W. Friedt

GCIRC General GCIRC General AssemblyAssembly, , FebruaryFebruary 2009 2009 ManesarManesar nearnear Delhi, Delhi, IndiaIndia

Improving Improving rapeseed (rapeseed (BrassicaBrassica napusnapus) as a highly ) as a highly productive productive ooilseed and source of valuable products ilseed and source of valuable products for nutritionfor nutrition

Wolfgang Friedt & Rod SnowdonPlant Breeding Department, IFZ Research CenterUniversity of Giessen, Germany

W. Friedt

CONTENT:

Yield potential (Heterosis)

Stability (stress tolerance)

Oil yield and content

Fatty acid composition

Extraction meal quality

Genomics approaches

Outlook & perspectives

W. Friedt

48.0

49.0

50.0

51.0

52.0

53.0

54.0

55.0

56.0

57.0

3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80

Seed yield (t/ha)

V23-HV24-H

V25-H

MarcantZerucaMaruca

Maplus

Express

Falcon

PantherTalent

Artus

Pronto

ErucicErucic test test hybridshybrids

High erucic cultivars

Standard cultivars (00) Oil

cont

ent(

%)

Seed yield (t/ha)

Oilseed rape: Seed and oil yield

W. Friedt

Investigating heterosis in oilseed rape

Heterosis field trial, Grund Schwalheim, May 2006

Question: Common QTL for heterosis in different oilseed rape crosses?

Material: 2 populations of 250 DH lines and their test hybrids from a common male sterile parent

W. Friedt

Field trials for analysis of yield heterosis

Performance trials in 8 environments (2 years, 4 locations)

Measurement of plant height, plot yield and yield components

Calculation of mid-parent heterosis

Heterosis field trial, 2006

W. Friedt

Mid-parent heterosis for seed yield

0

10

20

30

40

50

60

70

80

22.5-

24.0

24.0-

25.5

25.5-

27.0

27.0-

28.5

28.5-

30.0

30.0-

31.5

31.5-

33.0

33.0-

34.5

34.5-

36.0

36.0-

37.5

37.5-

39.0

39.0-

40.5

40.5-

42.0

V8 Express F1

250 DH lines(Express x V8)

Mean seed yield (dt/ha) 2007

Freq

uenc

y

250 test hybrids(DH lines x MSL-Express)

8 environments (4 locations,

2 years)Giessen,

Göttingen, Breeders

W. Friedt

Co-localising epistatic QTL (test hybrids)Na12H06CB10413E34M49_141E31M49_351E45M48_118E32M49_212E44M50_56E34M49_80E37M50_86E44M57_448E31M61_305BRAS056E43M51_108E35M48_148E34M49_444HMR443Na14G10Ol11G11E42M55_125Ol11B05BRMs043Na12E02CB10036E33M49_207Ra2A06E33M47_141E44M62_55E34M49_61CB10079E42M59_103E31M55_158E43M51_254

0

10

20

30

40

50

60

70

80

90

100

110

120

130

N3CB10545E43M52_253E32M51_329E32M51_325E33M50_48E44M49_261E32M49_149E34M59_94BRAS063HMR342Na10E02E43M59_132E36M47_151E36M51_300MD21CB10574E43M52_320E34M55_189CB10609E34M59_153E46M62_78GMR014Na12E01bE31M55_105E31M62_195E33M49_104E32M59_162E42M55_166E42M60_190

0

10

20

30

40

50

60

70

80

N5E33M47_216E46M59_160HMR588aE45M57_110BRAS039aCB10364HMR388CB10578HMR307E45M59_514HMR353aE45M51_344E38M55_386E31M53_515HMR582E45M51_302E44M48_414E32M51_301CB10629E39M59_106E39M61_243E43M62_222E39M47_181Na12B05E32M59_123E44M49_170Ra1F06E42M59_122

0

10

20

30

40

50

60

70

N8E44M60_472E32M48_677HMR572E33M47_73E44M60_432E33M60_378Na12D08E45M49_137E33M58_56E34M55_290E43M62_272E33M50_327E31M53_161HMR558HMR201HMR389E32M62_70E43M59_81Na12H07bE34M62_116E34M62_304E34M62_305

0

10

20

30

40

50

60

70

80

90

N6E39M49_307

E44M58_101HMR166MR153bRa2G08Na12B02Na12E11Ni2D03

0

10

20

30

40

N7

E35M48_183

E44M60_67E33M50_312MR26E36M47_696E36M47_689

Ol10F08E33M49_199E38M49_117Na12E01aMR54E44M51_331E34M54_45E32M51_225E45M48_151E31M55_372E31M49_112E32M51_350CB10632E43M62_168E39M41_200E45M48_404E32M59_273E32M59_285CB10211bE34M62_490BRAS048

0

10

20

30

40

50

60

70

80

90

100

110

120

130

N16

E35M60_324E37M59_135E37M59_140CB10587E33M50_415E33M50_414Ol13C10bE31M55_102E32M54_52E43M59_241E33M50_298E36M51_477HMR292CB10206E31M60_314E32M54_81E32M62_248E44M58_436CB10536aE44M50_316E44M48_97

0

10

20

30

40

50

60

70

80

N11

E45M51_80CB10266E35M62_232MR111cE32M49_405E45M49_173E33M47_256E44M62_104E34M51_252E32M62_386GMR013bE39M61_62E43M62_82E33M47_135E39M49_102E34M62_369E43M51_385E42M51_357HMR281Ol12F07Na12G04E44M48_121E32M49_578E32M49_386E45M54_500E36M61_358E32M50_374

0

10

20

30

40

50

60

70

80

90

100

110

120

N19

MR86bOl10E05MR111aE33M62_153E33M49_268HMR320E32M62_252E44M51_636E32M59_85E44M47_290E45M57_436E46M59_202E33M60_156E45M59_151E34M49_501E35M62_226MR153aOl10B07E46M62_122E39M47_352E43M59_291E35M56_176Na10G10aE44M47_237E38M55_460E31M61_195E42M53_334BRAS068E44M47_322MR49E46M62_120E45M51_52E44M47_239HMR322Na10C01aBRAS039bE36M51_45E43M62_147E31M62_310BRMs033Na12B07bE32M48_304E31M62_410

0

10

20

30

40

50

60

70

80

90

100

110

N13

Seed yield

Plant height

Seed weight

QTL forheterosis

Seed yield

Plant height

Seed weight

Additive QTL (DH lines)

Shoot weight

Cotyledon length

Leaf weight

Seed yield

Plant height

Seed weight

Epistatic QTL (test hybrids)

Leaf area (28d)

W. Friedt

CONTENT:

Yield potential (Heterosis)

Stability (stress tolerance)

Oil content and quality

Extraction meal quality

Genomics approaches

Outlook & perspectives

W. Friedt

ResynthesizedResynthesized rapeseedrapeseed as as donordonor of of resistanceresistance

Example:

B. rapa chinensis 56515 (Pak Choi, China)xB. oleracea acephala 8207 (Wild kale, Syria)

W. Friedt

Resynthesized (RS) Rapeseed as a Resourcefor Resistance Breeding

Example 1: Clubroot Resistance

- Race specific resistance transferred from B. oleraceainto RS Rapeseed (FU Berlin), by backcrossing

- Simple inheritance (1-2 resistance genes)?

- But, new virulent pathotypes may break resistance

Polygenic types of resistance required

Resistant cultivars for infested locations: Mendel (NPZ)and Tosca (SW Seed)

W. Friedt

- “Complete resistance” against Leptosphaeria maculans/Phomalingam of B. rapa was transferred to RS Rapeseed; cultivars developed by repeated backcrossing and resistance screening

- Monogenic, dominant inheritance- “Breakage” of resistance by virulent pathotypes observed in

Australia

Resistant Spring rape cv. ‘Surpass 400’

Example 2: Phoma Resistance

Resynthesized (RS) Rapeseed as a Resourcefor Resistance Breeding

W. Friedt

Resynthesized (RS) Rapeseed as a Resourcefor Resistance Breeding

Example 3: Novel Verticillium Resistance

Goal: Development of durable Verticillium resistance by combining different sources of resistance; marker-assisted pyramiding of resistance genes

W. Friedt

Screening of mapping parents

0.00

0.50

1.00

1.50

2.00

2.50S

228.

8.1

FS B

1/3.

3S

108.

1.1

K 19

9.16

.2

Sollu

x

R 53

FS 9

4.3

Oas

eSa

mou

rai 1

1.4

Lase

rS

45.2

.2

Gao

you

Expr

ess

YE1

schw

arz

DH-2

6-96

Man

shol

ts 5

.1

Falcon

Expr

ess

Falcon

Expr

ess

617

Lion

YE2

gelb

101

2-98

YE1

gelb

T-2

5629 V8

AU

DP

Cco

rr

Selected RS lines (B.oleracea x B.rapa)B. napus Check cultivars

B. napus Mapping parents

Segregation for Verticillium resistance in Express 617 x R53

‘R53’ (U Göttingen): B. oleraceaacephela (Winter cabbage) x B. rapa pekinensis (Chinese cabbage

Express 617

W. FriedtExpress x R53: 275 DHs, 191 loci (Map from Radoev et al. 2008)

QTL Analysis of Verticillium Reaction

AUDPC

*E32M48b0

*Na10G08b20

*Ol10B0231

*CB1061148

*E32M49u59*CB1006566

*CB1043576

*CB1060987

*MR95112*E32M51k117*E32M51o124*E32M49k129

N15

LOD=7.61Part. R2=29.6

Major QTL explainsapprox. 30 % of resistance phenotype

- DH population Express 617 x R53 (100 DH lines)

W. Friedt

307-406-1 x 307-230-2 Express 617 x R53

BRMS030

Ra2F11_b

AUDPC

AUDPC

N15

Comparative SSR marker analysis

LOD=10.06Part. R2=12.5

LOD=7.61Part. R2=29.6

Major Major resistanceresistance genegene fromfrom B. B. oleraceaoleracea

W. Friedt

Fine mapping of major QTL and marker developmentin Express 617 x R53 via Bulked-Segregant Analysis

FurtherFurther workwork: Gene : Gene isolationisolation

256 AFLP primer combinations tested for 2 parents and 2 bulks of 10 DHs each

W. FriedtRygulla et al. (2008) Phytopathology 98: 215-221

- DH population 307-406-1 x 307-230-2 (SW Seed)- Major QTL on N14 and N15 identified in four different experiments

Isolate mix MeanIsolate mix Exp. 3

Single spore isolate

Isolate mix Exp. 1Isolate mix Exp. 2

Pyramiding resistance loci of different regions

W. Friedt

CONTENT:

Yield potential (Heterosis)

Stability (stress tolerance)

Oil content and quality

Extraction meal quality

Genomics approaches

Outlook & perspectives

W. Friedt

VegetableVegetable oiloil: : TypicalTypical oiloil qualitiesqualities ((fattyfatty acidacidpatternpattern))

Omega-3 Omega-6Oleic acid 18:1

HOLLi Rapeseed~70% 18:1, ~3% 18:3

W. Friedt

FOODFOOD NON FOODNON FOOD

Human Human nutritionnutrition:: TechnicalTechnical oilsoils & & fatsfats::-- SaladSalad oiloil -- OleochemicalsOleochemicals-- Margarine Margarine -- HydraulicHydraulic oilsoils-- FryingFrying oiloil -- LubricatingLubricating oilsoils-- DietaryDietary nutritionnutrition -- CosmeticsCosmetics, , paintspaints, , plasticsplastics, etc., etc.

NutritionalNutritional and Nonand Non--food food UsagesUsages of of RapeseedRapeseed OilOil

W. Friedt

Fatty acids- Very long-chain, poly-unsaturated (LCPUFA)

Antioxidants- Tocopherols (content, pattern)- Resveratrol

Polar Lipids- Novel lecithine

Protein and meal quality- Glucosinolates, phenolics (sinapins, tannins)

=> Crushing & processing technology

NovelNovel FeedstuffsFeedstuffs fromfrom RapeseedRapeseed

NAPUS 2000

W. Friedt

Genetic modification of the tocopherolbiosynthetic pathway

Methyltransferase II

2-Methyl-6- phytylchinone

γ - Tocopherol

α - Tocopherol

2,3-Dimethyl-6- phytylchinone

Methyltransferase I

Cyclase

Shikimate Isopentenyl PP

HomogentisateDioxygenase

Phytyl PP

4- Hydroxyphenylpyruvate Geranylgeranyl PP

Reductase

Prenyltransferase

* *

*

* genes have been identified

cyclase

Arabidopsis thaliana

Zea mays

cyclase

HPPD2

Eucaryoticorganism

prenyl

Synechocystis sp.

HPPD1

Arabidopsis thaliana

W. Friedt

Cuphea lanceolata

http://www.mpiz-koeln.mpg.de

Brassica napus

LB p35S t35S pNap tNap pNap tNap RB

npt II clKAS IIIb mut clFATB3

npt II clKAS IIIb mut chFATB2

LB p35S t35S pNap tNap pNap tNap RB

npt II clKAS IIIb mut chFATB2

LB p35S t35S pNap tNap pNap tNap RB

LB p35S t35S pNap tNap RB

npt II clKAS IIIb wt

LB p35S t35S pNap tNap RB

npt II clKAS IIIb mut

T0-Seedling

T1-Plant

T2-Seeds

T2-Plant

T3-Seeds

T3-Plant

T4-Seeds

DevelopmentDevelopment of of transgenictransgenic rapeseedrapeseed plantsplantsand and propagationpropagation of of selectedselected progenyprogeny

Transfer of Transfer of selectedselected genes genes involvedinvolved in in seedseedlipidlipid and and tocopheroltocopherol biosynthesisbiosynthesis to to B. B. napusnapus

((NAPUSNAPUS 20002000 Project)Project)

W. Friedt

name R1 R2 R3 Rel. biolog. activity (%)

α-Tocopherol -CH3 -CH3 -CH3 100

γ-Tocopherol -H -CH3 -CH3 10

δ-Tocopherol -H -H -CH3 3

β-Tocopherol -CH3 -H -CH3 50

TocopherolTocopherol -- StructureStructure

R 3

R 2

OHR 1

CH3

CH3

O

CH3 CH3CH32 4 8

1

345

6

8

7

NAPUS 2000

W. Friedt

HPT= Nap_HPTTC= Nap_TCDouble= DC3_HPPD2,

Nap_HPTTriple= DC3_HPPD2,

Nap_HPT,Nap_TC

N = Number of T2 plants

* P <0,001 (Tamhane)

43344635344235374148401736109N =

1336.557

1336.735

1336.702

1336.699

1336.705

1335.696

1332.515

1332.451

1332.445

1334.691

1334.684

1333.677

1320.642

Kontrolle

1600

1400

1200

1000

800

600

400

200

0

402

Toco

pher

ol(m

g/kg

Öl)

T1-Nachkommenschaft

* * * * * * * * * * * * *100 = 770 ppm

HPT ---TC---- --Double- -------- Triple --------

177189

Variation of Variation of TocopherolTocopherol content in genetically content in genetically engineered engineered seedoilseedoil (pooled T3(pooled T3--seeds)seeds)

Raclaru M. et al. (2006) Molecular Breeding 18:93-107

W. Friedt

CONTENT:

Yield potential (Heterosis)

Stability (stress tolerance)

Oil content and quality

Extraction meal quality

Genomics approaches

Outlook & perspectives

W. Friedt

Influence of Influence of phenolicphenolic compounds compounds of rapeseed mealof rapeseed meal

These compounds contribute to

Dark colourBitter tasteAstringent flavourNon-specific reactions with proteins, enzymes or essential forms that create nutritionally-unavailable products

W. Friedt

GABI-Project: Functional Genomics approaches for the development of yellow-seeded, low sinapine(„YelLowSin“) oilseed rape/canola (B. napus)

Cooperation: German Breeders & Canadian Partners

- Reduced seed coat- Low fibre (lignin) content- Increased oil & protein

Rapeseed coat formation: Feed value

W. Friedt

Field trials: 5 locations 2003 - 2007 (458 DH-lines)

Genetic map: 166 DH-lines used for mapping 258 AFLP and SSR markers covering a total of 1,721 cM

‘1012-98’Canola quality

X‘Express 617’Canola quality

DH-population YE2: total 458 DH-lines

W. Friedt

Fibre genes in N9 QTL?Fibre genes in N9 QTL?Fibre genes in N9 QTL?

Major QTL for seed colourand seed fibre content

E32M62_1290.00

cb010373xx25.43

cb010476xx33.35E33M61_4637.13

E33M50_38343.46Ni4D0949.07SSR1_14452.46E32M50_41953.55E32M59_10557.81E33M59_11160.64E33M62_7362.48E33M61_15065.50E32M48_18369.15E33M62_17972.95E33M61_10475.98E33M50_8080.14

N9

CCR1-like gene, 65 kbp fromortholog of QTL-linked SSR

Cellulose synthase CES A9(embryo-expressed)

Mannitol dehydrogenase-likegenes, similar to CAD2/CAD3

(two homologous copies)

9.127

9.2879.292

9.338

10.187

10.251

E32M50_419

At2g21730At2g21770

At2g21890

At2g23910

Ni4D09

At2

Mbp CCR = Cinnamoyl CoA Reductase

W. Friedt

CONTENT:

Yield potential (Heterosis)

Stability (stress tolerance)

Oil content and quality

Extraction meal quality

Genomics approaches

Outlook & perspectives

W. Friedt

NDF: LOD 8.4 part. R² 44.3ADF: LOD 4.4 part. R² 47.8

Colour: LOD 6.1 part. R² 24.2Protein: LOD 3.1 part. R² 4.3

E32M48_1830

E33M61_1505E33M62_739E33M59_11111E32M59_10514

E32M50_41919SSR1_14420Ni4D0923

E33M50_38329BnCESA9-SSR31At2g22000-SCAR34At2g21660-SCAR38cb010476xx41ENA2145cb010373xx49

BnAHA10-12SSR55

N9N9

Colour: LOD 7.0 part. R² 21.1

ADL: LOD 3.8 part. R² 45.2

Ol12A040E32M61_1962E33M47_1786E33M47_18010E38M59_15914E33M59_28216E33M62_25118sn007410xx21E33M48_5824Ol10B1129E33M59_28133

N10N10

NDF: LOD 4.8 part. R² 11.0Oil: LOD 5.8 part. R² 11.3Cellulose: LOD 6.7 part. R² 14.7

E32M61_1990

E38M61_44414E35M60_15919Ol10D02_B20

E35M62_29326

Gi12b32E32M62_9936E33M59_13738E38M61_10840E33M49_14843Na10A08_B46Na10D1151Ra2A04_A53BnSGT-455E33M50_17656Ra2F1163E33M47_14666E33M47_14768E33M50_11469E32M62_26070E35M60_15373

E33M59_25881

Ol10D02_A86E32M48_30889

N15N15

Oil: LOD 11.0 part. R² 24.0Colour: LOD 3.6 part. R² 9.6ADL: LOD 9.6 part. R² 15.3Cellulose: LOD 10.7 part. R² 21.3

E32M47_700E33M47_1517E33M50_1519E35M62_15110E32M62_19712E33M50_30115E33M50_29916E32M47_28619E32M48_24520Ol12F1121mr000228xx23BnSGT-224BnTT1-SSR26E32M47_40827E33M50_15829E32M47_34630E33M48_57032E32M49_23033E35M62_59735E33M47_5337E35M62_26439E32M62_24941E38M62_15045E33M49_12547E40M60_7749E35M60_12653E32M59_10256

N11N11

NDF: LOD 3.8 part. R² 8.3Cellulose: LOD 6.7 part. R² 15.4Oil: LOD 5.4 part. R² 11.3

Gi12a0

E33M47_18624

E33M62_16131E32M47_57638E33M59_65439E33M62_25645E40M60_7248E38M59_10253E33M48_18655E32M49_57857Na12G0459E32M62_24761E35M62_16963E32M47_27965pk48x2467snrc0010xx72

E33M47_13382

E33M47_15688E32M61_42289

N19N19

Cellulose: LOD 4.8 part. R² 12.7

NDF: LOD 6.7 part. R² 5.8ADF: LOD 3.1 part. R² 3.6Oil: LOD 3.5 part. R² 8.7

Major seed quality QTLMajor seed quality QTLMajor seed quality QTL

Seed colourOil contentProtein contentCellulose

NDF

ADFADL

QTL for seed traits

Verticillium QTL!

W. Friedt

- Use of Arabidopsis-Brassica comparative genomics tools to identify At-regions with synteny to major seed colour QTL

- Isolation, sequencing, marker development and mapping of B. napus orthologs for potential syntenic genes

- Marker saturation in QTL regions of rapeseed

Gene identification: Gene identification: Positional and functional characterization of new seed colour (transparent testa) candidates by At-Bn synteny

Open question: Negative effect of alien germplasm on seed and oil yield in oilseed rape?

W. Friedt

Yellow Rapeseed: OutlookYellow Rapeseed: Outlook

- RT-PCR of CCR-like, CAD-like, CESA9 and TT10 candidates during seed development in black and yellow RILs

- Field trials: New segregating materials for genetic/ marker analysis

- GC-MS to identify major phenolic compound(s) contributing to differences in YE2-DH population

W. Friedt

Finding regulatory genes involved in heterosis:

Many QTL hotspots control heterosis for multiple traits

Epistatic interactions appear to play a role in regulation of QTL

eQTL analysis to identify regulatory candidate genes (e.g. transcription factors) with a key role in heterotic gene expression

Express, V8F1 (Express x V8)

mRNA from seedlings(controlled conditions,

14 & 28 days after sowing)

Selected DH lines withhigh and low heterosis for

cotyledon length and rapeseed yield

Ultradeepexpression

next-gen

EST-Tag profiling

(Illumina/Solexasequencing)

eQTL analysisterosis-relevant

gene expressionof he

W. Friedt

Crop Harvested area Average yield (t/ha) Rate

ha (x106) % arable 1961-1970 1991-2000 dt/year

Cereals 37.8 51 2.6 5.27 0.88

Wheat 18 24 2.4 5.54 1.02

Maize 4.2 6 3.19 8.32 1.69

Rapeseed 3.0 4 1.92 2.88 0.34

Sunflower 1.9 3 1.17 1.54 0.18

Ewert F. et al. (2005) Agriculture, Ecosystems & Environment 107:101-116

Land Land useuse and and selectedselected yieldyield statisticsstatistics forfor majormajorEuropean European graingrain cropscrops

W. FriedtPhoto: Norddeutsche Pflanzenzucht Hans-Georg Lembke KG

AcknowledgementsAcknowledgementsCanadian PartnersCanadian PartnersAAFC & PBI, SaskatoonAAFC & PBI, SaskatoonU of Alberta, EdmontonU of Alberta, EdmontonGenome CanadaGenome Canada

IPB Leibniz Inst, HalleIPB Leibniz Inst, Halle

University of GiessenUniversity of GiessenBenjamin Benjamin WittkopWittkopWilfriedWilfried LLüühshsPanjisaktiPanjisakti BasunandaBasunandaMalteMalte LuhLuh, Stavros , Stavros TzigosTzigosMechthildMechthild SchwarteSchwarte, , a.oa.o..

University of University of GGööttingenttingenHeikoHeiko BeckerBeckerMladenMladen RadoevRadoevWolfgang Wolfgang EckeEcke

Breeding CompaniesBreeding CompaniesDSV AG, LippstadtDSV AG, LippstadtKWS KWS SaatSaat AG, AG, EinbeckEinbeckNPZ NPZ LembkeLembke KG, KG, HohenliethHohenliethSW Seed GmbH, SW Seed GmbH, HadmerslebenHadmersleben

Funding:DFGGFPBMBF (GABI)BMELV