This document summarizes research on the effects of adult plant resistance genes in wheat on leaf rust severity in Uruguay. It found that the genes Lr68 and Lr34 individually reduced leaf rust severity by 30% and 13% respectively in one population, while combinations of these genes with Sr2 increased reduction to 47% and 57%. The relevance of combining multiple slow rusting genes to increase resistance was confirmed. Environmental conditions affected gene expression, so identifying appropriate gene combinations for target environments is important. Future work will analyze data from an additional year and location, as well as test other genes and populations.

1. mpsilva@inia.org.uy
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

2. § Introduction
§ Objective
§ Materials and methods
§ Results and Discussion
§ Conclusions
§ Future works
Outline

3. INTRODUCTION

4. 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)

5. 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

6. 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

7. 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
uster
2 LR
Cluster
3
none
only
Lr46
only
LrP
only
Lr34
Lr46+LrP
Lr46+Lr34
LrP+Lr34
Lr46+LrP+Lr34
Nogenes
Lr46
Lr68
Lr34
Lr46+Lr68
Lr46+Lr34
Lr68+Lr34
Lr46+Lr68+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
Nogenes
Lr46
Lr68
Lr34
Lr46+Lr68
Lr46+Lr34
Lr68+Lr34
Lr46+Lr68+Lr34
URUGUAY 2005 to 2007
ARGENTINA 2007
0
10
20
30
40
50
60
70
80
LR
Cluster
1 LR
Cluster
2
%
disease
severity
§ Lr46: no
effect
§ Lr68: most
effective
§ Lr34:
moderate
effect

8. 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

9. 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

10. MATERIALS AND METHODS

11. 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

12. 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

13. 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)

14. 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

15. 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

16. RESULTS AND DISCUSSION

17. Susceptible checks and parents
§ S checks: AUDPC 4500 or higher
§ Parula: highly resistance
§ LE2304: intermediate
§ ORL99192 : susceptible
fDS: final disease severity
AUDPC_DS
Thatcher Avocet Lalbahadur PARULA LE2304 ORL99192
fDS 89%
fDS 99%
fDS 99%
fDS 5%
fDS 58%
fDS 93%

18. 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

19. 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

20. ANOVA – Linear model

21. Contrasts: LE2304*2/Parula
§ Sr2 alone: no effect
§ Lr68 alone: 30% ALRR
§ Lr68 + Sr2: 47% ALRR
020004000
a
b
a
c
AUDPC_DS
Effect of single genes and gene combinations on LR AUDPC
Lr34 present in all lines
ALRR: AUDPC LR reduction

22. 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
0200040006000
b
a
c
d
e ee
f
AUDPC_DS
Effect of single genes and gene combinations on LR AUDPC

23. § 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

24. Future work in Uruguay
§ 2013 data
§ Yellow rust (Toluca, Mexico 2012)
§ INIA-CIMMYT-CSIRO:
• Effect of Lr46: Avocet Lr34 x Avocet Lr46
• Lr68 Mutants

25. Funding
GRACIAS
THANK YOU
Richard García
Fernando Pereira
Noelia Pérez
Ruth Scholz

26. 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

27. ANOVA – Mixed model
§ G x L: expression of resistance in both locations
§ LR underestimated on late maturing genotypes

28. Results and Discussion: Locations
La Estanzuela Young
AUDPC_DS
§ LR infection was severe in both locations
§ Higher disease pressure in Young relative than LE
fDS: 59%
fDS: 64%
fDS: final disease
severity

29. Checks and parents
AUDPC_DS
020006000
LE2304 ORL99192PARULA
fDS: 5%
fDS: 58%
fDS: 93%
fDS: 89%
fDS: 64%
fDS: 99%
fDS: 99%
fDS: 96%
fDS: 99%

30. LE2304*2/Parula ORL99192*2/Parula
AUDPC_DS
Populations
fDS: 71%
AUDPC_DS AUDPC_DS
fDS: 31%