Disease resistance. Leaf rust

Lr47

Alternative codominant PCR marker

A single PCR reaction including three primers at different concentrations can be used to generate a codominant marker for the Lr47 chromosome segment. It is recommended to adjust first the annealing temperature to each specific laboratory condition, by searching for the temperature that results in similar fragment intensities in a heterozygous individual (F1). 

   PCAPSR:  5'- CGT GAG ACT CGC CGT TAC CTT G -3'
   PS10L:   5'- TCT TCA TGC CCG GTC GGG T -3'
   PS10L2:  5'- GGG CAG GCG TTT ATT CCA G -3'

1. Denaturing step: 94° 3 min
2. 40 cycles of: [94° 45 seconds, 61° 30 seconds, 72° 30 seconds]
3. Extension step: 72° 7 min

Final volume: 25 µl

• 1X Taq DNA polymerase buffer (Promega)
• MgCl₂: 1.5 mM
• Primers:
  • PCAPSR 0.2 µM
  • PS10L 0.4 µM
  • PS10L2 0.1 µM.
• dNTP: 200 µM each
• DNA input: 120 ng/reaction
• Taq polymerase [5 U/ml]: 0.2 µl/reaction (1 U/reaction)

Conditions presented here should be consider only as a starting point of the PCR optimization for individual laboratories.

Expected digested product size: 224 (S allele) and 394 bp (A Allele). 

Lanes 1 and 5 are bread wheat varieties 'Kern' and 'Neepawa'. Lane 2: Isogenic line of Kern homozygous for Lr47. Lane 3: molecular marker. Lane 4: heterozygous line from the backcrossing program.