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 Must be in Slide Master mode to swap out photos.

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. https://iapreview.ars.usda.gov/Main/docs.htm?docid=9910 Kleinhofs et al, 2009 OREGON STATE UNIVERSITY

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

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

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

Results Cycle 1 Allele specific assays Sequencing Rpg5 in parents Line IT-M (GR)a CIb Genotype MC0181-11 120; (R) 2.5 Rpg1, rpg4/Rpg5 MC0181-31 -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 10.0691 10.1151 ------, 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

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 MC0181-11 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

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

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 542691409 292.7 Kb Disease resistance protein HORVU5Hr1G074400 543253172 268.9 Kb LRR receptor-like ST-K HORVU5Hr1G074410 543253592 269.4 Kb HORVU7Hr1G110200 7 629580430 1.1 Kb Receptor-like protein kinase 5 OREGON STATE UNIVERSITY

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 10.0777 10.0860   Parents Donors Cycle 1 All Resistant, All Rpg5. Donors UC-Davis UC1266 and UC1322 Resistant. UC1231L Susceptible. No amplification for Rpg5 or rpg5. “Susceptible” DH130939 and DH120412 Resistant!. No Rpg5 amplification. 10.0777 Resistant! rpg5 amplification. OREGON STATE UNIVERSITY

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 10.0777 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

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

Thanks!!! Special thanks to: Dr. Brian Steffenson, UMN Dr. David Marshall, USDA-ARS Financial support: USDA-NACA Awards 58-2072-5-037 and 58-2050-6-005.

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

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

Summary of TTKSK resistance experiments Female 04_028_36 10.0691 MC181-11 MC181-31 SB97197 SH98076 Violetta Male MC181-11 10.1151 Full Pint Tibet 37 Oscar 04-0.28_36 Violetta Karma MC0181-31 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 10.0777 DH130939 10.0860 DH130004 DH120412 n 358 Doubled Haploids F1 Anther culture Cycle 2 OREGON STATE UNIVERSITY