ACW Changins, Switzerland28 April - 1 May 2013p y

Advances in BiotecAdvances in Biotec‐‐ and Markerand Marker‐‐Assisted Assisted Breeding of Oilseed Rape (Breeding of Oilseed Rape (BrassicaBrassica napusnapus))Breeding of Oilseed Rape (Breeding of Oilseed Rape (BrassicaBrassica napusnapus))

Wolfgang FriedtDepartment of Plant Breeding, Justus‐Liebig‐University Giessen

HybridHybrid breedingbreeding schemescheme forfor rapeseedrapeseedSterile female

Hybrid Hybrid breedingbreeding schemescheme forfor rapeseedrapeseedSterile female+ Maintainer

Ms pool

Restorer lineRf-Pool

Test crossesInbred lines Inbred lines

GCASCA

Experimental

Ms line Rf lineSCA

hybrids

Source: NPZ Lembke

Hybrid cultivar

CreatingCreating diversitydiversity: Natural : Natural allopolyploidsallopolyploids bybyh b idi ih b idi i dd i ii i ii B iB ihybridizationhybridization andand speciationspeciation in in BrassicaceaeBrassicaceae

Ethiopean m stard Indian m stardEthiopean mustard Indian mustard

Oilseed rape

Doubled haploids of novel trigenomic BrassicaDoubled haploids of novel trigenomic Brassicaderived from various interspecific crosses

X. X. Geng et al. (2013) Plant Cell Tiss Organ Culture

unique interspecific crosses:

• Brassica rapa (genome AA) x B. carinata (BBCC)

• B nigra (BB) x B napus (AACC)B. nigra (BB) x B. napus (AACC), 

• complex cross between B. juncea (AABB), B. napus and B. i t l ti l t bl h b (2 54)carinata, relatively stable chromosome number (2n=54)

PloidyPloidy level in microsporelevel in microspore‐‐derived plants derived plants by flow by flow cytometrycytometry based on DNA content of controls with based on DNA content of controls with known DNA content. known DNA content. Plants were classified as Plants were classified as hexaploidhexaploid (6x), (6x), triploid (3x) mixed, or unknown, and doubling frequency (%) is the triploid (3x) mixed, or unknown, and doubling frequency (%) is the proportion of proportion of hexaploidshexaploids in the totalin the total (Geng X.X. et al. 2013)(Geng X.X. et al. 2013)

Hybrid code

Hexa‐ploid

Triploid Mixed Un‐known

Total  Doubling frequencycode ploid known  frequency 

(%)

H08 1 23 16 4 11 54 42 6H08‐1 23 16 4 11 54 42.6H11‐2 370 298 6 19 693 53.4H16‐1 241 185 3 7 436 55.3H24‐1 21 19 2 14 56 37.5

BroadeningBroadening geneticgenetic variationvariation via  via  „„ResynthesisResynthesis“ “ ofof rapeseedrapeseed

XXhttp://international.

t kf d

https://www.google.de/search?q=cabbage

B. oleracea2n=2x=18

B. rapa2n=2x=20

stockfood.com

BrassicaBrassica napusnapus2n=4x=382n=4x=38

WR

Extending Extending genetic genetic diversitydiversityRS diversity diversity in in BrassicaBrassica

RS

napusnapusSRSR

Principle coordinate analysis of 142 resynthesized lines and 57 B. napus L. genotypes of European and Asian origin. Symbols are for winter (circle), for spring‐type (square), 

for Asian B. napus (triangle) and for resynthesized lines (filled diamond); Girke et al., GRACE (2012)

New New markermarker typestypes forfor highhigh‐‐throughputthroughput MASMASRFLP Restriction fragment length polymorphismRAPD Random amplification of polymorphic DNASSR Simple sequence repeats (microsatellite markers)AFLP Amplified fragment length polymorphismDArT Diversity arrays technologySNP Chips e.g. Infinium Bead Chips, Affymetrix GeneChips RAD Restriction site associated DNA

Single nucleotide polymorphism (SNP)

RAD Restriction-site associated DNAGBS Genotyping-By-Sequencing markers

DA T Bead

1st generation 2nd generation 3rd generation

RFLPSSR

DArTGeneChips RAD GBS

BeadArrays,

SNP Chips

RAPD AFLP TaqMan,KASP

4th generation

Hybridization of labeled probe

PCR-based,gel-based

Genotyping by

1st generation 2nd generation 3rd generation

1974 1990 1992 1995 2004 2008 2011Genotyping

by

2006Hybridization-based,

gel-basedPCR-based,

gel-free

2002

4th generation

labeled probe to size-separatedrestricted DNA

gel-based by hybridization

by sequencing

Library cloning(probe design from sequences required)

gel-based

Complex dataprocessing required

gel-free(fluorescence)

FromFrom QTL QTL mappingmapping toto markermarker‐‐assistedassistedl il i (MAS) i OSR(MAS) i OSR b dib di

Papers listed in Google Scholar: 10 April 2013. Note: Google Scholar indexes papers, not journals. It includes journal and conference papers theses and dissertations academic books pre prints abstracts technical reports and

selectionselection (MAS) in OSR (MAS) in OSR breedingbreeding

80007220/2

013

includes journal and conference papers, theses and dissertations, academic books, pre-prints, abstracts, technical reports and other scholarly literature from all broad areas of research and thus includes doubles and overestimates the relevant literature.

6000

7000

e Scho

lar 4 QTL

MASQTL and MAS

4000

5000

33703560

sin Goo

gle

2000

3000

14301180

767

1380

2410

1110rofp

apers

0

1000

Rice (O. sativa) Maize (Z. mays) Arabidopsis (A. Brassica Brassica napus

767

108530 346

80363 221

Num

be

Rice (O. sativa) Maize (Z. mays) Arabidopsis (A. thaliana)

Brassica Brassica napus (B. napus)

Species name in title / terms in text of articles

FromFrom QTL QTL mappingmapping toto markermarker‐‐assistedassisted

100

selectionselection (MAS) in OSR (MAS) in OSR breedingbreedingG b t bli h d QTL d

80

90 Gap between published QTL and marker-assisted selection studiesin breeding of OSR (like other crops)

50

60

70

of articles

Mapping and QTL software

LD, association mapping

30

40

50

Num

ber o B. napus QTL

B. napus MAS

packages released (CIM)

pp gapproachespublished,

TASSEL 2007

10

20

0 1980198119821983198419851986198719881989199019911992199319941995199619971998199920002001200220032004200520062007200820092010201120122013

YearNumbers of articles with the terms “Brassica napus” in title and “quantitative trait locus“ or

“quantitative trait loci” (QTL) and “marker-assisted selection” (MAS) or similar somewhere within the document (excluding citations) by years (1980–2013) from Google Scholar (9 April 2013).

SNP Discovery in Rapeseed: Pod ShatterSNP Discovery in Rapeseed: Pod Shatter--Resistant Associated SNPsResistant Associated SNPs

Genetic linkage map of chromosome

Resistant Associated SNPs Resistant Associated SNPs

Genetic linkage map of chromosome A09 from rapeseed.

The numerals in left column indicate the genetic distance [cM]. SNP or SSR

k i di t d i th i htmarker names are indicated in the right column. Psr1 indicates the QTL region of pod shatter-resistance *)of pod shatter resistance. )

*) genetic contribution rate 47%.

Hu Z et al. (2012) PLoS ONE 7(4): e34253.u et a ( 0 ) oS O ( ) e3 53

Major OSR Major OSR traitstraits forfor whichwhich molecularmolecular markersmarkersll dd bb (G )(G ) B dB dareare currentlycurrently usedused byby (German) (German) BreedersBreeders

Trait of interest Type of markerTrait of interest Type of marker

Genetic distance SSR, SNP, DArTCl fi ld® HT SNPClearfield® HT  SNP 

MSL hybrid system InDel, SNP, SSR 

Ogu‐Rf hybrid system SNP, InDel, SSR 

Dwarf growth type  SNP 

High oleic/Low linolenic SNP 

Phoma resistance SSR InDelPhoma resistance  SSR, InDel 

Clubroot resistance SSR 

Markers for quantitative traits to be used for MAS

QTL QTL mappingmapping ofof resistanceresistance toto fungalfungal diseasesdiseasesPathogen Brassica

speciesConsensus QTL(number of populations)

Leptosphaeria maculans(Phoma stem canker, blackleg)

B. napusB. napusB. napus

A7, A2, A10 (race‐specific)A4 (race non‐specific)A1 (4 of 5), A10 (3 of 5), C1 (2 of 4)blackleg) B. napus

B. napusA1 (4 of 5), A10 (3 of 5), C1 (2 of 4)A10

Plasmodiophora brassicae( l b )

B. rapaB

A1, A6, A8A2 A3(clubroot) B. rapa

B. napusB. oleracea

A2, A3A2, A3, A8, C3, C5, C3,C5

Albugo candida(white rust)

B. napusB. rapa

A2 (2 of 2)A4 (race non‐specific), A2 (race‐sp.)

S l ti i l ti B A3 (2 f 3)Sclerotinia sclerotiorum(Sclerotinia rot)

B. napusB. napusB. napus

A3 (2 of 3)C7 (3 of 3)C12 (2 of 3)

Verticillium longisporum(Verticillium disease)

B. napus C5, C4 (major QTL) A6, C8, C1 (minor QTL)

BreedingBreeding forfor resistanceresistance toto VerticilliumVerticillium longilongi‐‐dd kk d l td l tsporumsporum andand markermarker developmentdevelopment

1. Screening in B. rapa and B. oleracea for1. Screening in B. rapa and B. oleracea for Verticillium resistance

2. Creation of resynthesized B. napus (RS)WUR

y p ( )

3. Production of DH mapping populations (elite lines x RS lines)lines x RS lines)

4. Genetic mapping and analysis of resistance

5. Comparative QTL analysis: identification of common QTL

6. Development of new QTL-linked markers

7 Oil quality analysis of DH lines7. Oil quality analysis of DH lines

=> Obermeier C. et al., Poster

C

A A highhigh‐‐densitydensity mapmap applyingapplying GBS GBS markersmarkers

C1 C5

11 marker 56 marker 24 marker 55 marker

Express 617 x R53106 DH lines

=> Obermeier C. et al., Poster

C

QTL QTL mappingmapping ofof V. V. longisporumlongisporum resistanceresistance

C1 C5

QTL for V. longisporum

R2=13

g presistance

R 13-15% R2=20

- 30%

Express 617 x R53

11 marker 56 marker 24 marker 55 marker

p ess 6 53106 DH linesNormalized AUDPCfrom 3 experiments

C

Development Development ofof newnew markersmarkers forfor markermarker‐‐assistedassisted selectionselection

C1 C5Two QTL regions on

assistedassisted selectionselection

QTL for V. longisporum

R2=

R2=13.7%

R2=13

g presistance

R2=12.6%

R2=18 7%

R2=20- 30%

R 13-15%

R2=18.7%

Express 617 x R53

11 marker 56 marker 24 marker 55 marker

Express 617 x R53106 DH linesNormalized AUDPCfrom 3 experiments

Breeding technology pipeline from past to Breeding technology pipeline from past to present to futurepresent to futurepresent to futurepresent to future

Example: Verticillium longisporum resistance

Adapted from

Fernie &in oilseed rape? Fernie & Schauer

(2008)

Fernie AR, Schauer N (2008). Metabolomics-assisted breeding: a viable option for crop improvement? Trends in Genetics 25:39-48

Stress Stress tolerancetolerance andand yieldyield stabilitystabilitydue due toto strong strong rootroot systemsystem

• Better availability andacquisition of water andnutrients

• Stress (drought) and• Stress (drought) anddisease resistance (e.g. V ti illi ) b ttVerticillium),  better cropstability

• Adequate yield (output) atlower input (fertilization, p ( ,pesticides, etc.)

Thanks:German Breeders (DSV, NPZ, KWS, GFP)Dr. Christian Obermeier, M. Hossain, a.o.Prof. A. v. Tiedemann, U Göttingen (Germany)

Thank you for yourThank you for yourThank you for your Thank you for your attention!attention!attention! attention!