Quality traits. Waxy mutants

PCR markers for detecting the null alleles of waxy genes (4)

McLauchlan et al. published a paper reviewing and validating a number of PCR markers for the different alleles of the waxy genes (4).

Primer sets #3 and #4 amplify all the three loci and can be used to detect null alleles. They amplify fragments located on exons 4 and 6. Primer pair #4 was used in a waxy wheat breeding program (4). 

For the breeding program at UC Davis we tested primer pairs #3 and #4. We obtained better and more reproducible results with pair #4. For scoring this marker the authors use radioactive primers and the products are analyzed with a capillary sequencer. As a cheaper alternative for non high-throughput programs we tried agarose 3% and the bands can be clearly discriminated.

This PCR protocol was originally described in Ref. 4.

     Left primer:  5'-AAG AGC AAC TAC CAG T-3'
     Right primer: 5'-TCG TAC CCG TCG ATG AAG TCG A-3'

The MgCl₂ concentration is 1.5 mM.

PCR program:

• Denaturing step: 95ºC, 3 min
• Touchdown step:
  • 94ºC, 1 min
  • 64ºC to 58ºC (1ºC/2cycles), 1 min
  • 72ºC, 1 min
• Amplification step (25 cycles):
  • 94ºC, 1 min
  • 58ºC, 1 min
  • 72ºC, 30 sec
• Extension step: 72ºC, 5 min
• Hold at 4ºC

More information for these markers can be found on ref. 4.

Some labs reported problems to reproduce this protocol. Recently, Marcelo Helguera (mhelguera@correo.inta.gov.ar) introduced a few modificatons that yield stronger bands:

• The MgCl₂ concentration was increased to 3mM.
• The amplification step has 35 cycles at 58ºC, instead of 25 cycles.
• Final primer concentration: 0.2 µM each.

The picture on the left shows a typical result for the modified protocol. Gamenya and Haelberg are WxB1b lines.

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