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Insect resistance. Hessian fly.

Gene H13

Contributed by Herbert Ohm and Lingrang Kong

Microsatellite markers

H13 is a dominant resistance gene with a high and stable level of resistance against many Hessian Fly biotypes. Gene H13 was previously mapped in chromosome arm 6DL (1). However, Liu et al.(2) recently determined that its location is on the distal part of chromosome arm 6DS. In a population of 192 F2:3 families, microsatellite locus Xcfd132 is completely linked to H13.

In case of lack of polymorphism for Xcfd132, a close marker is microsatellite locus Xgdm36, located 2.7 cM distal to H13. Microsatellite locus Xgdm36 is located in a distal position close to H13. The amplification product from the resistant parent has a 170-bp size, and the susceptible parent amplifies a 130-bp band. Two other flanking markers useful for breeding are Xcfd42 and Xgdm141.

    allele sizes
marker distance resistant parent susceptible parent
Xgdm36 2.7 cM, distal 170-bp, 130-bp
Xcfd42 5.8 cM, distal 180-bp, 170-bp
Xgdm141 6.0 cM, proximal 135-bp, 130-bp

Other susceptible parents can yield products of different sizes, or not be polymorphic with Molly. For more details and other markers, see the paper of Liu et al. (2).

Locus Xcfd132-6B

Primers
CFD132-F:  5'- CAA ATG CTA ATC CCC GCC -3'
CFD132-R:  5'- TGT AAA CAA GGT CGC AGG TG -3'
Final concentrations of the products used in the PCR reaction:
  • 1X Termophilic DNA polymerase buffer (10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100)
  • dNTP: 0.2 mM each
  • MgCl2: 1.8-2.0 mM
  • primers: 0.4 µM each
  • Taq polymerase: 1 U 9Promega)
  • DNA input: 100 ng/reaction

Final volume: 25 µl

PCR conditions (times for each step might vary according to the Thermocycler model):
  • Denaturing step: 94°C, 3 min
  • Amplification step (40 cycles)
    • 94°C 1 min
    • 55°C 1 min
    • 72°C 2 min
  • Extension step: 72°C 10 min
Expected products

The PCR reaction amplifies a band of 160-bp from the resistant parent (Molly), and a 160-bp band from the susceptible parent, PI 372129.

RAPD marker

The RAPD primer UBC Set 4,399 amplifies a region linked to H13.

Primer

H13: UBC Set 4,399: 5'- TTG CTG GGC G -3'

PCR conditions

Reaction mixture (50 µl total volume):

  • Tris-HCl ( pH9.0 at 25°C): 10 mM
  • KCl: 50 mM
  • MgCl2: 1.8 mM 
  • dATP, dTTP, dCTP, and dGTP: 200 µM each
  • 10-mers primer: 0.2 mM  (singly, or when two primers were used together, a concentration of 0.2 mM of each)
  • Template DNA: 100ng
  • Taq DNA polymerase: 2.0 units

The reaction mixture was overlaid with 50 µl of light mineral oil if needed. Amplifications were carried out in a MJ Research PTC-100 thermocycler programmed for 40 cycles of 1 min at 94°C, 50 s at 36°C, 1 min at 72°C, and ending with 6 min at 72°C. The primers were used singly and in pairwise combination. The PCR products were fractionated either on 1.2% agarose gels in 0.5 × TBE buffer or by denaturing gradient-gel electrophoresis in 12% polyacrylamide with a denaturant gradient range of 10-50%.

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

References

1. Chromosomal mapping of Hessian fly-resistance gene H13 in the D genome of wheat. Gill BS, Hatchett JH, Raupp WJ. In: Journal of Heredity, 1987, 78(2):97-100. [link]

2. Hessian fly resistance gene H13 is mapped to a distal cluster of resistance genes in chromosome 6DS of wheat. Liu XM, Gill BS, Chen MS. In: Theoretical and Applied Genetics, 2005, 111: 243-249. DOI:10.1007/s00122-005-2009-5

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