1. Department of Crop and Soil Science
Stem rust resistance in barley: complexity of introgressing rpg4/rpg5 into TTKSK- susceptible germplasm
Javier Hernandez
Phd student
Patrick Hayes
Professor
Barley project
Oregon State University
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2. Introduction Barley Barley stem rusts
Fourth most important cereal in the world.
Multiple uses / Growth habits / Morphology.
North America ~ 1 million ha.
Barley stem rusts
Puccinia spp. a major threat.
Puccinia graminis f. sp. tritici (Pgt) and f. sp. secalis.
Rpg1 widely used to control many races of Pgt.
New race TTKSK virulent to Rpg1.
95% of barley germplasm tested is susceptible.
rgp4/Rpg5 complex (5H) from Q21861 confers seedling and adult plant resistance.
Kleinhofs et al, 2009
OREGON STATE UNIVERSITY

3. The rpg4/Rpg5 complex Breeding considerations:
Kleinhofs et al, 2009
Breeding considerations:
No systematic introgession.
Marker assisted selection (MAS)?
Potential interaction with genetic background?
Pyramiding with new sources of TTKSK resistance?
OREGON STATE UNIVERSITY

4. Hypotheses
The rpg4/Rpg5 locus will confer resistance to TTKSK in a range of genetic backgrounds.
The Rpg1 gene will interact with rpg4/Rpg5 to confer higher levels of resistance to TTKSK.
There are undiscovered sources of TTKSK resistance.
OREGON STATE UNIVERSITY

5. Summary of TTKSK resistance experiments
Phenotyping
Controlled environment and field.
Multiple diseases.
Genotyping
Infinium iSelect barley 9k SNP array chip and allele specific assay for Rpg5 and Rpg1.
Genotyping and phenotyping
Chi-square and genome wide analysis.
Phenotyping
Controlled environment and field.
Multiple diseases.
Genotyping
Infinium iSelect barley 50k SNP array chip and allele specific assay for Rpg5.
Genotyping and phenotyping
Chi-square and genome wide analysis.
5 Resistant DHs selected
OREGON STATE UNIVERSITY

6. Results Cycle 1 Allele specific assays Sequencing Rpg5 in parents
Line
IT-M (GR)a
CIb
Genotype MC
120; (R)
2.5
Rpg1, rpg4/Rpg5 MC
-3 (MS)
3.3
SH98076
0;-1 (R)
0.5
SB97197
2-3 (MR)
3.2
TR02272
1.8
Full Pint
2;-3 (S)
3.4
------, c
04_028_36
3+3 (S)
4.1
rpg1, Rpg4/rpg5
Violetta
3 (S)
4.0
------, Rpg4/rpg5
Karma
23 (S)
3.7
Oscar
-3 (S)
3.5
------, rpg4/Rpg5 d
Tibet 37
Allele specific assays
Resistant donors: Rpg1 and Rpg5.
Susceptible parents: No Rpg1 or Rpg5.
Oscar exception.
Sequencing Rpg5 in parents
Resistant donors = Q21861 sequence.
Oscar = C insertion causing stop codon.
OREGON STATE UNIVERSITY

7. Results Cycle 1 Chi-square tests Phenotype Genotype
Number of DH lines
1:1
1:3
Pedigreea
Total
Resistant
Susceptible χ2
Prob(>χ2)
All
110 22 88 39
1.4
0.24
MC x__ 13 4 9
1.9
0.16
0.2
0.63
SH98076 b x__ 57 15 42
0.1
0.81
SB97197 x__ 40 3 37 29
6.5
0.01
Chi-square tests
Phenotype
Overall: 1(R) : 3(S) ratio.
Two or more independent genes?
Genotype
1:1 for both markers.
Genotype + Phenotype
All R lines have Rpg5.
Some S lines have Rpg5.
Rpg1 not related with resistance.
Number of DH linesa
1:1
Number of DH lines
Type
Rpg5
rpg5
null χ2
Prob (>χ2)
Rpg1
rpg1
Resistant 21 9 2 10
Susceptible 43 40 13 51 24
Total 64 53 1
0.31 60 57
0.78
OREGON STATE UNIVERSITY

8. Results Cycle 1 Association mapping
rpg4/Rpg5
Association mapping
Significant SNPs on 5H at the Rpg5 region.
Two QTLs: 5H and 7H.
5H QTL and Rpg5 unlinked (~10Mb apart).
5H QTL
7H QTL
OREGON STATE UNIVERSITY

9. Results Cycle 1 Rpg5 + 5H + 7H Candidate genes near QTLs
Rpg5 necessary but not sufficient to achieve resistance.
Rpg5 + 2 QTLs < CI than other combinations.
1(R) : 3(S) ratio.
Candidate genes near QTLs
Gen
Chr bp
Dist
Function
HORVU5Hr1G074260 5
292.7 Kb
Disease resistance protein
HORVU5Hr1G074400
268.9 Kb
LRR receptor-like ST-K
HORVU5Hr1G074410
269.4 Kb
HORVU7Hr1G110200 7
1.1 Kb
Receptor-like protein kinase 5
OREGON STATE UNIVERSITY

10. Results Cycle 2 Parents Donors Cycle 1 Donors UC-Davis “Susceptible”
Name
Type IT CI
Class
Rpg5
rpg5
DH140279
Donor_I
0;1-
0.75
Resistant +
DH140030 0;
0.5
DH140213
1-0;
1.25
DH140512
DH140278
00;
0.13
UC1266
Donor_II
10;
1.63 --
UC1231L
3-2
3.38
Susceptible
UC1322
DH130939
Recurrent
0;1
0.88
DH130004
23-1
3.05
Mod Suscep
DH120412
Parents
Donors Cycle 1
All Resistant, All Rpg5.
Donors UC-Davis
UC1266 and UC1322 Resistant.
UC1231L Susceptible.
No amplification for Rpg5 or rpg5.
“Susceptible”
DH and DH Resistant!.
No Rpg5 amplification.
Resistant!
rpg5 amplification.
OREGON STATE UNIVERSITY

11. Results Cycle 2 Progeny 331 Resistant : 20 Susceptible. Rpg5 x --
All progeny resistant.
New allele?
Some segregation.
Two linked genes?
Rpg5 x rpg5
Expected segregation.
Parents
Progeny
Female
Pheno / Geno
Male N R S
Rpg5
rpg5 --
Obs
DH140512
R / Rpg5 x
UC1322
R / -- 26 21 5
DH140279
DH120412 67 66 1 40 27
1S: --
DH140278
DH130939 46 25
All --: R
R / rpg5 23 13 10
All_rpg5: R
10.086
MS / rpg5 44 39 24 19
All_S: rpg5 / 14R: rpg5
DH130004
MS / -- 75 71 4 34 41
2S:Rpg5 / 2S: --
DH140030
UC1231L
S / -- 36 30 6 15
1S: Rpg5 / 5S: --
UC1266
DH140213 18 16
4S: --
OREGON STATE UNIVERSITY

12. Javier Hernandez, Oregon State University, USA
Stem rust resistance in barley: complexity of introgressing rpg4/rpg5.
Javier Hernandez, Oregon State University, USA
SUMMARY
H1: The rpg4/Rpg5 locus will confer resistance to TTKSK in a range of genetic backgrounds.
The rpg4/Rpg5 locus is necessary but not sufficient for resistance to TTKSK.
H2: The Rpg1 gene will interact with rpg4/Rpg5 to confer higher levels of resistance to TTKSK.
Rpg1 not related with TTKSK resistance.
H3: There are undiscovered sources of TTKSK resistance.
Two QTLs interact with Rpg5 to confer resistance.
1:3 ratio for phenotype and multi-locus model.
Donors from UC Davis: A new resistance allele at Rpg5 or at a tightly linked gene?
“Susceptible” (actually resistant!) parents: a new variant of rpg5 or a tightly linked gene?
QUESTIONS
Cycle 1:
What are the genes responsible for the 5H and 7H QTLs?
Cycle 2:
Is there a new resistance gene tightly linked to Rpg5?
Are there new alleles at Rpg5 and/or rpg5 ?
OREGON STATE UNIVERSITY

13. Thanks!!! Special thanks to: Dr. Brian Steffenson, UMN
Dr. David Marshall, USDA-ARS
Financial support: USDA-NACA Awards
and

14. Questions
Cycle 1:
What are the genes responsible for the 5H and 7H QTLs?
Cycle 2:
Is there a new resistance gene tightly linked to Rpg5?
Are there new alleles at Rpg5 and/or rpg5 ?
OREGON STATE UNIVERSITY

15. Summary Cycle 1 germplasm
The rpg4/Rpg5 locus is necessary but not sufficient for resistance to TTKSK.
Rpg1 not related with TTKSK resistance.
Two QTLs interact with Rpg5 to confer resistance.
1:3 ratio for phenotype and multi-locus model.
Cycle 2:
Donors from Cycle 1: all resistant and have Rpg5.
Donors from UC Davis: A new resistance allele at Rpg5 or at a tightly linked gene?
“Susceptible” (actually resistant!) parents: a new variant of rpg5 or a tightly linked gene?
OREGON STATE UNIVERSITY

16. Summary of TTKSK resistance experiments
Female
04_028_36
MC181-11
MC181-31
SB97197
SH98076
Violetta
Male
MC181-11
Full Pint
Tibet 37
Oscar
_36
Violetta
Karma MC
SH98076
TR02272
Phenotyping
Controlled environment and field.
Multiple diseases.
Genotyping
Infinium iSelect barley 9k SNP array chip and allele specific assay for Rpg5 and Rpg1.
Genotyping and phenotyping
Chi-square and genome wide analysis. F1 n
Cycle 1
123
Doubled Haploids
Anther culture
Phenotyping
Controlled environment and field.
Multiple diseases.
Genotyping
Infinium iSelect barley 50k SNP array chip and allele specific assay for Rpg5.
Genotyping and phenotyping
Chi-square and genome wide analysis.
5 Resistant DHs selected
Female
DH140512
DH140030
UC1266
DH140248
DH140279
Male
UC1322
UC1231L
DH140213
DH130939
DH130004
DH120412 n
358
Doubled Haploids F1
Anther culture
Cycle 2
OREGON STATE UNIVERSITY