Gpc-B1 gene from Triticum turgidum ssp. dicoccoides. Stripe rust resistance gene Yr36

Detection of the Gpc-B1 gene from Triticum turgidum ssp. dicoccoides

This section provides experimental details for Xuhw89, a recently developed high-throughput PCR marker that does not require digestion with restriction enzymes. Additional markers, useful for non-polymorphic backgrounds or to reduce linkage drag can be found following this link.

Xuhw89

This PCR marker is located very close (0.1-cM) of the Gpc-B1 gene, and can de used in high-throughput systems (8). It reveals a 4-bp deletion linked to the Gpc-B1 gene from Triticum turgidum ssp. dicoccoides, and is absent in most common and durum wheat lines. None of the 117 varieties included in a screening with Xuhw89 showed the 4-bp deletion (8).

Primers to detect the 4-bp deletion:

   UHW89-BF  5'- TCT CCA AGA GGG GAG AGA CA -3'
   UHW89-R   5'- TTC CTC TAC CCA TGA ATC TAG CA -3'

Final concentrations of the products used in the PCR reaction:

• dNTP: 200 µM each
• primers: 0.2 µM each
• 1X Taq buffer (Mg²⁺ free)
• MgCl₂ 1.5 mM
• Taq polymerase: 2.5 U
• DNA input: 50-100 ng/reaction

Final volume: 25 µl

PCR and restriction conditions:

1. Denaturing step: 5 min at 94°C
2. 37 cycles of: [94°C 30s, 59°C 30s, 72°C 45s]
3. Extension step: 5 min at 72°C

Expected products:

Amplification products were separated on a 6% polyacrylamide gel.

Marker Xuhw89. The red arrowhead shows the band amplified in non Gcp-B1 germplasm (126-bp) . The blue arrowhead shows the position of the fragment containing the 4-bp deletion linked to the Gpc-B1 gene from Triticum turgidum ssp. dicoccoides.

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