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)
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
(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.
Institute for Resistance Research and Stress Tolerance
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
platform/ simultaneous
analysis
platform/ simultaneous
analysis
platform/ simultaneous
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
Institute for Resistance Research and Stress Tolerance
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.
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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
SCRI_RS_149683 SCRI_RS_232577
15.0
BOPA2_12_11011 BOPA2_12_30933
SCRI_RS_179144 17.3
BOPA1_7372-1253 22.1
SCRI_RS_165792 23.7
SCRI_RS_164150 SCRI_RS_119312
BOPA2_12_30817 SCRI_RS_148733
BOPA2_12_30950 BOPA2_12_30918
25.3
GMS021 28.8
SCRI_RS_130600 SCRI_RS_205669
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
SCRI_RS_171221 SCRI_RS_4891
BOPA1_4226-570 SCRI_RS_239784
BOPA1_6081-850 SCRI_RS_217160
48.7
SCRI_RS_116548 SCRI_RS_161702
GBM1042 Bmag872
BOPA1_5346-1587
54.6
SCRI_RS_170878 SCRI_RS_153785
SCRI_RS_170869 SCRI_RS_189197
BOPA1_5381-1950
61.8
SCRI_RS_205816 SCRI_RS_140837
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_116685 SCRI_RS_158003
SCRI_RS_172170 BOPA2_12_31134
SCRI_RS_189898 SCRI_RS_229932
SCRI_RS_163127 SCRI_RS_7813
SCRI_RS_189920 BOPA2_12_11209
SCRI_RS_160466 SCRI_RS_130590
SCRI_RS_219043 BOPA2_12_31272
SCRI_RS_189248 SCRI_RS_141598
BOPA1_1294-473 SCRI_RS_122889
127.8
BOPA2_12_30694 BOPA2_12_30243
BOPA1_5798-867 SCRI_RS_14834
SCRI_RS_118168 SCRI_RS_198546
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_182431 SCRI_RS_192779
SCRI_RS_121978 BOPA2_12_30821
SCRI_RS_130666 SCRI_RS_236104
BOPA1_1857-64 SCRI_RS_21483
SCRI_RS_211274 SCRI_RS_152795
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
SCRI_RS_120605 SCRI_RS_133886
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
SCRI_RS_195238 SCRI_RS_159201
BOPA2_12_30298
174.9
BOPA1_4665-882 BOPA2_12_30742
BOPA1_1670-369 BOPA2_12_30147 176.0
SCRI_RS_169123 SCRI_RS_153727
SCRI_RS_196439 BOPA1_6142-1544
SCRI_RS_1473
177.1
SCRI_RS_160234 SCRI_RS_183588
BOPA1_233-1327 SCRI_RS_188360
BOPA1_713-972 SCRI_RS_156208
BOPA1_5772-1176 BOPA2_12_11267
SCRI_RS_219726 SCRI_RS_823
SCRI_RS_151244 SCRI_RS_181239
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
SCRI_RS_188909 SCRI_RS_136856
SCRI_RS_213539 SCRI_RS_78578
SCRI_RS_236160 SCRI_RS_235968
BOPA1_2711-234 BOPA1_1270-1263
SCRI_RS_143810
215.5
SCRI_RS_206759 SCRI_RS_215740
SCRI_RS_106752 HvHVA1
SCRI_RS_106754 SCRI_RS_232650
BOPA2_12_30191
231.0
SCRI_RS_162 233.1
SCRI_RS_176063 BOPA1_1625-303
SCRI_RS_192730 SCRI_RS_161388
BOPA1_4962-1295 BOPA2_12_30532
SCRI_RS_197337
235.5
BOPA1_10360-563 236.7
SCRI_RS_157953 SCRI_RS_238125
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
SCRI_RS_48925 SCRI_RS_196910
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_165600 SCRI_RS_10956
SCRI_RS_141322 SCRI_RS_130891
SCRI_RS_156009 SCRI_RS_155407
SCRI_RS_150563 SCRI_RS_150786
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
Marker based harnessing of genetic resources: P. hordei
D. Perovic et al. (in prep.)
Institute for Resistance Research and Stress Tolerance
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
Marker based harnessing of genetic resources: P. hordei
D. Perovic et al. (in prep.)
Institute for Resistance Research and Stress Tolerance
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-Pick15 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-Pick272 HB HB HB HB HB HB HB HV HV
DH-Pick275 HV HB HB HB HB HB HB HB HB
DH-Pick276 HB* HV HV HV HV HV HV HV HV
DH-Pick279 HB HB HB HB HB HB HB 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
DH-Pick383 HV HV HV HV HB HB HB HB HB
DH-Pick391 HB HB HB HB HB HB HB HV HV
DH-Pick403 HV HV HV HV HB HB HB 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 87 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 88 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
QBS 91 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
QBS 92 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_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 - - - - - - - - - - - - - - - -
QBS 93 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
% 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
Institute for Resistance Research and Stress 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
QBS90_contig121643 149.256.373.938 625575040 2 contig_43474 148.158.640.226.629 2 morex_contig_65625
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
QBS91_contig1005709 149.362.606.232 625975560 2 contig_45939 148.158.640.226.629 2 morex_contig_38887
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
QBS93_contig40900 121.104.815.864 11111640 3 contig_47904 149.150.141.643.059 2 morex_contig_52954
Ryd203S11Hb
Berges pers.
com.
Perovic et al. unpublished
Marker based harnessing of genetic resources: BYDV-tolerance
Institute for Resistance Research and Stress Tolerance
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
Perovic et al. unpublished
Marker based harnessing of genetic resources: BYDV-tolerance
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
Yang et al., in preparation
Development of allele specific markers
Institute for Resistance Research and Stress Tolerance
Construction of a high resolution mapping population is in progress
rym7
Gene isolation via mapped based cloning: rym13
Institute for Resistance Research and Stress Tolerance
5HS
Gene isolation via mapped based cloning: BaYMV/BAYMV-2
Perner et al. unpublished
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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
Institute for Resistance Research and Stress Tolerance
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