Disease resistance. Leaf rust

Lr47

PCR-specific primers for the T. speltoides S genome allele of Xabc465

DNA extraction: RNAsa treatment is recommended to see the PCR products in agarose gels

Primers sequences:

PS10R:   5' GCT GAT GAC CCT GAC CGG T 3'

PS10L:   5' TCT TCA TGC CCG GTC GGG T 3'

PCR amplification conditions:

1. Denaturing step: 94°, 3 min
2. Touchdown from 70° to 64°. Seven cycles of: denaturation at 94 ° for 30 seconds, in every cycle the annealing temperature steps down  {70, 69, 68, 67, 66, 65, 64 °C} for 30 seconds and extension at 72 ° for 30 seconds
3. 35 cycles of: [94° 30 seconds, 63° 30 seconds, 72° 30 seconds]
4. Extension step: 72° 7 min

Final volume: 25 µl

Final concentrations of the reagents used in the PCR reaction:

• 1X Taq DNA polymerase buffer (Promega)
• MgCl₂: 3.0 mM
• primers: 0.2 µM each
• dNTP: 200 µM each
• DNA input: 120 ng/reaction
• Taq polymerase [5 U/ml]: 0.3 µl/reaction (1.5U/reaction)

Electrophoresis: Agarose 2% gels

Expected PCR product size: 282 bp

Dvorak, J; Mcguire, P E; Cassidy, B. (1988). Apparent sources of the A genomes of wheats inferred from polymorphism in abundance and restriction fragment length of repeated nucleotide sequences. Genome 30(5):680-689

Weining, S; Langridge, P. (1991) Identification and mapping of polymorphisms in cereals based on the polymerase chain reaction. Theoretical and Applied Genetics, 82(2):209-216