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P. Silva, V. Calvo-Salazar, F. Condón, M. Quincke, C. Pritsch, L. Gutiérrez, A. Castro, S. Herrera-Foessel, J. von Zitzewitz and S. Germán
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Effects and interactions of wheat leaf rust adult plant resistance genes in Uruguay P. Silva, V. Calvo-Salazar, F. Condón, M. Quincke, C. Pritsch, L. Gutiérrez, A. Castro, S. Herrera-Foessel, J. von Zitzewitz and S. Germán
BGRI Workshop 19-22 August 2013 New Delhi , India
§ Introduction
§ Objective
§ Materials and methods
§ Results and Discussion
§ Conclusions
§ Future works
Outline
INTRODUCTION
URUGUAY
Uruguay - South America (34ºS, 55ºW)
Wheat area Regional: 6 million has
Uruguay: 0.5 million has
54 % of S, MS and I cultivars
DIEA, 2013
Leaf rust: most important and
widespread wheat rust
§ High dynamism of the pathogen population § Short duration of resistance § Cultivar replacement
Genetic resistance is the best strategy to control LR Increase use of slow rusting resistance (durable)
Slow rusting genes
For leaf rust:
§ Lr34/Yr18/Pm38/Sr57/Bs: 7DS
§ Lr46/Yr29/Pm39/Sr58: 1BL
§ Lr67/Yr46: 4BL
§ Lr68: 7BL
For stem rust:
§ Sr2/Yr20/Lr27: 3BS
Pleiotropic effect or linkage
Combinations of 4 - 5 of these genes results in near immunity
Most reported to have differential expression at different temperatures
Environmental effect on LR resistance
Principal component analysis of leaf rust severity of Avocet-S x Parula population in South America and Mexico
Germán et al. 2010
PC1:59.8% PC2:13.0%
Differential expression of resistance genes present in Parula under different environments
Mexico Southern Cone
Environmental effect on LR resistance
0
10
20
30
40
50
60
70
80
LR Cluster 1 LR Cluster 2 LR Cluster 3
% disease severity
none
only Lr46
only LrP
only Lr34
Lr46+LrP
Lr46+Lr34
LrP+Lr34
Lr46+LrP+Lr34
0
10
20
30
40
50
60
70
80
LR Cluster 1 LR Cluster 2 LR Cluster 3
% disease severity
none
only Lr46
only LrP
only Lr34
Lr46+LrP
Lr46+Lr34
LrP+Lr34
Lr46+LrP+Lr34
No
gene
s Lr
46
Lr68
Lr
34 Lr46
+Lr6
8 Lr
46+L
r34
Lr68
+Lr3
4 Lr
46+L
r68+
Lr34
MEXICO 1997, 1998
§ Lr46, Lr68: moderate effect
§ Lr34: most effective
Modified from Lillemo et al. 2011
0
10
20
30
40
50
60
70
80
LR Cluster 1 LR Cluster 2 LR Cluster 3
% disease severity
none
only Lr46
only LrP
only Lr34
Lr46+LrP
Lr46+Lr34
LrP+Lr34
Lr46+LrP+Lr34N
o ge
nes
Lr46
Lr
68
Lr34
Lr
46+L
r68
Lr46
+Lr3
4 Lr
68+L
r34
Lr46
+Lr6
8+Lr
34
URUGUAY 2005 to 2007 ARGENTINA 2007
0
10
20
30
40
50
60
70
80
LR Cluster 1 LR Cluster 2 LR Cluster 3
% disease severity
none
only Lr46
only LrP
only Lr34
Lr46+LrP
Lr46+Lr34
LrP+Lr34
Lr46+LrP+Lr34
§ Lr46: no effect
§ Lr68: most effective
§ Lr34: moderate effect
The expression of the slow rusting genes vary under different environments
Environmental effect on LR resistance
Which are the genes and specific gene combinations that are most appropriate to reduce LR in different target environments
Objective
Investigate the presence, relative effects and interactions of durable resistance genes present in Parula on leaf rust severity by using linked molecular markers in two BC1F6 populations in Uruguay
MATERIALS AND METHODS
LE2304*2/Parula: 73 BC1F6 lines – Population 1 ORL99102*2/Parula: 69 BC1F6 lines – Population 2
Resistant donor:
Parula - México (CIMMYT)
Adapted - previously described as susceptible to LR - presence of slow rusting genes: unknown
LE2304 – Uruguay (INIA)
ORL99192 – Brazil (OR-Sementes)
Lr34, Lr46, Lr68 and Sr2 ( Singh et al., 2011; Herrera-Foessel et al., 2012)
Plant Material
Phenotypic characterization of LR in the field
Plots : 1m row Spreaders rows P. triticina race : TFT-10,20
Virulent to: parents, Lr14b, Lr27+31 (seedlings)
Lr13 (adults)
Two locations: § La Estanzuela
(34.3° S, 57.7° W, 70 masl)
§ Young (32.7° S, 57.6° W, 76 masl)
Experimental design: § incomplete
augmented block with two reps
§ nine repeated checks
Phenotypic characterization of LR in the field § Disease severity (DS) was scored using the Modified Cobb Scale § Four DS scores were taken every
7 to 14 days § The area under the disease
progress curve (AUDPC) was calculated using the following equation:
Peterson et al. 1948
AUDPC= ∑n i=1 [(LRSi + LRSi+1)/2] × (ti+1 – ti)
Genotypic characterization with molecular markers
CIMMYT Protocols, 2005
APR gene
Primer name Marker Type Reference
Lr34 csLV34 + LR34PLUS STS Lagudah et al. 2006; 2009
Lr46 csLV46G22 CAPS (BspEI ) Lagudah, pers comm
Lr68 cs7BLNLRR CAPS (HaeIII ) Herrera-Foessel et al. 2012
Sr2 csSr2 CAPS (BspHI) Mago et al. 2010
Statistical analysis
Mixed model – Software R (Package LME4): LR AUDPC means Locations, genotypes and days to heading: fixed effects
§ LR underestimated on late maturing genotypes
Block and replication: random effects
Linear model: § Gene individual effect § Gene interaction
§ Population x gene interaction
Contrasts:
§ among average LR AUDPC per gene combination (class) § p-value <0.05
RESULTS AND DISCUSSION
Susceptible checks and parents
§ S checks: AUDPC 4500 or higher
§ Parula: highly resistance
§ LE2304: intermediate
§ ORL99192 : susceptible
fDS: final disease severity
AU
DP
C_D
S
Thatcher Avocet Lalbahadur PARULA LE2304 ORL99192
fDS 89%
fDS 99%
fDS 99%
fDS 5%
fDS 58%
fDS 93%
Genotypic characterization: parents
§ LE2304*2/Parula: Lr68 and Sr2 polymorphic
§ ORL99192*2/Parula: Lr34, Lr68 and Sr2 polymorphic
+ positive allele, - negative allele
§ Parents: Lr46 present. Monomorphic
Genotypic characterization: Classes
Four genotypic classes
Eight genotypic classes
11 31 53 50
38 65 82 80 55 63 92 88
% fDS
Average AUDPC: 1324 fDS: 31%
Average AUDPC: 2640 fDS: 71%
fDS: final disease severity
ANOVA – Linear model
Contrasts: LE2304*2/Parula
§ Sr2 alone: no effect
§ Lr68 alone: 30% ALRR § Lr68 + Sr2: 47% ALRR
0 20
00
4000
a
b
a
c
AU
DPC
_DS
Effect of single genes and gene combinations on LR AUDPC
Lr34 present in all lines
ALRR: AUDPC LR reduction
Contrasts: ORL99192*2/Parula
§ Sr2 alone: - 8% ALRR
§ Lr34 alone: 13% ALRR
§ Lr34 + Sr2: 26% ALRR
§ Lr68, Lr34+68, Lr68+Sr2: 35% ALRR
§ Lr34+68+Sr2: 57% ALRR
0 20
00
4000
60
00
b a
c d
e e e
f
AU
DPC
_DS
Effect of single genes and gene combinations on LR AUDPC
§ Local higher effect of Lr68 than Lr34 in reduction of LR
§ Sr2 genomic region affected LR AUDPC in certain gene combination depending on genotypic background
§ The relevance of combining several slow rusting genes was confirmed
§ Increasing the frequency and combing these genes in new breeding lines will be valuable to increase LR resistance in future Uruguayan cultivars
Conclusions
Future work in Uruguay
§ 2013 data
§ Yellow rust (Toluca, Mexico 2012)
§ INIA-CIMMYT-CSIRO:
• Effect of Lr46: Avocet Lr34 x Avocet Lr46
• Lr68 Mutants
Funding
GRACIAS THANK YOU
Richard García Fernando Pereira Noelia Pérez Ruth Scholz
Phenotypic characterization of seedling infection type
Singh, 2003
Race: TFT-10,20
§ Four lines showed an intermediate score (IT 2) (Roelfs et al.1992)
• LE2304*2/Parula – Lr68+Sr2: 6%DS
• LE2304*2/Parula – Lr68+Sr2: 5%DS
• ORL99192*2/Parula – “no genes”: 74%DS
• ORL99192*2/Parula – Lr34+Sr2: 42%DS
ANOVA – Mixed model
§ G x L: expression of resistance in both locations
§ LR underestimated on late maturing genotypes
Results and Discussion: Locations
La Estanzuela Young
AU
DP
C_D
S
§ LR infection was severe in both locations § Higher disease pressure in Young relative than LE
fDS: 59%
fDS: 64%
fDS: final disease severity
Checks and parents A
UD
PC
_DS
0
20
00
6000
LE2304 ORL99192 PARULA
fDS: 5%
fDS: 58%
fDS: 93%
fDS: 89%
fDS: 64%
fDS: 99%
fDS: 99%
fDS: 96%
fDS: 99%
LE2304*2/Parula ORL99192*2/Parula
AU
DP
C_D
S
Populations
fDS: 71%
AUDPC_DS AUDPC_DS
fDS: 31%