1. Molecular Markers for the Oat Stem Rust Resistance Gene Pg16. Peter Eckstein 1, Tom Fetch 2, Donna Hay 1, Tom Zatorski 1, Brian Rossnagel 1, Graham Scoles 1 1 Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, SK, CANADA, S7N 5A8 2 Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB, CANADA, R3T 2M9 References: Brown, P.D., Forsberg, R.A., McKenzie, R.I.H., and Martens, J.W. 1986. The use of disomic alien addition lines in the transfer of oat stem rust resistance to hexaploid oats. In: Lawes, D.A. and Thomas, H. (eds.). Proc. 2 nd International Oat Conference, Dordrecht, Netherlands. pp 16-20. Results Five RAPD markers co-segregated perfectly with Pg16 type stem rust resistance. A robust PCR based marker for Pg16 has been developed for use in MMAS. Segregation ratios for the disease reaction in both crosses did not support single gene disease resistance. However, perfect linkage with a number of markers, and the fact that all of the cis linked fragment amplified only from lines carrying Pg16, suggests a single resistance gene located on a segment of unique DNA introgressed (inserted) from A. barbata. Slightly impeded transmission of the gene has been previously noted by Brown et al (1986). Segregation ratios for disease reaction in OT399 x OT2030 may have been further skewed through previous selection for a number of agronomic traits. Attempts to locate the gene to an oat genetic map have failed since none of the polymorphic fragments were amplified in either the Kanota/Ogle or the Marion/Terra mapping populations. Attempts to design a co-dominant marker, or a marker for the alternate allele, have failed since the six polymorphic fragments failed to amplify from DNA other than from resistant lines (W99162, OT2030). Materials, Methods Introduction Plant Materials W99162 (resistant) x Ronald, 162 F 4 derived F 5 RILs. Ninety-three lines involved in marker discovery, 69 additional lines used for linkage confirmation. OT399 x OT2030 (resistant), 58 F 5 RILs used for linkage confirmation. PCR Conditions for Pg16-1100 20 mM Tris-HCl, 50 mM KCl, 2 mM MgCl 2, 400 µM dNTP, 200 nM each primer, 1 unit Taq polymerase, 50 ng template DNA. Cycling; 94ºC (3 min), followed by 35 cycles of 94ºC (30 sec), 57ºC (45 sec), 72ºC (60 sec). PCR Primers Random 10-mer primers originated from the University of British Columbia (UBC) and Operon Corporation (O series). SCAR primer sequences; 5’ - GGACCCTTACATCAC 5’ - GGACCCTTACTCTGG Table 1. Polymorphic fragment sizes for five random primers and the Pg16- 1100 SCAR marker, and their segregation (+ = present, - = absent) among progeny of W99162 x Ronald. Homo.R and Homo.S = lines homozygous for each disease reaction, Seg = lines segregating for the disease reaction. Table 2. Segregation, and concordance with the disease reaction, of marker Pg16-1100 among progeny of W99162 x Ronald (162 RILs) and OT399 x OT2030 (58 RILs). Homo.R and Homo.S = lines homozygous for each disease reaction, Seg = lines segregating for the disease reaction. The marker indicated genotype (+ or -) for Pg16-1100 is shown on the lower line. The ratio of homozygous resistant to homozygous susceptible lines deviated from the expected (1:1) with a predominance of susceptibles and a greater than expected number of segregating lines in both crosses (Tables 1 and 2). Five 10-mer primers (OPB20, UBC110, UBC172, UBC297, UBC331) amplified six polymorphic fragments (Figure 1). All polymorphic fragments amplified from DNA of the resistant parent (+) and failed to amplify from the susceptible parent (-), and all co-segregated in W99162 x Ronald. All polymorphic fragments showed 100% linkage (cis) with the disease reaction of lines involved in marker discovery that were homozygous for resistance/susceptibility (Table 1). Linkage between SCAR marker Pg16-1100 and resistance was identical to that of the RAPD fragments (Table 1). Linkage of SCAR marker Pg16-1100 with disease resistance in additional lines from W99162 x Ronald, and OT399 x OT2030 was absolute (Table 2). All polymorphic fragments were isolated from W99162 and sequenced. None were similar to each other. The 1100bp OPB20 fragment was chosen for conversion to a PCR-based MMAS friendly marker. Primers complementary to the ends of the fragment produced a robust single band from DNA of resistant genotypes, while failing to amplify fragments from susceptible genotypes (Figure 2). Discussion and Summary Marker DiscoveryRAPD analysis of bulked-segregant DNA ( resistant and susceptible DNA bulks, each from eight constituent lines). Puccinia graminis Pers. f. sp. avenae Eriks.& E. Henn. is the causal agent of stem rust which occurs commonly in Manitoba and eastern Saskatchewan. Infection level depends largely on the amount, race structure, and time of arrival of inoculum carried on southerly winds from the USA. Several genes conferring resistance have been identified of which Pg16 is effective against many races of Puccinia graminis f. sp. avenae, including race NA67. This gene was stably introgressed into hexaploid oat from Avena barbata Pott. collection D203 by Brown et al (1986), and is the same source of Pg16 resistance used in current breeding. Individual stem rust resistance genes are often difficult to screen for since their effects may be masked by the presence of others. The ability to screen using molecular markers-assisted selection (MMAS) is therefore desirable. OPB20 UBC110 UBC172 UBC297 UBC331 * * * * * * Ronald W99162 R W Figure 1. Composite image of banding patterns generated by five random primers on DNA of Ronald and W99162. Polymorphic fragments are identified by an asterisk. RonaldW99162 8 Resistant RILs 8 Susceptible RILs OT399 OT2030 1 Kb Figure 2. Banding pattern of SCAR marker Pg16-1100. A single fragment of 1058bp is amplified (+) from DNA of lines carrying Pg16 (resistant). PhenotypingTwelve plants per RIL were seeded in 5” fibre pots and grown in a greenhouse at 22 +/- 2°C, 14 hour photoperiod. Seven day old seedlings were inoculated with a 5 mg/ml urediniospore suspension of P. graminis f. sp. avenae race NA67, using a rust inoculator and air pump. Inoculated plants were placed in a dew cabinet set at 19°C with near 100% humidity and dark for 16 hours, then returned to the greenhouse. Disease severity was rated at 13 days post-inoculation, on a 0-4 scale where 0-2 is indicative of low infection type (IT) (resistance) and 3-4 of susceptibility (Gordon, 1933). Acknowledgements: We would like to thank our industry partners for their continued support of oat research; Pepsi-Quaker Tropicana Gatorade Canada Can-Oat Milling Cargill Inc.