Novel characterization techniques the Phenomics and Genomics approach

Ben Vosman

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

The pest insects

Cabbage whitefly (Aleyrodes proletella)

Cabbage aphid (Brevicoryne brassicae)

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

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

Five Tasks

1. Germplasm screen 2. Metabolomics 3. Sequencing the transcriptome 4. Transcriptomics 5. Identifying candidate genes and markers

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

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

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

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

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

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)

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

EPG analysis

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

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

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

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

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

Transcriptomics Experiment

● Cultivar Rivera

● Four time points (6-8-10-12 weeks) ● Treatment: Induced (whiteflies) and non

induced ● 3 Biological replicates

PCA according to time category

Data analysis ongoing

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

Characterization of resistance in white cabbage

Resistant Susceptible

White cabbage (Brassica oleracea var. capitata)

Rivera F1 hybrid

Christmas Drumhead Open pollinated variety

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

Oviposition behaviour

Rivera Christmas Drumhead

Broekgaarden et al. 2012 EEA 142:153–164

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

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

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

Mapping

Data set

● 171 F2 plants ● 140 markers

QTLs for

● Whitefly oviposition ● Wax layer ● Leaf thoughness

Confirmation in F3 ongoing

Chr. 1

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

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