novel characterization techniques the phenomics and
TRANSCRIPT
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Novel characterization techniques the Phenomics and Genomics approach
Ben Vosman
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The Problem:
Direct damage by ● Phloem feeding ● Secretion of honeydew ● Sooty mold growth
Reduced marketability of crop
Problems increase
● Ban on insecticides ● Climate change
Host plant resistance
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The pest insects
Cabbage whitefly (Aleyrodes proletella)
Cabbage aphid (Brevicoryne brassicae)
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WP1: Phenomics and Genomics
This WP will demonstrate how novel phenomics, genomics and transcriptomics tools can be used to speed up plant breeding Brassica crops
Resistance towards insects
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Aims of WP1
Finding host plant resistance to the cabbage whitefly and cabbage aphid via a germplasm screen ● Crop wild relatives ● Landraces
Elucidating resistance mechanism Providing tools for breeding whitefly and aphid resistant
varieties ● Characterization of resistant and susceptible plant
material ● Markers for introgression of QTLs
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Five Tasks
1. Germplasm screen 2. Metabolomics 3. Sequencing the transcriptome 4. Transcriptomics 5. Identifying candidate genes and markers
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Task 1: Germplasm screen
Field evaluations for resistance towards Cabbage aphid (UoB) and whitefly (DLO) Greenhouse confirmation of the whitefly resistance. EPG analysis was carried out to get insight into the
feeding behavior of aphids on resistant and susceptible accessions Material for crossings selected
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Germplasm screen
Field experiment (432 accessions), Landraces, crop wild relatives ● Non choice test, clip cages with 5 adult females,
counting ● Survival after 7 days ● Number of eggs produced
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432 accessions
249 accessions
104 accessions
183
48 accessions
Cycle 1 1 clip cage
145
R
S
Cycle 2 1 clip cage
Cycle 3 4 clip cages
Putative resistant
S
R R
Schematic overview of the selection
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Whitefly resistant accessions per species
Species Total number Putative resistant accessions
B. bourgeaui 2 - B. cretica 10 - B. fruticulosa 15 - B. incana 10 2 B. insularis 1 - B. macrocarpa 2 - B. maurorum 2 - B. montana 7 2 B. oleracea landraces 325 31
wild 44 5 B. rapa 3 - B. rupestris 3 2 B. spinescens 1 - B. villosa 7 6
432 48
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Result germplasm screen
No resistance found in Brussels sprouts and kale Strong resistance in some landraces and varieties of
white cabbage (B. oleracea var. capitata) Strong resistance in some Crop Wild Relatives
● B. villosa ● B. incana
B.villosa
B.oleracea acephala
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Tjallingii, 1988
Data Analysis
Data acquisition, display and recording of EPG signals
Electrodes
Faraday’s Cage
EPG probe
Gold wire
Amplifier (8-channels)
Electrical penetration graph (EPG)
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1 h
np np np
path path path path path
probe probe probe
xylem phloem phloem
E1
E1 E2
G E1 E2
x y l e m p h l o e m (sieve element)
5 sec 5 sec 5 sec
DETAILS ACTIVITIES
Sieve element salivation (E1)
Water ingestion (G)
G
Xylem Phloem
Phloem sap Ingestion (E2)
EPG waveforms and activities
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EPG analysis
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Task 2: Metabolomics
Metabolites are believed to play an important role in resistance towards insects ● Glucosinolates ● Terpenoids
We carried out metabolomics analysis on resistant and susceptible accessions using LCMS and GCMS Unfortunately, analysis did not result in the identification
of a metabolite that may explain resistance Resistance factor may be a protein or
a morphological adaptation
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Task 3: Sequencing the transcriptome
Information on which genes are expressed in the different Brassica species and the difference between them (SNP markers) All material (15 accessions) that was selected for
sequencing has been sequenced ● Includes one representative of each (sub) species ● Parents of the mapping populations
Data analysis has just started. First aim is the development of an Axiom array, based on
SNPs that will be identified. Array to be produced early 2014
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Task 4: Transcriptomics
Assess the differences in gene expression in Brassica in response to aphids and whiteflies Three different experiments carried out Arabidopsis array used Will deliver information on gene networks and candidate
genes for resistance
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Effect of plant age on whitefly performance
2 plant ages ● 6-wk old ● 12-wk old
No-choice test
● Females in clip cages ● Adult surv. after 1
wk
● Nymph surv. after 3 wks
Greenhouse experiment
12-wk-old 6-wk-old
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Whitefly performance depends on plant age
0
10
20
30
40
50
60
70
80
6-wk old 12-wkold
Nym
ph s
urvi
val (
%)
Rivera
ChristmasDrumhead
0102030405060708090
100
6-wk old 12-wkold
Adul
t sur
viva
l (%
) ns
ns
***
*
Broekgaarden et al. 2012 EEA 142:153–164
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Transcriptomics Experiment
● Cultivar Rivera
● Four time points (6-8-10-12 weeks) ● Treatment: Induced (whiteflies) and non
induced ● 3 Biological replicates
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PCA according to time category
Data analysis ongoing
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Task 5: Candidate genes and markers Combining the information from the Tasks 1–4 will give a
first indication of the candidate genes for insect resistance. For validation, seggregating populations are used:
For cabbage whitefly resistance: ● Christmas Drumhead (s) x Rivera (r) ● B. villosa (r) x B. oleracea (s) ● B. incana (r) x B. oleracea (s) For cabbage aphid resistance: ● B. fruticulosa (r) x B. fruticulosa (s) B.villosa
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Characterization of resistance in white cabbage
Resistant Susceptible
White cabbage (Brassica oleracea var. capitata)
Rivera F1 hybrid
Christmas Drumhead Open pollinated variety
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Results Non choice test
Rivera Christmas Drumhead
0102030405060708090
100
2008 2009
**
***
Adult survival (%)
**
0102030405060708090
100
2008 2009
Nymph survival (%)
**
Year 1 Year2 Year 1 Year 2
Broekgaarden et al. 2012 EEA 142:153–164
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Oviposition behaviour
Rivera Christmas Drumhead
Broekgaarden et al. 2012 EEA 142:153–164
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Results EPG analysis
0
50
100
150
200
250
300
Tota
l tim
e (m
in)
RiveraChristmas Drumhead
*
ns
ns
ns
Broekgaarden et al. 2012 EEA 142:153–164
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Screening F2 population for resistance to cabbage whitefly
Cross: Rivera (R) x Christmas Drumhead (S) ● 180 F2 plants
Phenotyping
● Cabbage whitefly (clip cages) ● Developmental stage (head formation) ● Leaf; wax layer and toughness
● Leaf material for DNA isolation
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SNP genotyping (KASPer markers)
150 unique SNPs were selected ● 15 per chromosome,
n=9 ● reference genome,
B. rapa Rivera G:G Christmas Drumhead A:A Heterozygous G:A
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Mapping
Data set
● 171 F2 plants ● 140 markers
QTLs for
● Whitefly oviposition ● Wax layer ● Leaf thoughness
Confirmation in F3 ongoing
Chr. 1
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Conclusions
Resistance to cabbage whitefly and cabbage aphid is present in Brassica spp. Whitefly resistance is found in several landrace
accessions of B. oleracea var. capitata as well as in several CWR species The whitefly resistance identified in accessions of the
CWR B. villosa, B. incana and B. montana is expressed both in six and twelve week old plants, which makes them novel sources of resistance for breeding Cabbage aphid resistance from B.fruticulosa looks very
promising
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Acknowledgements
WUR Plant Breeding Koen Pelgrom Roeland Voorrips Colette Broekgaarden Greet Steenhuis-Broers Johan Bucher
ServiceXS Bart Janssen Wilbert van Workum
University of Birmingham Garima Sharma Brian Ford-Lloyd Jeremy Pritchard University of Nottingham Sean May Stanley Adobor Marcos Castellanos-Uribe