Quality traits. Pre-harvest sprouting tolerance (PHS)

Contributed by Anne K. Sturbaum (anne.sturbaum@ars.usda.gov) and Ed Souza (souza.6@osu.edu)

Viviparous-1 alleles for PHS

Yang et al. (11) identified two new allelic variants of Viviparous-1 related to PHS tolerance on chromosome arm 3BL of bread wheat (Triticum aestivum) and designated them as Vp-1Bb and Vp-1Bc. Sequence analysis showed that Vp-1Bb and Vp-1Bc had an insertion of 193-bp and a deletion of 83-bp fragment, respectively, located in the third intron region of the Vp-1B gene. The authors also developed a co-dominant marker, Vp1B3, that amplifies an 845 or 569-bp fragment from many tolerant cultivars carrying the insertion or the deletion, and a 652-bp fragment from the wild type, susceptible lines.

Primers sequences:

  Vp1BF: 5'- TGC  TCC TTT CCC AAT TGG -3'
  Vp1BR:	5'- ACC  CTC CTG CAG CTC ATT G -3'   

PCR amplification conditions:

1. Denaturing step: 94° 5 min
2. 36 cycles of: [94° 1 min, 61° 1 min, 32° 1 min]
3. Extension step: 72° 10 min

Final concentrations of the reagents used in the PCR reaction:

• 0.8 U Taq (GoTaq, Promega)
• MgCl₂: 2.0 mM
• primers: 0.4 µM each
• dNTP: 200 µM each
• DNA input: 100 ng/reaction

Reaction volume: 20 µl

This protocol is very dependent on the PCR equipment. Mark Sorrells (personal communication) indicated that in his laboratory amplification was only detected using an MJ Research Tetrad machine.