Marker Assisted Selection in Wheat Marker Assisted Selection in Wheat - HOME CSREES-USDA

Quality traits. Grain Texture

Contributed by Gabriela Tranquilli (gtranqui@cirn.inta.gov.ar)

Methods for Pinb-D1 allelels:

Pinb-D1b: To trace the specific allele for the glycine-to-serine mutation (Pinb-D1b) there are two PCR-based markers. The first one, developed by Giroux and Morris (3,4) uses specific primers for the mutation. However, discrimination of these primers is based on a single base pair difference at the 3’ end of the primers and occasional false positives are observed when PCR conditions are not perfectly optimized. To avoid this problem a codominant PCR-CAPs marker, based on the same point mutation, was developed (12).
This PCR-CAPs marker is used in this Marker Assisted Selection Program.

After amplification of a 447-bp segment of the Pinb-D1 gene using the primers designed by Gautier et al. (11), the PCR product is digested with the restriction enzyme Bsr BI. This enzyme recognizes the sequence with the glycine-to-serine mutation but not the sequence without the mutation present in all soft varieties. After digestion, fragments of 320bp are expected for genotypes lacking the glycine to-serine mutation, while fragments of 200bp are expected for those genotypes carrying the allele for hard texture.


Primers: 
PinB-D1-F:  5'- ATG AAG ACC TTA TTC CTC CTA -3'
PinB-D1-R:  5'- TCA CCA GTA ATA GCC ACT AGG GAA -3'
Final concentrations of the products used in the PCR reaction:
  • 1X Taq buffer (Mg free)
  • dNTP: 250 uM each
  • MgCl2: 1.5 mM
  • primers: 1 ng/ µL each
  • Taq polymerase: 2.5 U
  • DNA input: 50-100 ng/reaction

Final volume: 50 µl


PCR conditions (times for each step might vary according to the Thermocycler model):
  • Denaturing step: 94°C, 3 min
  • Amplification step (37 cycles)
    • 94°C 90 sec
    • 55°C 90 sec
    • 72°C 2 min
  • Extension step: 72°C 10 min
Restriction:

To perform the restriction, 5U of Bsr BI is added to 25µL of the PCR product and incubated at 37 °C for 4hs. No extra buffers are added.


PinB-D1
w = wild-type sequence, m = glycine to serine mutation (allele Pinb-D1b), hard texture, h= heterozygous


Pinb-D1c: A CAPs marker is available to introgress this allele (5). Amplification is performed as indicated for the Pinb-D1b allele. To detect the Pro-60 mutation, the PCR product is digested with 2 U of the restriction enzyme Pvu II.

PinB-D1c
w = wild-type sequence, m = Leucine to proline mutation (allele Pinb-D1c), hard texture, h= heterozygous


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