Anther culture for doubled haploid (DH) production Stem rust resistance in barley: complexity of introgressing rpg4/Rpg5 into TTKSK-susceptible germplasm Javier Hernandez1, Brian J. Steffenson2, Kelly Vining1, Tanya Filichkin1, Scott Fisk1, Laura Helgerson1, Brigid Meints1, David S. Marshall3 and Patrick M. Hayes1, 1Oregon State University, Corvallis, OR, 2University of Minnesota, St. Paul, MN, 3USDA-ARS, Raleigh, NC Introduction Stem rust (Puccinia graminis f. sp. tritici, Pgt) race TTKSK is a major threat to barley because most cultivars are susceptible. Rpg1, a widely-used gene which confers resistance to most North America races, is not effective against TTKSK. The rgp4-mediated resistance locus (RMRL), which confers resistance to TTKSK, was discovered in Q21861. At this complex locus, which maps to 5H, there are at least four genes related with resistance: of these, polymorphism at Rpg5 is most commonly used to identify resistant and susceptible germplasm. Introgression of rpg4/Rpg5 into diverse genetic backgrounds is a first line of defense against the eventual arrival of TTKSK to the Americas. Marker assisted selection (MAS) could accelerate this process. Materials and Methods Steffenson et al 2017. TTKSK Resistant (rgp4/Rpg5) Lines N= 119 Cycle 1 Population F1’s TTKSK Susceptible Lines (Food, Malt, Feed) Genotyping Barley 9K iSelect SNP Chip Genotyping Rpg1 and Rpg5 Re-sequencing Rpg5 Phenotyping Stem rust race TTKSK rated as Infection Type (IT) Plant Growth Biosafety Level – 3 Facility, UMN Seedling stage IT converted to Coefficient of Infection (CI) Anther culture for doubled haploid (DH) production Results TABLE 1. Parental accessions reaction to seedling inoculation with Pgt race TTKSK TABLE 3. Chi-square tests for allele-specific markers targeting Rpg5 and Rpg1 in the TTKSK Cycle I population rpg4/Rpg5 Fig. 1. GWAS using CI means in Cycle I. The continuous line indicates the 5% FDR. The dotted line indicates the coincident threshold levels at FDR 10% using all SNPs and at 5% using a subset of SNPs. Line IT-M (GR) CI Genotype MC0181-11 210; (R) 2.5 Rpg1, rpg4/Rpg5 MC0181-31 3- (MS) 3.3 SH98076 0; (R) 0.5 SB97197 23- (MR) 3.2 TR02272 1-20; (R) 1.8 Full Pint 2;3- (S) 3.4 ------, ------------- 04_028_36 33+ (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 Tibet 37   # DH lines 1:1 Type Rpg5 rpg5 null χ2 Prob (> χ2) Rpg1 rpg1 Resistant 21 9 2 10 Susceptible 43 40 13 51 24 Total 64 53 1.03 0.31 60 57 0.1 0.78 5H QTL 7H QTL TABLE 4. AOV for main and interaction effects for race TTKSK Source of variation Df Sum of squares F value Rpg5 1 2.18 8.17** QTL5 8.57 34.31*** QTL7 1.05 4.20* Rpg5*QTL5 5.73 22.94*** Rpg5*QTL7 2.12 8.48** Residuals 113 28.22   Resistant donors: all Rpg1 and Rpg5 Overall: 1(R) : 3(S) ratio: Two unlinked loci All R lines Rpg5. Some S lines are Rpg5 Rpg1 not related to phenotype Rpg5 + QTL alleles at 5H and 7H: Lowest CI. Fits 1(S) : 3(R) ratio Significant interaction between Rpg5 and other two QTL alleles TABLE 2. Classification of DHs as resistant or susceptible based on a threshold of CI = 2.8. Chi-square tests for CI.   # DH lines 1:1 1:3 Pedigree Total R S χ2 Prob (>χ2) All 110 22 88 38.76 0.00 1.35 0.24 MC0181-11 x__ 13 4 9 1.92 0.16 0.23 0.63 SH98076 x__ 57 15 42 12.79 0.05 0.81 SB97197 x__ 40 3 37 28.90 6.53 0.01 Conclusions In the Cycle I genetic backgrounds The rpg4/Rpg5 complex is necessary, but not sufficient, to confer resistance to TTKSK Rpg1 is not related with resistance to race TTKSK At least two QTLs interact with rpg4/Rpg5 to confer resistance Effective MAS for rpg4/Rpg5-mediated TTKSK resistance requires consideration of genetic backgrounds and phenotypic validation Fig. 2. Multi-locus haplotype at Rpg5 and QTL on 5H and 7H for CI. “+” and “-” refer to resistance and susceptibility alleles, respectively. Bars represent the mean for each haplotype plus or minus standard error. Bibliography Arora et al. 2013. Brueggeman et al. 2002. Mascher et al. 2017. Steffenson et al. 2009, 2017. Wang et al. 2013. Financial support: USDA-NACA Awards 58-2072-5-037 and 58-2050-6-005.