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

§ 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%