Quality traits. Waxy mutants

Codominant markers for the normal and null allele of the Wx-D1 locus

Baihuomai is a Chinese landrace with a null allele, Wx-D1b, at the Wx-D1 locus. This line could be the same as Bai-Huo, which we are using for some of our breeding programs. Vrinte et. al (7) and Shariflou et al. (10) developed markers for this allele. Both methods are described below.

PCR protocol for the marker developed by Shariflou et. al (10)

Primers:

     Wx-D1-2-F	5'-ACA GGA TCT CTC CTG GAA G-3'
     Wx-D1-2-R 	5'-GCA AGG AAA ATA GTG AAG C-3'

• primers: 0.5 µM each
• MgCl₂: 1.5 mM
• dNTP: 200 µM each
• DNA: 100 ng/reaction
• Taq Polymerase (Promega): 1U/reaction (0.2µl/reaction)

final volume: 25 µl

• Denaturing step: 95ºC, 3 min
• Touchdown step (8 cycles):
• 94ºC, 1 min
• 63ºC to 55ºC 1ºC/cycle, 1 min
• 72ºC, 1 min
• Amplification step (33 cycles):
• 94ºC, 1 min
• 55ºC, 1 min
• 72ºC, 1 min
• Extension step: 72ºC, 5 min
• Hold at 4ºC

The null allele of cultivar Bai-Huio yields a band of 279 bp, while the amplification product from the normal allele is larger (see figure).

PCR protocol for the marker developed by Vrinte et. al (7)

Primers:

     Wx-D1-1-F	5'-GAG ATG GTC AAG AAC TGC AT-3'
     Wx-D1-1-R 	5'-TAG TGC GTC CAG ACT CAC AG-3'

• primers: 0.5 µM each
• MgCl₂: 1.5 mM
• dNTP: 200 µM each
• DNA: 100 ng/reaction
• Taq Polymerase (Promega): 1U/reaction (0.2µl/reaction)

final volume: 25 µl

• Denaturing step: 95ºC, 3 min
• Touchdown step (8 cycles):
• 94ºC, 1 min
• 65ºC to 57ºC 1ºC/cycle, 1 min
• 72ºC, 1 min
• Amplification step (25 cycles):
• 94ºC, 1 min
• 55ºC, 1 min
• 72ºC, 1 min
• Extension step: 72ºC, 5 min
• Hold at 4ºC

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