PCR Reagents and Conditions

Protocol S1

PCR reagents and conditions

Bacterial Artificial Chromosome Contig Development. Reagents for screening included 1 ml DNA (50ng/ml), 1 ml 10´ PCR Buffer, 0.5 ml 2mM dNTPs, 0.3 ml MgCl2 (50mM), 0.15 ml each primer (50 mM) and 0.05 ml Taq DNA polymerase (Invitrogen #10342-053, Carlsbad, CA). PCR conditions were 2 min at 94°C followed by 35 cycles (45 s at 94°C, 30 s at the appropriate annealing temperature, and 45 s at 68°C) and a final incubation for 2 min at 72°.

Amplification of Rpp4 candidate genes from the resistant genotype (PI459025B).PCR reagents for amplification included 1 ml PI459025B DNA (50ng/ml), 1 ml 10´ PCR Buffer, 0.5 ml 2mM dNTPs, 0.3 ml MgCl2 (50mM), 0.15 ml each primer (50 mM) and 0.05 ml Taq DNA polymerase (Invitrogen # 10342-053). PCR conditions were 2 min at 94°C followed by 35 cycles (45 s at 94°C, 30 s at 55°C, and 1 min at 68°C) and a final incubation for 2 min at 72°C. PCR products were cloned according to the manufacturer's recommendations using a TA cloning kit (Invitrogen # K4500-01) and One Shot TOP10 Chemically Competent E. coli (Invitrogen # C404003).

SSR marker development and screening.Primers were tested by both a touchdown method (4 min at 95°C followed by 10 cycles of 30 s at 95°C, 30 s at 65°C (-1°C /cycle), and standard PCR method (1.5min at 72°C followed by 10 cycles of 30 s at 95°C, 30 s at 55°C and 1.5min at 72°C). Six primer pairs, polymorphic between PI459025B (Rpp4) and BRS184 (rpp4), were mapped using bulk segregant analysis in an F2:3population as previously described by Silva et al. (2008).

Expression analyses of the Rpp4 candidate genes in resistant and susceptible interactions with ASR.Cycling conditions for semi-quantitative PCR included 45 min at 42°C for reverse transcription, 10 min at 95°C to disable any remaining Superscript, then 40 cycles of 30 sec at 95°C, 1 min at 55°C, 30 sec at 72°C. The second step of RT-PCR using the Ambion RETROscriptâ (Applied Biosystems #AM1710) was completed using the following components: 1 µl cDNA, 2 µl 10´ Buffer, 2 µl (2mM) dNTP, 1.2 µl (50mM) MgCl2, 0.3 µl each primer (50µM), 0.15 µl Platinum Taq (Invitrogen # 11304-011), and 14.5 µl water for a 20µl reaction. Reaction conditions were 2 min at 94°C followed by 35 cycles (45 s at 94°C, 30 s at 55°C, and 1 min at 68°C) and a final incubation for 2 min at 72°C.