O2
AsA
MDHA
+H2O
DHA
GSH
GSSG
FlavonoidFlavonoid
.
+ H2O
APx MDHAR DHAR GR
POX
CAT
SOD
43
2
1
-2-3
-4
Healthy Infected Br, t=14
Davey, MW No 2010
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To understand the regulation of antioxidant metabolism in fruits, and to
specifically identify regulatory genes responsible for maintaining tissue
antioxidant pools during fruit development and ripening, focusing in
particular on vitamin C
� AsA and GSH accumulation during fruit development/ripening
�In vivo labelling studies with non-radio and radio-labelled precursors of AsA
biosynthetic pathways
�Quantification of enzymes involved in AsA/GSH cycle, and other
antioxidant enzymes
�Gene expression studies of AsA related genes
Vit C Regulation Tomato
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Plant material
�‘Ailsa Craig’ – model variety
�‘Santorini’ – promising variety
Very high AsA levels during fruit ripening compared to other widely
grown varieties-excellent model to investigate the genetic basis of AsA
production
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Fruit sampling
Pool: 10 fruits/ stage/ variety
B-1 B+1
Breaker
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Relative Rate of totAsA biosynthesis
Mature green fruits
L-galactose pathway Alternative pathways
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Conclusions
1. Santorini fruits had significantly higher AsA levels compared to
those of Ailsa Craig, however…
2. Ailsa Craig fruits had better biosynthetic and recycling capacity
than those of Santorini
Differences in AsA content between the 2 varieties can not be
explained by biosynthetic or recycling capacity of the tissues
•Oxidation??
•Transport??
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Biofortification
Breeding Nutritionally-Rich Crops
Davey, MW No 2010
6
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BIOFORTIFICATION
- Breeding for ‘nutrient-dense’ crops. ‘functional foods’
- Differs from ‘traditional’ breeding strategies- no easy to score phenotype
- expensive- quantitative traits- complex biology
- Well Suited to MAS approaches
Increasingly important as sustainable manner to improve dietary intakes of target populations.
Crop Biofortification
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CRITERIA FOR NUTRITIONAL ENHANCEMENT
- non-toxic, i.e. no danger of over-consumption
- real perceived health benefit to consumer
- non-detrimental to plant/crop species
- knowledge database
POSSIBLE TARGETS
- Fe - deficiency, afflicts 30% of world population, impaired development,
anaemia's etc
- Vitamin A, afflicts 13% of world population, anticancer, antioxidant pigments,
carotenoids, “golden rice”- folate, essential during pregnancy, NTD
- vitamin E, fat-soluble antioxidant, CVD, anticancer, no true deficiency disorder- flavonoids, antioxidants, uncertain mechanism, bioavailability
- vitamin C
Targets for Biofortification
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Biofortification; vit A
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Global vit A Deficiencies
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Severe Consequences:
– Blindness
– Weakened immune system
– (In pregnant women) Night blindness
– Increases risk of maternal mortality
Facts:
– Between 100 and 140 million children are vitamin A deficient
– About 250,000 to 500,000 become blind, (1/2 dying within 12 months of losing sight)
– 600,000 women die from childbirth-related causes each year
Why vit A?
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Biofortification; vit A Musa
Davey, MW No 2010
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http://www.harvestplus.org
AIMS: • Reduce micronutrient malnutrition in developing countries• Improve food security, production and quality of life through low cost
biofortified staple crops
TARGETS: • Provitamin A carotenoids (pVACs)• Fe• Zn
MUSA: • Bananas & plantains – staple food• Cooking, roasting, frying, beer• Central& West Africa < 400 kg/person
VITAMIN A: ‘HarvestPlus’ Program, KUL
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“Fast-tracking”
“Fast-Tracking” - exploiting existing germplasm to identify biofortified Musa cultivars
– >1000 Musa cultivars recognised
– Most “popular” bananas have low pVACs contents (white flesh)
– Orange-fleshed varieties have high pVACs content
• Lois Englberger et al
• Micronesian Fe’i types
“Karat” (Fe’i)“Cavendish-type”
~2,500 µg/100 gfw BCE’s – 8,500 BCE’sHawaiian varieties
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0
20
40
60
80
100
120
140
160
180
200
Figue P
omm
e 2
Silk
IDN 0
77
Sam
ba
Pa (syn
. Patth
along)
Khom
Mo en
ang
Akon dro
Main
ty
Wam
bo
Sab
a
P isang B
angka
hulu
Yangambi k
m5
Thon g Det
Lacatan
Figue
Fa m
ille
Pis
ang Glin
tong
Hom (S
akon N
akhon)
Auko 9
83 (Auko)
Tomolo
Vunapope
Boung-fu
Medou
Pitu
Aiv
ip
Who-g
u
Kenar
Mal
a
Cavendis
hA
gul
Awonda
eke
Ngenge
Pis
ang P
apan
Am
biri
Pelip
i ta
Padji
Ebib
i
Mb
wazirum
e
Ato 8
20
Ebang
Mbet
a 2E
nar
French S
ombre
Maoli
'Ele
'ele s
ub var H
inupu a'
a
Gro
s Mic
hel
Mnalo
uki
Mak
embe N
oirBir
a
Crb
p 39
Bungaois
an
Pop o'u
lu L
ahi
Buto
be
Wik
ago
Banane Tig
rée
Bata
rd
Topala
Chek P
orng M
oan
Ihole
na Lel
e sub v
ar 'L
o ng Ped
uncular'
Cultivar
t-B
C e
qu
ivale
nts
in
pm
ol/
gd
w
Figue Pomme 2 (AAB)
Grande Naine (AAA)Cachaco (ABB)
Mbwazirume (AAA)
Batard (AAB)
Bantol Red (UNK)
Butobe (AAB)
0
20
40
60
80
100
120
140
160
180
200
Figue P
omm
e 2
Silk
IDN 0
77
Sam
ba
Pa (syn
. Patth
along)
Khom
Mo en
ang
Akon dro
Main
ty
Wam
bo
Sab
a
P isang B
angka
hulu
Yangambi k
m5
Thon g Det
Lacatan
Figue
Fa m
ille
Pis
ang Glin
tong
Hom (S
akon N
akhon)
Auko 9
83 (Auko)
Tomolo
Vunapope
Boung-fu
Medou
Pitu
Aiv
ip
Who-g
u
Kenar
Mal
a
Cavendis
hA
gul
Awonda
eke
Ngenge
Pis
ang P
apan
Am
biri
Pelip
i ta
Padji
Ebib
i
Mb
wazirum
e
Ato 8
20
Ebang
Mbet
a 2E
nar
French S
ombre
Maoli
'Ele
'ele s
ub var H
inupu a'
a
Gro
s Mic
hel
Mnalo
uki
Mak
embe N
oirBir
a
Crb
p 39
Bungaois
an
Pop o'u
lu L
ahi
Buto
be
Wik
ago
Banane Tig
rée
Bata
rd
Topala
Chek P
orng M
oan
Ihole
na Lel
e sub v
ar 'L
o ng Ped
uncular'
Cultivar
t-B
C e
qu
ivale
nts
in
pm
ol/
gd
w
Figue Pomme 2 (AAB)
Grande Naine (AAA)Cachaco (ABB)
Mbwazirume (AAA)
Batard (AAB)
Bantol Red (UNK)
Butobe (AAB)
Hi-resolution HPLC Screening
RP-HPLC Analysis 171 genotypes• >3 fruit/ genotype• standardised sampling
MEAN Vit A nutritional contents
Europe
Uganda
Cameroon
Ghana
Davey et al 2009, Food Chem
•New variety introduction•Breeding material
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Breeding in Musa
3n landrace,
2n landrace,
Superior (biofortified)
cv’s
2n (wild) x 3n landrace,
2n landrace,
2n (improved)
2n
(improved)x
4n (hybrid)x2n
(wild or improved)
3n (hybrid)
• Majority of bananas cv’s
are sterile, parthenocarpic,
triploid hybrids, ..
• From crosses with wild or
semi-wild (improved)
diploid parents
• few seeds, embryo rescue,
in vitro culture,
infrastructure,..
• lengthy
Breeding should focus on parental (2n) improvement
Exploitation of genetic diversity of wild-type germplasm
madav 1011 madav
Biofortification; vit C
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Vitamin C (L-ascorbic acid, ascorbate)
Synthesised by all higher plants: • all tissues except possibly dormant seeds• all cellular compartments cytoplasm, chloroplast, vacoule apoplast &
mitochondria• fruit tissue concentrations vary from 0.1 – 73 µmol/gfw
Synthesised by most animals except:• primates (humans),
• some species of bird, • guinea pig, bats,
• some fish
Yeasts and some fungi preferentially synthesise D-erythroascorbate a C5
analogue
AsA
Davey, MW No 2010
8
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Reviews - Smirnoff et al 2001, Annu Rev Plant Physiol Plant Mol Bio, 52, p231;
Davey et al 2000,J. Sci Food Agr 80 p825;
1. Animals:
D-Glc ���� UDP-Glucose ���� UDP-Glucuronate ���� D-Glucuronate ���� L-Gulonate ���� L-Gulonolactone ���� L-AsA
2b. Plants (Uronic acids):
D-Galactose ���� D-Galacturonate ���� L-Galactonate ���� L-Galactonolactone ���� L-AsA
D-Glucose ���� D-Glucuronate ���� L-Gulonate ���� L-Gulonolactone ���� L-AsA
Myo-inositol ���� D-Glucuronate ���� L-Gulonate ���� L-Gulonolactone ���� L-AsA
2a. Plants (L-galactose)
D-Glc ���� GDP-D-Mannose ���� GDP-L-Galactose ���� L-Galactose ���� L-Galactonolactone ���� L-AsA
What pathway?
Fruit biosynthesis?
AsA Biosynthesis
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OXIDATIVE-STRESS - RELATED DISEASE PREVENTION AND CURE
• Non-toxic up to 6g/d
• Deficiency can cause scurvy (rare)
• Cardiovascular disease - (50% reduction in mortality with 50 mg/d L-AA)
• Anticancer - effective against stomach, oesophageal, oral and
pharyngeal cancers. Also cervical, rectal, pancreatic, breast, ovarian,
colon cancers
• Cataracts - 45% decrease found with ascorbate supplements
• Immune system - 23% reduction of incidence of common cold
with megadoses of ascorbate, etc.
• Prevention of ROS-related pathogenic diseases e.g.
Parkinson’s, Alzheimers etc.
AsA & Human Health
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Vitamin C (AsA) and glutathione (GSH) are the most abundant plant antioxidants but also diverse cellular functions including;
•enzyme co-factors
•cell expansion and growth
•redox signalling and development•resistance to fruit storage disorders
•regeneration of oxidised vit E.•detoxification of xenobiotics
•fruit colour development
•enzymatic and non-enzymatic radical defence•stomatal opening
•systemic aquired disease resistance
L-AA content
25% WT
AsA & Plant Health
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Cyt b
L-AA
MDHAMDHA
L-AA
DHA DHA
MDHAR
oxalateCa - oxalate
DHAR
2GSH
GSSG
NADP
NADPH
GR
2CO2 + H2O2
O2
H2O
H2O2
H2O
H2O
monolignol.
monolignol
OxO
AO
APx
H+
ATP
ADP
CytoplasmWall
Smirnoff (1996) Annals of Botany 78: p661
O3
Cyt b
L-AA
MDHAMDHA
L-AA
DHA DHA
MDHAR
oxalateCa - oxalate
DHAR
2GSH
GSSG
NADP
NADPH
GR
2CO2 + H2O2
O2
H2O
H2O2
H2O
H2O
monolignol.
monolignol
OxO
AO
APx
H+
ATP
ADP
CytoplasmWall
Smirnoff (1996) Annals of Botany 78: p661
O3
AsA & cell Wall Metabolism
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Chloroplastic ROS production
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L-Ascorbate
COOH
OH
HO
O
OH
CH2OHGlycoaldehyde
CH2OH
CHO
CH2OH
COOH
OH
HO
+
L-Tartaric acid
D-xylo-5-Hexulo-sonic acid
OH
OH
OH O
CH2OH
O=C
COOH
COOH
CO2 + H2O2
Oxalic acid
COOH
COOH
OH
HOL-Threonic acid
C4/C5 cleavage(vitaceous plants)
grape
C2/C3 cleavage(geraniaceous plants)
L-Glyceric acid + CO2
( rev. Loewus 1999, Phytochem 53, p193)
AsA Substrate for Oxalate & Tartrate
Davey, MW No 2010
9
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O
O
OHHO
HO CH2OH
O
O
OO
HOCH2OH
O
O
OO
HOCH2OH
H
L-AA MDHA DHA
H,e- H,e-
2,3-DKG
CH2OH
OHCOOH
OO
HO
H2O
MDA
O
O
OO OH
HO
O
O
OHHO
HO CH2OH
O
O
OHHO
HO CH2OH
O
O
OO
HOCH2OH
O
O
OO
HOCH2OH
O
O
OO
HOCH2OH
H
O
O
OO
HOCH2OH
H
L-AA MDHA DHA
H,e- H,e-
2,3-DKG
CH2OH
OHCOOH
OO
HO
CH2OH
OHCOOH
OO
HO
H2O
MDA
O
O
OO OH
HOO
O
OO
O
O
OO OH
HOvit C mutants
- null mutants lethal- slow growth- early senescence- ozone sensitive
Conkin et al 1997, Plant Physiol
Keller et al 1999, Plant J
Chemistry of AsA
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In comparison to other fruits, concentrations of L-AA in fruit are relatively low
Adapted: Davey et al 2000, J. Sci Food Agr 80 p825;
Source mg/100g µmol/gfw
Acerola (west indian cherry) 1300 73.00
Apples 2-20 0.11 -1.13
Apricot 7-10 0.39 -0.56Blackberry 15 0.84
Banana 10-30 0.56-1.68aCauliflower 64-78 3.63-4.38
Cauliflower (cooked) 55 3.09Cherry 5-8 0.28-0.45
Blackcurrant 200-210 11.2-11.8
Redcurrant 40 2.25
Kiwi 60 3.41Orange 50 2.84
Pepper (green) 128 0.72Plum 3 0.17
Pear 3-4 0.17 -0.23
Spinach 51 2.86Strawberry 59 -60 3.37
Tomato 20 -25 1.14 -1.40
Variability in fruit AsA contents
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0
200
400
600
800
1000
1200
1400
1600
1800
Sunris
e
Gra
vens
tein
Retin
a
Prima
Del
bare
Estival
Jam
es G
rieve
Alk
men
eArle
tG
ala
Elsta
r
Liber
ty
Mer
lijn
Prisci
lla
HL248
Ango
ld
Gold
en
Kanzi
Cox
Del
bare
Jubile
Mic
rom
alus
Gol
drush
Florin
a
Idar
ed
Rome
Beauty
Glo
ster
Green
star
Jona
gold
Fuji x
NEG
Ontario
x P
riscill
a
Bra
eburn
Pink
Lady
Variety
me
an
L-A
A i
n n
mo
l/g
fw
Harvest
Shelf-life
3-months
tot AsA contents: harvest v 10days shelf-life v 3-months cold storage
Davey et al 2004, JSciFoodChem
Vit C in Apple & Impact of Storagemadav 1011 madav
Variation in apple vit C contents (BE)
L-AA DHA tot L-AA% DHA
nmol/gfw S.D. nmol/gfw S.D. nmol/gfw S.D.
harvest
Mean 644.1 ±242.3 97.6 ±143.5 741.67 ±274.44 10.7
Range 296.5 – 1283.1 0 – 265.7 400.9 – 1448.2 0 – 25.1
Fold
variation4.3 - 3.6 -
shelf-life
Mean 442.2 ±326.2 43.2 ±35.2 488.39 ±340.15 11.4
Range 83.7 – 1227.2 0 – 128.5 108.5 – 1314.4 0 – 34.5
Fold
variation15.3 - 12.1 -
cold-
storage
Mean 565.6 ±371.1 40.2 ±22.6 567.89 8.2
Range 134.2 - 1314.6 2.9 – 86.3 157.2 – 1589.1 0.3-12.0
Fold
variation11.3 29.8 10.1 40.0
Davey et al 2004, JAgrFoodChem
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Harvest date vs mean apple AsA content
• later-harvesting (slow-ripening) varieties tend to have higher AsA contents than early
varieties
.
y = 10.158x + 92.462
R 2 = 0.8247
y = 11.527x - 227.54
R 2 = 0.827
-200
0
200
400
600
800
1000
1200
0 20 40 60 80 100
Days after Aug 1
nm
ol to
t L
-AA
/gfw
Harvest
Shelf-Life
08-Aug. 25-Oct.
Davey et al 2004, JAgrFoodChem
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VitC (AsA) in apple and hard fruit
�Marker of fruit quality
� Stress tolerance in crops
�X-tolerance and induced resistance
� Link to post-harvest fruit quality
� Storage disorders
�Antibrowning agent
Davey, MW No 2010
10
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F1-Mapping Population • Telamon x Braeburn• 257 individuals, • M9 rootstock, own roots• commercial harvest date, • IPM
Segregating Populations
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Marker Type # markers on map
Telamon Braeburn Common
SSR’s 51 53 37
Polymorphic AFLP’s 242 245 32
Total # Markers 293 298 65
Length in cM 1099.9 1300.5
Mean length of LG/cM 64.7 76.5
Mean # markers/LG 17.2 17.5
Markers/cM 0.27 0.23
1 marker/x cM 3.75 4.36
Largest gap/cM 26.63 (LG9) 29.0 (LG3)
(Kenis & Keulemans 2005, Mol Breed, 15, p205-219)
Genetic Linkage Maps
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0
5
10
15
20
25
30
35
40
0-20
21-4
0
41-6
0
61-8
0
81-1
00
101-
120
121-
140
141-
160
161-
180
181-
200
201-
220
221-
240
241-
260
mean fruit weight/g
nu
mb
er
of
ind
ivid
uals 2005
2006
M 2005
M 2006
0
5
10
15
20
25
30
35
40
0-20
21-4
0
41-6
0
61-8
0
81-1
00
101-
120
121-
140
141-
160
161-
180
181-
200
201-
220
221-
240
241-
260
mean fruit weight/g
nu
mb
er
of
ind
ivid
uals 2005
2006
M 2005M 2005
M 2006M 2006
0
5
10
15
20
25
0-50
51-1
00
101-1
50
151-2
00
201-2
50
251-3
00
301-
350
351-
400
401-4
50
451-
500
501-
550
551-6
00
601-6
50
651-7
00
701-7
50
751-
800
801-
850
851-9
00
901-9
50
flesh mean L-AA content in nmol/gfw
nu
mber
of
indiv
iduals 2005
2006
M 2005
M 2006
0
5
10
15
20
25
0-50
51-1
00
101-1
50
151-2
00
201-2
50
251-3
00
301-
350
351-
400
401-4
50
451-
500
501-
550
551-6
00
601-6
50
651-7
00
701-7
50
751-
800
801-
850
851-9
00
901-9
50
flesh mean L-AA content in nmol/gfw
nu
mber
of
indiv
iduals 2005
2006
M 2005M 2005
M 2006M 2006
0
5
10
15
20
25
30
250-
400
551-
700
851-
1000
1151
-130
0
1451
-160
0
1751
-190
0
2051
-220
0
2351
-250
0
2651
-270
0
>285
0
peel mean L-AA content in nmol/gfw
nu
mb
er
of
ind
ivid
uals 2005
2006
M 2005
M 2006
0
5
10
15
20
25
30
250-
400
551-
700
851-
1000
1151
-130
0
1451
-160
0
1751
-190
0
2051
-220
0
2351
-250
0
2651
-270
0
>285
0
peel mean L-AA content in nmol/gfw
nu
mb
er
of
ind
ivid
uals 2005
2006
M 2005M 2005
M 2006M 2006
Davey et al 2006, Plant Physiol 142, p343
•HPLC Phenotyping
•Quantitative traits•Transgressive segregation
Molecular Maps & vit C QTL’smadav 1011 madav
TraitBraeburn 2006 Telamon 2006
QTL LG LOD cM %Variation Marker QTL LG LOD cM %Variation Marker
L-AA Flesh
1 11 3.70 32.1 22.3 EACMCAT122 1 17 3.22 15.9 13.8 EAGGMCCG73
2 17 2.85 11.2 8.9 Hi02f12 2 11 2.56 6.2 9.5 CH04g07
3 16 2.69 21.0 8.7 EAAGMCAC192 3 16 2.58 10.7 8.1 EACCMCGG94
39.9 31.4
DHA Flesh
1 11 3.33 19.4 28.6 EAGCMCAT122 1 16 3.19 15.2 31.1 EACAMCCA59
- - - - - - 2 11 2.93 10.0 13.9 ECACMCCT64
- - - - - - 3 15 2.66 5.1 12.0 CH02c09
28.6 57.0
Tot L-AA Flesh
1 11 4.65 31.1 27.1 EAGCMCAT122 1 11 4.78 18.2 15.2 Ch04G07
2 16 3.01 9.1 10.4 ECAAMCGA174 2 16 4.94 29.9 13.8 EAACMCGG94
3 17 2.47 5.3 7.3 Hi02f12 3 10 3.08 8.9 8.4 ECAAMCGA385
44.8 37.4
% DHA flesh
1 16 4.46 9.4 31.8 ECATMCAT283 1 17 4.09 10.8 20.4 ECAAMCG123
2 7 3.99 22.0 15.5 EAGAMCCG85 - - - - - -
3 14 2.87 9.7 11.4 EACGNCCG109 - - - - - -
58.7 20.4
VITAMIN C: QTLs
madav 1011 madav
Davey et al 2006, Plant Physiol 142, p343
Lo
gari
thm
of
Od
ds
Map Position LG 15
QTL flesh DHA
Map Position LG 10
Lo
gari
thm
of
Od
ds
QTL flesh total AA
Vit C QTL’s
madav 1011 madav
Vit C 2005
Vit C 2006
Browning
• QTL’s for DHA and % DHA
• Cortex & peel• Flesh browning
• On Telamon map• Stable over 2 years
Vit C QTL’s
Davey, MW No 2010
11
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Royal Gala x Braeburn population
• Genetic linkage map with 500+ SNP markers
Trait Phenotyping
• 120 trees phenotypedin 2008 (Clyde)
• 170 trees phenotypedin 2010 (Clyde)
• 6 fruit per tree harvested at maturity
• Flesh and skin samples analysed separately
• HPLC analysis of “total ascorbate” (AsA + DHA)
• QTL analysis: LOD threshold based on PT
QTL mapping for vit C (PFR)madav 1011 madav
Population distributions AsA, PFR
0.0
10.0
20.0
30.0
40.0
50.0
60.0
fre
qu
en
cy
mg/100gfw
Flesh2008
2010BR 2010
RG 2010
RG 2008
BR 2008
0.0
10.0
20.0
30.0
40.0
50.0
60.0
fre
qu
en
cy
mg/100gfw
Skin2008
2010
BR 2010
RG 2010
RG 2008
BR 2008
•Join Map 3.0
•MapQTL v4.0, rMQM analysis
Peel Flesh
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QTLs for vitamin C, PFR NZ.
� 5 QTL regions detected: LG’s 1, 6, 8, 13 & 17
� 2010: high % population variability explained (<48%, 3 QTLs)
� No QTLs detected for RG in 2008 (high AsA!)
� Common across tissues
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� 3 QTL regions in common between RG and Br
� LG-1, LG-6 & LG-8
� LG-6 stable across 2 seasons in BR in both tissues
2010 data
2008 data
Vit C QTLs Br x RG (NZ)
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QTLs in different mapping populations (NZ v BE)
2010 data
2008 data
� 2 QTL regions common across populations & environments
� LG-6 & LG-17
Davey et al 2006, PlantPhysiol
madav 1011 madav
QTLs different mapping populations (NZ & BE)
2010 data
2008 data
Davey, MW No 2010
12
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CGs & Regulation of vit C contents
• Biosynthesis (Smirnoff et al, 2001, Linster and Clark, 2008)
•L-gal, uronic acid, myo-inositol
• Recycling (Smirnoff et al, 2001)
•AsA-GSH pathway
• Degradation (Green and Fry, 2005)
•Biosynthetic precursor (DeBolt et al 2007)
• Transport (Horemans et al, 2000, Hancock et al 2008, )
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Alignment to Golden Delicious genome (NZ)
� GMP/vtc1 ☺☺☺☺ (biosynthesis)
� DHAR ☺☺☺☺ (recycling)
� GPX ☺☺☺☺ (recycling)
(cM) (cM)(Mbp)
Recycling
L-galactose
Uronic acids
CGs AsA Metabolism
D-Man-1-P D-Man-6-PGDP-D-Man
GDP-L-Gal
L-Ascorbate
L-Gal
L-Gal-1-P
GDP-L-Gulose
L-Gulose
L-Gulose-1-P
L-GuLL-GaL
UDP-GalUA
D-GalUA-1-P D-GalUA L-GalA
MeGalUA
GDP-L-Fuc
Polysaccharides/
N-Glycosylation
myo-Inositol
D-GlcUAL-GulA D-GlcUA-1-P
UDP-GlcUA
MDHA
oxalate
2GSH
GSSG
polysaccharides
D-Glc-6-P
NADPHthreonate
DHA
NADP
tartrate2,3 DKG
CO2 + H2O2
Biosynthesis
AO
DHAR
MIPS
VTC1
APx
GPx
2H2O
H2O2
madav 1011 madav
Next Generation Sequencing (GAII)
Sequencing By
Synthesis:
per cyclus 1 base inbouwen en
een image nemen
elke base heeft een andere fluorescente
kleur
76 cycli76 images76 basen / cluster
madav 1011 madav
Next Generation Sequencing (GAII)
De 76 bp sequentie van een cluster (cDNA) wordt afgelezen als de kleur (base) in opeenvolgende images (76 cycli) op dezelfde positie
Er worden 300 miljoen clusters geanalyseerd in 1 experiment
madav 1011 madav
reads (76 bp)
De novo assembly transcript sequences
Uit de overlap tussen korte reads wordt een doorlopende sequentie samengesteld (contig)
1-3 contigs per gen (allel) 70.000 contigs per genotype
samen het transcriptoom
overlap
contig (400-1000 bp)
Davey, MW No 2010
13
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Read mapping and polymorfisms
Korte reads onderling vergelijken (de novo) of tegen referentie genoom: SNPs en Indels
deletie SNPsequentie fout
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NGS-SNP Chip Strategy- Outputs
Advantages:
– In depth sequencing of 14 apple cv’s – confirms gene structure and
genome-wide identification of >100,000 SNPs
– Chip allows HTP-genotyping of individuals with SNPs distributed over
the genome
– Establish population structure and relatedness of individuals
– Relatively cheap and very fast!
– Suitable for LD association studies
“Disadvantages”
– Extensive bioinformatics required
– LD in apple unknown
– Transferability of SNP markers to ‘old’ accessions uncertain
– Linkage of other SNP markers to architectural gene set.
– Has to be done in collaboration with other partners.
madav 1011 madav
SUMMARY:
ANTIOXIDANT BREEDING
• Many potential benefits for entire production chain; yields, nutrition, quality
• Complex! new development, complex quantitative traits, requires knowledge base
• Approach depends on crop; transgenic v classical and molecular breeding approaches
• Introgression of alleles from unusual and wild-type germplasm
• Availability of molecular maps, markers and physical genome
maps important
• MAS allows the early screening for genotypes with the optimum combination of genes
madav 1011 madav
Thanks!
Molecular markers
Linkage Maps
Abiotic stress
Transgenics
