Genomics based marker development and breeding for

www.jki.bund.de

Genomics based marker development and

breeding for resistance in barley

Dragan Perovic, Thomas Lüpken, Sandra Lehmann, Katja Perner, Ping Yang,

Christina Silvar, Ilona Krämer, Antje Habekuß, Doris Kopahnke, Andreas Graner,

Nils Stein and Frank Ordon

Institute for Resistance Research and Stress Tolerance

Introduction: Barley

Europe 2012: 24.372812 ha

World 2012: 51.005801 ha

India 2012: 780000 ha

In-breeding species with 2n=2x=14 chromosomes

Genome size about 5.3x109 bp

Acreage in 2012 Germany

http://faostat.fao.org/ u. Beschreibende Sortenliste 2013

Barley , 1683000

Wheat, 3061000

Rye, 710000 Triticale, 371400

Oat, 146000

Maize, 526000

Maize (green), 2036000

Rape seed,

1300000

Sugar Beet,

402000

Potato, 238300

Legumes, 79000

Animal feed Human Nutrition

home.arcor.de www.manager-magazin.de

Use

lactogenicdiet.blogspot.com

http://blogs.oregonstate.edu/deliciousnessw09/files/2009/02/bb01.jpg

http://www.melissas.com

Development of winter barley yield

in Germany (1952-2008)

http://faostat.fao.org/


Introduction: Barley diseases and molecular markers

Pathogene No. of genes tagged by

molecular markers

B. graminis 13

P. hordei 15

P. striiformis 2

P. graminis 4

R. commune 8

P. teres 10

C. sativus 1

T. incarnata 1

P. graminea 2

U. nuda 1

S. paserrinii 1

H. avenae 2

BSMV 1

BYDV 3

BaMMV/BaYMV 16

Barley Genetics Newsletter, Coordinator´s Report: Disease and Pest Resistance Genes


(ryd2, ryd3, QTL-) (Ryd2, Ryd3, QTL+)

winter barley, Quedlinburg, April 2008

QTL + (211 bp)

QTL - (218 bp)

QTL + (211 bp)

QTL - (218 bp)

Ryd3 (180 bp)

ryd3 (188 bp)

Ryd3 (180 bp)

ryd3 (188 bp)

ryd

2(2

53 b

p)

Ryd

2(3

11 b

p)

ryd

2(2

53 b

p)

Ryd

2(3

11 b

p)

QTL Post 2H

(HVCSG)

Ryd3

(HVM74)

Ryd2

(YLP + HSP92 II)

BYDV – Pyramiding

´RIL K4-56´ (Ryd3) x ´DH21-136´ (Ryd2 + QTL Post)

rrr rrs rsr srr ssr srs rss sss

No. DH-lines 89 49 45 99 49 77 38 29

Segregation Ryd2/ Ryd3/ Post-QTL

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Ryd2Ryd3QTL+

Ryd2Ryd3QTL-

Ryd2ryd3

QTL+

Ryd2ryd3QTL-

ryd2Ryd3QTL+

ryd2Ryd3QTL-

ryd2ryd3

QTL+

ryd2ryd3QTL-

DH21-136

(Ryd2ryd3

QTL+)

RIL K4-56

(ryd2Ryd3QTL-)

Rub-ina

(ryd2ryd3

QTL-)

ELIS

A e

xtinction

Allele combination

G F D C E CD A B

Riedel, C., A. Habekuss, E. Schliephake, R. Niks, I. Broer, F. Ordon, 2011 Theor. Appl. Genet. 123, 69-76.


Marker type RFLPs Genomic SSRs AFLPs EST

SNPs/SSRs DArTs BOPAs/OPAs iSelect Genotyping by

sequencing

Throughput single marker

application single marker

application few marker application

single marker application 6K 1,5K 9K 50K

Multiplexing no mutiplexing few markers multiplexing

low multiplexing

few markers multiplexing

platform/ simultaneous

analysis


analysis


analysis


analysis

simultaneous multiplexing NGS/GBS

Amount of D N A Large amount low amount low amount low amount low amount low amount low amount low amount low amount

Quality of D N A very good average average average very good very good very good very good very good

Introduction: Molecular tool box

0

2 5 0 0 0

5 0 0 0 0

7 5 0 0 0

1 0 0 0 0 0

1 2 5 0 0 0

1 5 0 0 0 0

1 7 5 0 0 0

2 0 0 0 0 0

2 2 5 0 0 0

3 4 0 3 4 5 3 5 0 3 5 5 3 6 0 3 6 5 3 7 0 3 7 5 3 8 0

G B S 5 6 4 _ M B R 1 0 1 2 . E 0 4 _ 1 1 0 4 1 1 1 4 P 3

S i z e ( n t )

Dye S

ignal

340 349,69

352,43

360

367,35

368,90

370,07

380

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 4 0 3 4 5 3 5 0 3 5 5 3 6 0 3 6 5 3 7 0 3 7 5 3 8 0

G B S 5 6 4 _ S c a r l e t t . F 0 4 _ 1 1 0 4 1 1 1 4 P 5

S i z e ( n t )

Dye S

ignal

340 360 364,57

367,32

368,17

380

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 4 0 3 4 5 3 5 0 3 5 5 3 6 0 3 6 5 3 7 0 3 7 5 3 8 0

1 _ 2 _ 0 0 1 _ _ 1 _ 2 _ 0 9 6 _ _ 1 _ 5 _ 0 0 3 . A 0 1 _ 1 1 0 4 1 1 1 8 Q L

S i z e ( n t )

Dye S

ignal

349,33

352,30

367,28


Introduction: Molecular tool box


Marker-trait associations for powdery mildew

Chromosomes:

7864 SNPs from the 9K iselect Chip, 3967 SNPs with mapping information, 2805 MAF>5%

Marker Development: Association genetic studies

Traits

63 six-rowed 48 two-rowed

winter barley cultivars winter barley cultivars

Min Max Mean Min Max Mean

Powdery mildew (1-9) 1.84 4.79 3.39 1.52 4.27 2.98

Rynchosporium (1-9) 1.28 3.21 1.87 1.33 3.72 1.96

Net blotch (1-9) 1.95 4.24 3.26 2.35 3.85 3.05

Leaf rust (1-9) 1.17 6.83 3.65 1.78 4.67 2.94

Ramularia (1-9) 3.67 7.50 5.64 3.80 6.78 5.40

J. Lex pers. com.


MBR1012 Scarlett

Cultivar Gold Cebada Capa Bowmann Scarlett H.sp 680 MBR1012 L 94

Isolat / gene Rph4 Rph7 Rph15 Rph 3/Rph 9/Rph 12 Rph16 Rph MBR1012 susc.

R 8-1 3-4 0C 0 0 0N 0 3-4

R 8-2 3-4 0C 0 0 0N 0 3-4

R 14-1 3-4 0C 0 0 0N 0 3-4

R14-2 3-4 0C 0 0 0N 0 3-4

R 16-1 3-4 0C 0 0 0N 0 3-4

R 34-3 3-4 0C 0 0 0N 0 3-4

R 54-3 3-4 0C 0 0 0N 0 3-4

I 80 3-4 0C 0-2- 3 0N 0-2- 3-4

30-1 * 3-4 0C 0-2- 0 0N 0, 0-1 3-4

30-1/2* 3-4 0C 0 0, 0-2- 0N 0, 0-2- 3-4

23- 3 3-4 0C 0 0 0N 0 3-4

23-1/2/3* 3-4 0C 0 0 0N 2- 3-4

23-1+3* 3-4 0C 0 0 0N 0, 0-2- 3-4

MBR1012 is resistant to the most wide spread virulent leaf rust

pathotypes present in Europe.

Marker based harnessing of genetic resources: P. hordei


QBS2 0.0

GBMS187 GBS546 2.5

RphMBR1012 3.3 GMS021 9.3

GBM1007 17.6

Bmag872 28.8 GBM1042 28.9

GBM1032 38.7

Bmag347 Bmag211 41.3 GBM1070 42.6 GBM1336 45.3

Bmac63 47.0

GBS528 61.1

Bmac32 76.7

GBS582 96.2

HvHVA1 119.0

1H - SSR and SNP map

BOPA2_12_30969 SCRI_RS_180989

SCRI_RS_138156 BOPA2_12_10420

BOPA1_8670-388 SCRI_RS_204276

BOPA1_7174-365 BOPA2_12_30653

SCRI_RS_138128

0.0

GBS626 GBS546

BOPA1_3220-723 GBR534

Rph20 GBS564

BOPA2_12_30944 GBMS187

4.7

GBR218 8.1

BOPA2_12_31144 SCRI_RS_207257 11.6

SCRI_RS_66630 SCRI_RS_139538

SCRI_RS_60145 BOPA1_3101-111


15.0

BOPA2_12_11011 BOPA2_12_30933

SCRI_RS_179144 17.3

BOPA1_7372-1253 22.1

SCRI_RS_165792 23.7


BOPA2_12_30817 SCRI_RS_148733

BOPA2_12_30950 BOPA2_12_30918

25.3

GMS021 28.8


SCRI_RS_182656 SCRI_RS_130592 32.2

SCRI_RS_152464 34.0

BOPA2_12_30948 SCRI_RS_139220 35.6 GBM1007 42.8

SCRI_RS_150500 BOPA1_6792-1945

BOPA1_2901-1137 SCRI_RS_124926

SCRI_RS_122490 BOPA2_12_31276


BOPA1_4226-570 SCRI_RS_239784

BOPA1_6081-850 SCRI_RS_217160

48.7


GBM1042 Bmag872

BOPA1_5346-1587

54.6



BOPA1_5381-1950

61.8


SCRI_RS_189637 64.1

GBS582 89.9

BOPA2_12_30268 BOPA1_6720-641

SCRI_RS_9480 SCRI_RS_141140 117.6

BOPA1_3710-852 BOPA2_12_30683

BOPA1_2265-363 BOPA2_12_10314

SCRI_RS_151764 GBM1032

BOPA1_2407-1771

118.7

SCRI_RS_85918 BOPA1_ABC02639-1-4-

SCRI_RS_85917 BOPA1_5019-879

SCRI_RS_139785

122.2

BOPA2_12_10506 GBM1029

BOPA1_8613-278 Bmag211

Bmag347

123.3

GBM1070 SCRI_RS_189586

SCRI_RS_151874 BOPA2_12_10938

BOPA2_12_31208 BOPA1_3217-929

BOPA2_12_30592 SCRI_RS_236235

SCRI_RS_143927 BOPA1_C-GBS0371-2

SCRI_RS_155439 BOPA1_ABC11913-1-1-

BOPA1_4020-643 SCRI_RS_213103

125.6


SCRI_RS_172170 BOPA2_12_31134



SCRI_RS_189920 BOPA2_12_11209


SCRI_RS_219043 BOPA2_12_31272


BOPA1_1294-473 SCRI_RS_122889

127.8

BOPA2_12_30694 BOPA2_12_30243

BOPA1_5798-867 SCRI_RS_14834


GBM1336 SCRI_RS_119743

SCRI_RS_148600 BOPA2_12_30499

BOPA2_12_30750 BOPA1_5297-796

SCRI_RS_17256 BOPA1_4793-777

BOPA1_3689-1101 BOPA2_12_30672

BOPA1_7800-594 BOPA2_12_30786

BOPA1_4716-1205 BOPA2_12_30478

BOPA2_12_30406 BOPA2_12_30110

BOPA1_8743-197 SCRI_RS_125407

SCRI_RS_170542

130.1

BOPA1_6118-595 132.4

Bmac63 134.7

SCRI_RS_167877 SCRI_RS_161643 137.0

BOPA2_12_10198 138.2

SCRI_RS_1445 143.8

SCRI_RS_207335 SCRI_RS_193401 145.1

SCRI_RS_100503 147.3

SCRI_RS_225107 SCRI_RS_145305 148.5

SCRI_RS_229636 BOPA2_12_30115

SCRI_RS_213455 BOPA1_5790-558 153.1


SCRI_RS_121978 BOPA2_12_30821


BOPA1_1857-64 SCRI_RS_21483


156.6

BOPA2_12_10201 BOPA1_1190-86 161.2

SCRI_RS_237999 162.3

SCRI_RS_145336 SCRI_RS_231735 163.5

SCRI_RS_170810 SCRI_RS_204611 164.6

SCRI_RS_85391 166.1


BOPA1_11603-445 SCRI_RS_175487

SCRI_RS_180349 BOPA1_7299-183

SCRI_RS_219685 BOPA2_12_11062

BOPA1_ABC16273-1-1-

169.2

SCRI_RS_157039 Bmac32 172.6

BOPA2_12_30505 BOPA1_2036-1027


BOPA2_12_30298

174.9

BOPA1_4665-882 BOPA2_12_30742

BOPA1_1670-369 BOPA2_12_30147 176.0


SCRI_RS_196439 BOPA1_6142-1544

SCRI_RS_1473

177.1


BOPA1_233-1327 SCRI_RS_188360

BOPA1_713-972 SCRI_RS_156208

BOPA1_5772-1176 BOPA2_12_11267



178.2

SCRI_RS_181353 183.3

SCRI_RS_125339 188.4

BOPA2_12_11144 SCRI_RS_157246

BOPA1_568-1651 BOPA1_34-472

BOPA1_2881-935 SCRI_RS_168562

SCRI_RS_181300 BOPA2_12_31160

SCRI_RS_201866

191.1

SCRI_RS_236623 192.6

GBS528 195.6

BOPA1_9757-582 BOPA1_3201-603

SCRI_RS_170389 SCRI_RS_121048 199.1

SCRI_RS_189168 207.2

BOPA1_8867-459 SCRI_RS_188218

BOPA1_2935-1634 BOPA2_12_31163

SCRI_RS_197263

209.8

SCRI_RS_199689 BOPA2_12_31319

BOPA1_ABC11290-sfp4 SCRI_RS_194371

SCRI_RS_138010

210.9

SCRI_RS_142282 212.0

BOPA1_13589-175 SCRI_RS_199972




BOPA1_2711-234 BOPA1_1270-1263

SCRI_RS_143810

215.5


SCRI_RS_106752 HvHVA1


BOPA2_12_30191

231.0

SCRI_RS_162 233.1

SCRI_RS_176063 BOPA1_1625-303


BOPA1_4962-1295 BOPA2_12_30532

SCRI_RS_197337

235.5

BOPA1_10360-563 236.7


BK_01 SCRI_RS_91596

BOPA1_9105-497 SCRI_RS_218473

SCRI_RS_155997

243.9

BOPA1_557-1297 249.2

SCRI_RS_127646 BOPA2_12_21172 253.2

SCRI_RS_150607 BOPA2_12_11443


BOPA1_4027-1814 257.8

SCRI_RS_216088 SCRI_RS_169881 267.6

SCRI_RS_153450 SCRI_RS_167578 273.5

BOPA1_ABC05061-1-1- SCRI_RS_147318





SCRI_RS_180081

277.0

SCRI_RS_13869 SCRI_RS_170857 279.2

1H map - 311 markers

SCRI_RS_xxxx 0.0

BOPA2_12_xxxx 0.4

SCRI_RS_xxxx 0.5

QBS0002 2.6

BOPA2_12_xxxx 4.6

SCRI_RS_6xxx SCRI_RS_6xxx

BOPA1_3xxx-111 SCRI_RS_2xxx 4.8

BOPA2_12_3xxxx 4.9

GBS626 GBS564 5.0

GBR534 GBMS187

BOPA2_12_30xxx

BOPA1_3xxxxx

5.1

SCRI_RS_2xxxx 5.5

SCRI_RS_1xxxx 5.9

SCRI_RS_1xxxx 6.0

RphMBR1012 6.2

BOPA2_12_xxxx SCRI_RS_1xx4 7.2

BOPA1_7xxx-1xxx 10.7

GMS021 13.0

SCRI_RS_1xxxxx 14.2

GBM1007 21.1

SCRI_RS_xxxxx3 SCRI_RS_xxxxx9 BOPAx_xx_3x9x9 BOPAx_xx9x-xx37 SCRI_RS_xx374x BOPAx_xx_3x7xx BOPAx_3xxx-7x3 SCRI_RS_x3xxxx BOPAx_xx_xx4xx BOPAx_xx_3xxx3 BOPAx_xx7xxxxx SCRI_RS_xxx9x9 BOPAx_xxxx SCRI_RS_xxxx SCRI_RS_xxxxx BOPAx_xx_xxxx SCRI_RS_xxxx SCRI_RS_xxxxx SCRI_RS_xxxx3 BOPAx_xx_xxxxx SCRI_RS_xxxx7 SCRI_RS_xxxx BOPAx_xxxxx SCRI_RS_xxx BOPAx_xxxxx BOPAx_xx_xxx

GBMS187 GBS546 GBR534 GBS564

RphMBR1012 BOPAx_xx_xxx

SCRI_RS_xxx7

GBSxxx

SCRI_RS_xxx

SCRI_RS_xxx

BOPAx_xxxxxx SCRI_RS_xxxx BOPAx_737xxxx

QBS0002

SCRI_RS_x79x44

SCRI_RS_xx4x74 SCRI_RS_xx4xxx

SCRI_RS_x9x9

BOPAx_xx_3xx49 BOPAx_xx_3x9xx SCRI_RS_xx93xx SCRI_RS_xxx79x SCRI_RS_x4xxxx GMS021 SCRI_RS_xx4xxx BOPAx_xx_3xxx7 BOPAx_xx_3x9xx SCRI_RS_x4x733 BOPAx_xx_3xxxx SCRI_RS_x3xxxx SCRI_RS_xxxxxx SCRI_RS_x3xx9x SCRI_RS_xx77x7 SCRI_RS_xxxxx9 SCRI_RS_333x SCRI_RS_xxx4x4 SCRI_RS_x39xxx BOPAx_xx_3x94x

BOPAx_xx9xx-xx3 BOPAx_x49x-x9xx SCRI_RS_xx9xxx

GBM1007

BOPAx_xxxxx

0,0 0,7 3,8 4,1 5,7 7,2 7,9

8,2

8,4

8,6

9,0

9,2

9,4 11,2

12,4

13,0 13,2 13,7 13,9 14,8 14,9 15,6 15,9 16,4 17,0 17,7 17,8 17,9 20,1 22,3 23,3 23,9 24,8 25,6

26,3

27,5 27,6 29,9 30,0 31,5 32,0 32,1 32,9 33,5 33,6

39,1 39,4

42,2

1HS Consensus map

~50 new markers

for saturation


D. Perovic et al. (in prep.)


contig64079SC180989 0.0

SCRI_RS_xx56 0.1

SCRI_RS_xx28 0.2

BOPA1_86xx_ BOPA2_xxxx69

BOPA2_1xx_xx20 0.3

BOPA_12_xxxxx 2.9

BOPA2_1x_xxxx1 SCRI_RS_xx79144 3.0

contig50849 3.2

SCRI_RS_xx39538 3.3

BOPA1_xx-1xx SCRI_RS_xxx66630

SCRI_RS_x60145 SCRI_RS_xx32577

contig145384

GBR218_QBS002

3.4

SCRI_RS_xx07257 BOPA2_x_3xx44 3.6

GBRxxx4-QBS063 GBSxx

GBMS187

RphMBR1012

GBSxx4

3.7

GBSxx6

3.8

BOPA1_xxx BOPA2_xx_3xx4 3.9

contig1008249s133 contig1031142 4.0

contig53937 4.2

contig1019464 4.3

Contig 2160218_BOPA21230944 contig100824 4.4

SCRI_RS_xx83 4.7

BOPA2_xxx3 SCRI_RS_xx6

BOPA1_xxx4-365 SCRI_RS_xx89 4.8

contig121251 6.2

contig247169 6.9

BOPA1_xx_ 9.8

SCRI_RS_xx692 11.0

GMS21 11.7

iSelect

markers

ZIPPER

markers

Flanking markers

Locus Zipper

loci Position

cM contig64079_SC1989 12 0.0 SCRI_RS_xx56 - 0.0 SCRI_RS_xx28 - 0.2 BOPA1_86xx- 28 0.2 BOPA_12_xx39 28 0.2 BOPA_12_xx10 435 0.2 BOPA_12_xx33 - 2.9 BOPA_12_xx 77 3.0 SCRI_RS_xx4 85 3.0 contig50849 530 3.2 SCRI_RS_xx38 69 3.3 BOPA1_3xxx 64 3.4 SCRI_RS_6xxx0 61 3.4 SCRI_RS_xxxx5 57 3.4 SCRI_RS_xx377 1198 3.4

contig145384 165 3.4 GBR218_QBS002 - 3.4 SCRI_RS_xx057 824 3.6 BOPA_12_xx4 53 3.6 GBRxxx-QBS063 - 3.7 GBSxxx - 3.7 GBMS187 - 3.7 GBS6xx - 3.7 GBSxx4 - 3.7 Rph_MBR1012 - 3.8 BOPA1_3xxx3 38 3.9 BOPA_12_xx30944 38 3.9 contig1008249 33 3.9 contig1031142 45 4.0 contig53937 24 4.2 contig1019464 50 4.3 ctg2160218BOPA21230944 38 4.4 contig1008249 33 4.4 SCRI_RS_xx43 57 4.7 BOPA_12_xx33 12 4.8 SCRI_RS_xx046 - 4.8 BOPA1_71xx-xx - 4.8 SCRI_RS_xx8 12 4.8 contig121251 75 6.2 contig247169 22 6.9 BOPA1_xxxx- 100 9.8 SCRI_RS_xx657 - 11.0 GMS21 - 11.7


D. Perovic et al. (in prep.)


Identification of BYDV-tolerance

H. bulbosum line 203S11/M2/M2 = H. bulbosum A17 x Emir

FISH karyogram of H. bulbosum

line 203S11/M2/M2

Photo Pickering

Marker SSR1 SSR2 CAPS1 CAPS2 SSR3 CAPS3 CAPS4 SSR4 CAPS5

DH line Bin12 Bin12 Bin12 Bin13 Bin13 Bin14 Bin14 Bin14 Bin15

DH-Pick14 HV HV HV HV HB HB HB HB HB


DH-Pick22 HB - HV HV HV HV HB - HB

DH-Pick167 HB HB HB HB HB HB HB HV HB

DH-Pick168 HB HB HB HB HB HB HB HV HV

DH-Pick200 HV HV HV HB HB HB HB HB HB

DH-Pick204 HV HV HV - HV HV HV HV HB

DH-Pick214 HV HV HV - HV HV HV HV HB

DH-Pick245 HV HV HV HV HV HV HV HB HB

DH-Pick267 HV HV HV HV HV HV HB HB HB


DH-Pick275 HV HB HB HB HB HB HB HB HB

DH-Pick276 HB* HV HV HV HV HV HV HV HV


DH-Pick287 HB HB HV HV HV HV HV HB HB

DH-Pick307 HB HB HB HB HV HV HV HV HV

DH-Pick311 HB HB HB HB HB HB HB HB HV

DH-Pick313 HB HV - HV HB HB HB HB HB

DH-Pick323 HB HB HB HB HB HB HV HV HV

DH-Pick364 HV HV HV HV HV HV HV HB HB




Out of 212 DHs a set of 118 carried the H. bulbosum

fragment

27 recombinants the in introgressed fragment (9 PCR

based markers: 4 SNPs and 5 SSRs covering ~25 cM)

Group Consensus Em

ir

DH

-Pic

k2

04

DH

-Pic

k2

14

DH

-Pic

k2

60

DH

-Pic

k2

76

DH

-Pic

k3

07

DH

-Pic

k3

23

DH

-Pic

k2

79

DH

-Pic

k1

68

DH

-Pic

k1

67

DH

-Pic

k2

72

DH

-Pic

k3

64

DH

-Pic

k1

37

DH

-Pic

k3

13

DH

-Pic

k2

75

HB

-20

3S

11

DH

-Pic

k3

91

DH

-Pic

k3

11

DH

-Pic

k1

4

DH

-Pic

k1

5

DH

-Pic

k2

00

DH

-Pic

k4

03

DH

-Pic

k3

83

DH

-Pic

k2

2

DH

-Pic

k2

87

DH

-Pic

k2

67

DH

-Pic

k2

45

GBM1047-2H-Bin12 a a a b a b b b b b b a a a b b b b a a a a a a b a a

SCRI_RS_230951 a a a a a b b b b b b a a a b - b b a a a a a a a a a

BOPA1_4240-749 a a a a a - - - - - - a a a - - - - a a a a a a a a a

SCRI_RS_740 a a a a a b b b b b b a a a b - b b a a a a a a a a a

BOPA1_3910-1648 a a a a a - - - - - - a a a - - - - a a a a a a a a a

SCRI_RS_114969 a a a a a - - - - - - a a a - - - - a a a a a a a a a

SCRI_RS_114673 a a a a a b b b b b b a a a b b b b a a a a a a a a a

SCRI_RS_156819 a a a a a b b b b b b a a b b - b b a a a a a a a a a

SCRI_RS_194653 a a a a a b b b b b b a a b b - b b a a a a a a a a a

SCRI_RS_116590 a a a a a a b b b b b a a b b - b b b b a a a a a a a

Bmag749-2H-Bin13 a a a a a a b b b b b a a b b b b b b b b b b a a a a

BOPA2_12_31506 a a a a a a - - - - - a - - - - - - - - - - - a a a a

QBS 81 a a a a a a b b b b b a b b b b b b b b b b b a a a a

QBS 82 a a a a a a a b b b b a b b b b b b b b b b b a a a a

QBS 83 a a a a a a a b b b b a b b b b b b b b b b b a a b a

SCRI_RS_142982 a a a a a a a a b b a b b b b - b b b b b b b a a b b

QBS 84 a a a a a a a a b b a b b b b b b b b b b b b a a b b

QBS 85 a a a a a a a a b b a b b b b b b b b b b b b a a b b

QBS 86 a a a a a a a a b b a b b b b b b b b b b b b b a b b



QBS 89 a a a a a a a a a a a b b b b b b b b b b b b b a b b

SCRI_RS_156075 a a a a a a a a a a a - - - - - - - - - - - - - - - -

QBS 90 a a a a a a a a a a a b b b b b b b b b b b b b b b b

GBM1036-2H-Bin13 a a a a a a a a a a a b b b b b b b b b b b b b b b b

SCRI_RS_206020 a a a a a a a a a a a b b b b - b b b b b b b b b b b



SCRI_RS_160512 a a a a a a a a a a a b b b b - b b b b b b b b b b b

SCRI_RS_8637 a a a a a a a a a a a - - - - - - - - - - - - - - - -


% grain weight / plant 6,0 7,8 7,4 6,3 10,3 15,2 12,7 14,0 9,5 10,0 17,8 19,1 32,1 28,0 28,1 53,7 56,6 54,9 49,4 44,7 51,5 31,2 41,1 30,4 34,2 33,0 41,4

Perovic et al. unpublished

The 9K iSelect chip detected

50 polymorphic SNPs

13 genome zipper based

markers were mapped

Fragment conferring

resistance was reduced from

25 cM to ~3 cM

Marker based harnessing of genetic resources: BYDV-tolerance


marker_id cM_IBSC bp_IBSC

chr_

IBSC FPcontig_IBSC cM_POPSEQ

chr_PO

PSEQ morex_contig_ALL

QBS88_contig248859 149.256.373.938 625575040 2 contig_43474 149.150.141.643.059 2 morex_contig_453346

SCRI_RS_156075 NA NA NA NA 147.946.175.637.394 2 morex_contig_2549570


GBM1036 149.256.373.938 625575040 2 contig_43474 148.158.640.226.629 2 morex_contig_1561486

SCRI_RS_206020 149.362.606.232 626560840 2 contig_45937 148.158.640.226.629 2 morex_contig_41516


QBS92_RS_138848 149.362.606.232 627567720 2 contig_45938 149.150.141.643.059 2 morex_contig_158818

SCRI_RS_160512_C2 149.362.606.232 627567720 2 contig_45938 148.158.640.226.629 2 morex_contig_44687

SCRI_RS_8637_C2 149.362.606.232 627950880 2 contig_44013 148.158.640.226.629 2 morex_contig_54095


Ryd203S11Hb

Berges pers.

com.




A set of 12 diff. expressed

genes from chromosome

2HL were down regulated

all having SNPs. co

ntig

log2

(fold

_cha

nge)

sign

ifica

nt

#Pol

ymor

phis

m

asse

mbl

y in

fo

1 -3,4 yes 3 2HL

2 -6,4 yes 2 2HL

3 -3,0 yes 3 2HL

4 -5,1 yes 3 2HL

5 -4,8 yes 6 2HL

6 -4,5 yes 11 2HL

7 -3,7 yes 3 2HL

8 -3,5 yes 2 2HL

9 -3,7 yes 4 2HL

10 -3,3 yes 2 2HL

11 -6,0 yes 2 2HL

12 -2,6 yes 4 2HL

Genotype Variant time

Emir BYDV_PAV 12 h pi

203 S11 BYDV_PAV 12 h pi

Emir K_ aphids 12 h pi

203 S11 K_ aphids 12 h pi

Emir Mock 12 h pi

203 S11 Mock 12 h piI




W. Huth

BaMMV, BAMMV-SIL, BaMMV-Teik, BaYMV, BaYMV-2

BaYMV/BaMMV- Yield Cultivar Reaction t/ha relative

Asorbia (6-rowed) resistant 5.33 100 Corona (6-rowed) susceptible 3.53 65

Romanze (2-rowed) resistant 4.20 100 Marinka (2-rowed) susceptible 2.38 57

10 m2 plots, 3 replications; LSD (5%) = 0.25 t/ha and 0.35 t/ha; yield in ATM

Yuka (6-rowed) resistant 7.66 100 Grete (6-rowed) susceptible 4.10 54

Duet (2-rowed) resistant 6.30 100 Angora (2-rowed) suceptible 4.24 67

Barley yellow mosaic virus disease

3H

1H(5)

100

cM

Chikurin Ibaraki 1

6H

5H(7)

7H(1)

2H

4H

rym7

rym4 rym5 rym6 rym10

Rym14HB

rym15

rym3

mod. Graner et al. 2000

Rym16HB

rym11 rym1 rym18 rym13 rym9 rym8

Ordon, F. 2009: Barley Genetics Newsletter 39, 58-69, Kai et al. 2012.

Rym17


Gene isolation via map based cloning: rym4/5

Identification of homozygous recombinant plants

Phenotypic analysis of homozygous recombinants (BaMMV, BaYMV, BaYMV-2)

F2

F3

F4

discard

Wicker et al. 2005. The Plant Journal, 41, 184-194

OP-AF18H971 E45*M69

MWG838

MWG010 BMac029

rym4

P11M36 P21M68

P20M46 B368N12

OP-Z04

0.10

0.97

0.82

0.34

0.05

A

MWG838+

E37*M36

E37*M63

E35*M81 E39*M39 STSE31*M41+

MWG010+

BMac029*

OP-AF18H971+

E45*M69

rym5

E62*M67

0.88

0.34

0.05

0.10

0.05 0.10

B

Y57C10+

0.66

0.034

~3500 F2-plants

2048 AFLP primer combinations

1200 RAPDs Pellio, B., S. Streng, E. Bauer, N. Stein, D. Perovic, A. Schiemann, W. Friedt, F. Ordon, A. Graner 2005. Appl. Genet. 110, 283-293.

Stein, N., D. Perovic, J. Kumlehn, B. Pellio, S. Stracke, S. Streng, F. Ordon, A. Graner, 2005. The Plant Journal 42, 912-922

Co-segregation proximal BAC-contig

rym5 rym4

../../Dokumente und Einstellungen/stein/Desktop/halle_041216/evo_rym4_locus5.html


1. Mapping of the gene

of interest on low to

medium resolution

2. Construction of a

high resolution

mapping population

4. Further marker saturation

by using sequence information

from rice, sorghum

brachypodium and NGS data

from barley (GenomeZipper)

3. Phenotyping of

segmental RILs (F4)

and marker saturation

using available high

density maps

5. Identification of

a BAC contig and

candidate genes

based on the

physical map of

barley or re-

sequencing of the

target interval

6. Identification

of rym13 by

transformation,

TILLING, RNAi

0

0.09

0.53

0.96

1.47

1.64

2.59

3.40

4.22

5.03

5.20

5.25

GBM1015

k0xxx6

k0xxx7

k0xxx5

k0xxx1

k0xxx2

rym13

k0xxx3

k0xxx4

k0xx10

k0xx11

HVM67

rym13

WMS06

GBM1015

HVM67

7,25 cM

5.97 cM

Humbroich et al. 2010

Map based cloning

Gene isolation via map based cloning: rym4/5


Gene isolation via map based cloning: rym11

HvPDIL5-1

Lüpken et al. 2013. Theor. Appl. Genet. 126: 1201-1212

Yang et al. 2014. www.pnas.org/cgi/doi/10.1073/pnas.1320362111


Functional verification by three approaches

T1 - E1 family T1 - E2 family Control

Transgene rym11 Transgene rym11 W757-612

(Res.)

Igri

(Sus.)

Maris Otter

(Sus.)

Res. 19† 21 3† 10 19 4† 2†

Sus. 30 0 45 0 0 22 28 † escapes the artificial BaMMV-inoculation under greenhouse condition.

T1- E1-13

Yang et al., PNAS, 2014

1. TILLING for EMS-induced mutants (Resistance)

2. Test for allelism among naturally occurring rym11 alleles (F1 plants,

resistance)

ZmUbi HvPDIL5-1 ter

3. Transgene-induced complementation (Susceptibility)

Gene isolation via map based cloning: rym11


365 Wild barley (H. spontaneum)

847 Landrace (H. vulgare)

559 Cultivar (H. vulgare)

5 H. agriocrithon

Total = 1,816 accessions

1 4 1 28 Number of accessions carrying different alleles

Yang et al. 2014. www.pnas.org/cgi/doi/10.1073/pnas.1320362111

Allele mining: rym11


Yang et al., in preparation

Development of allele specific markers


Construction of a high resolution mapping population is in progress

rym7

Gene isolation via mapped based cloning: rym13


5HS

Gene isolation via mapped based cloning: BaYMV/BAYMV-2

Perner et al. unpublished


Humbroich et

al. 2010

154 DH- lines

WMS06

GBM1015

HVM67

7.27 cM

Partial map of

Chromosome 4H

based on analysis

of 5191 F2-plants

5.74 cM rym13

Partial map of

Chromosome 4H

based on analysis

of 420 RILs

(4084 F2)

1 2 1

1

2

Gene isolation via mapped based cloning: rym13

GBM1015 0.0 SCRI_RS_xxx 0.1 k08xxx 0.2 k072xx B094_xxx 0.6 k00xxx 0.8 B296_C16xxxx 1.0 Bradi7xxxx0 1.1 B365_C87xxx 1.2 SCRI_RS_xxx 1.5

BOPA_xxx 3.1

k00xxx 3.5 contig_27xxx 3.8 contig_43xxx3 3.9 NiASHvxxxx1 4.1 Bradi7xxx0 4.2 2_1xxx 4.3 B250_Cxxx 4.7 k03xxx 4.9

S164_Cxxx1 5.4 k03xxx9 5.5 k00xxx 5.6 S357_Cxxx 5.8 S116_Cxxx 5.9 k08xxx 6.1 S829_Cxxx1 6.5 HVM67 6.6

4HL

Fp_contig 3xxx 22 genes

Allele-specific

sequencing of

interesting

candidate genes


Summary and future prospects

The availability of dense marker maps, high throughput genotyping

platforms, physical maps and genome sequences of barley itself and

related species facilitates an enhanced marker development and

isolation of resistance genes thereby leading to a deeper

understanding of resistance and the transfer of marker based

selection to the allele level.

This will lead to a more directed use of PGR in barley breeding

thereby broadening the genetic base of resistance.

Fernie, A.R., N. Schauer, 2008: Trends in Genetics 25, 39-

48


Thanks

BaMMV/BaYMV

Prof. Dr. Wolfgang Friedt

Dr. Andrea Schiemann

Dr. Bettina Pellio

Dr. Kay Werner

Dr. Antje Habekuß

Dr. Ilona Krämer

Thomas Lüpken

Sandra Lehmann

Katja Perner

Prof. Dr. Andreas Graner

Dr. Nils Stein

Dr. Dragan Perovic

Dr. Ping Yang

Funding

DFG

GFP

EU

BMELV

BMBF

BYDV

Dr. Winfried Huth

Dr. Konstanze Scheurer

Dr. Antje Habekuß

Dr. Edgar Schliephake

Dr. Rients Niks

Dr. Christine Riedel

Dr. Dragan Perovic

Leaf rust

Janine König

Dr. Doris Kopahnke

Dr. Brian Steffenson

Dr. Dragan Perovic

Documents

Genomics based marker development and breeding for