Disease resistance. Stem and Leaf Rust Resistance.

Sr39 / Lr35

Contributed by J. Douglas Procunier

Agriculture & AgriFood Canada

PCR-based marker for Sr39/Lr35

Sr39/Lr35 is tightly linked to the SCAR marker

   Sr39F :    5'- AGA GAG AGT AGA AGA GCT GC -3'
   Sr39R :    5'- AGA GAG AGA GCA TCC ACC -3'
   

• Genomic DNA: 40-60 ng
• Primers: 125 ng each
• dNTPs: 250 µM each
• Taq DNA polymerase: 2.5 U
• 1x PCR buffer (10 mM Tris-HCl, pH 8.3)
• KCl: 50 mM
• MgCl₂:1.5 mM

Total volume: 50 µl

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

Electrophoresis in 1.4% agarose gels and ethidium bromide staining was used to resolve the 900 bp amplification product.

This SCAR primer pair amplified a single 900 bp band from DNA of the resistant plants, but no amplification occurred with DNA from susceptible plants (Figure 1). Additionally, this SCAR marker was present in the six other wheat lines that carry Sr39/ Lr35 resistance genes on the translocation from Ae. speltoides (Figure 2).

Figure 1. SCAR marker screening of DNA from selected F2 segregants of RL6082/Thatcher cross. RR, homozygous resistant, Rr, heterozygous resistant, rr, susceptible. Far left and right lanes show a 100 base pair marker (BRL Life Technologies). Amplified DNA is resolved by agarose gel (1.4%) electrophoresis and ethidium bromide staining. The fragment of 900 base pairs (bp), indicated by the arrow, was found only in individuals homozygous (RR) or heterozygous (Rr) for the Sr39 resistance gene.

Figure 2. Ethidium bromide-stained 1.4% agarose gel shows SCAR marker presence in several diverse wheat lines bred for Sr39/Lr35 resistance. Far left and right lanes show a 100 base pair marker. Lane a, RL5711 (resistant(R) with Sr39 and Lr35); b, RL5910 (R); c, Marquis K (MqK, susceptible, (S)); d and e, segregants of RL6082 x BW148 (R and S respectively; leaf rust tested); f and g, segregants of RL6082 x BW173 (R and S respectively; leaf rust tested); h, p8810 (R); i, BL31 (S); j and k, segregants of RL6082 x MqK (R and S respectively; stem and leaf rust tested).

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