Disease resistance. Leaf Rust Resistance

Lr29 - Lr25

Contributed by J. Douglas Procunier

Agriculture & AgriFood Canada

PCR-based markers for Lr29 and Lr25

SCAR markers for both Lr29 and Lr25 are completely linked to these genes and can be used simultaneously by multiplex touchdown PCR

   Lr29F18   5'- GTG ACC TCA GGC AAT GCA -3' 
   Lr29R18   5'- GTG ACC TCA GAA CCG ATG -3' 
   Annealing: 60 °C

   Lr29F24   5'- GTG ACC TCA GGC AAT GCA CAC AGT -3' 
   Lr29R24   5'- GTG ACC TCA GAA CCG ATG TCC ATC -3' 
   Annealing: 65 °C
   
   Lr25F20   5'- CCA CCC AGA GTA TAC CAG AG -3' 
   Lr25R19   5'- CCA CCC AGA GCT CAT AGA A -3' 
   Annealing: 58 °C
   

• Genomic DNA: 100 ng
• Primers: 125 ng each
• dNTPs: 250 µM each
• Taq DNA polymerase: 2.5 U
• 1x PCR buffer (*)

Total volume: 50 µl
* : 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl₂ , 0.1% Triton X-100
Each tube was overlaid with 50 µl of mineral oil (Sigma).

Various annealing temperatures (58, 60 or 65 °C) were employed in order to retain the polymorphism between the resistant and susceptible lines.

• Denaturing step: 94°C, 3 min
• Amplification step (35 cycles):
  • 94°C, 1 min
  • 58, 60 or 65°C, 1 min
  • 72°C, 2 min
• Extension step: 72°C, 10 min

"Touchdown" PCR, was used to amplify genomic DNA containing both Lr29 and Lr25 resistance genes. Four primers, Lr29F24, Lr29R24, Lr25F20 and Lr25R19 were used in a single PCR. The PCR conditions were as described except that the annealing temperature was 65°C for the first 15 cycles and 58°C for the next 25 cycles.

Amplification products were electrophoresed on 1.2% agarose gels. DNA bands were stained by ethidium bromide and detected under UV light.

Fig. 1. Touchdown PCR for detecting Lr29 and Lr25. The Lr25 amplified band (1800bp) and Lr29 band (900bp) are shown.

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