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Insect resistance. Hessian fly.

Gene H31

Protocol contributed by Christie Williams and Herb Ohm

The H31 gene was identified in an accession of Triticum turgidum Desf. (durum wheat), CI3984, from Tunisia. Williams et al. (1) transferred this gene to hexaploid wheat, the resulting line was P921696 (PI 633876), a soft red winter wheat. H31 is located near the terminus of chromosome arm 5BS, this is the first Hessian fly resistance gene reported on that arm. The two other genes conferring resistance to biotype L, H9 and H13, were mapped on chromosome arms 5AL and 6DL respectively. Because of the diverse locations of the three loci and the availability of molecular markers for all of them, they are a convenient target for pyramiding.
 

STS marker Xupw4148 (12)

The locus Xupw4148 is located at 3.3 cM from H31. It is not known if this locus is proximal or distal to H31 and there is no other flanking marker available for this gene.

Primers

 STS-E41M48-Forward   5'- TCC TAC CTC CAT TCC CCT TT -3'
 STS-E41M48-Reverse   5'- TCA AAA TGA ATC GGA AGG GT -3' 

PCR conditions

DNA was amplified as follows in a PTC-100 thermal cycler (MJ Research, Waltham, MA).

  • Denaturing step: 4 min at 94°C
  • 35 cycles of: [1 min at 94°C, 1 min at 55°C, 2 min at 72°C ]
  • Extension step: 7 min at 72°C

Final concentrations of the products used in the PCR reaction (50 µl total volume):

  • 10 mM Tris-HCl ( pH9.0 at 25°C)
  • 0.1% Triton X-100
  • 50mM KCl
  • 1.5 mM MgCl2
  • 150 µM of each nucleotide
  • 20 pmols of the STS-E41M48 forward and reverse primers
  • 100 ng template DNA
  • 1.25 units of Taq DNA polymerase (Promega)

Expected products

PCR products were visualized on a standard 1% agarose gel with etidium bromide staining.

Amplification of the H31-linked STS-E41M48 marker.
The ethidium bromide-stained gel contains samples from ten resistant and ten susceptible individual F2 plants. P921696 and Ning 7840 were the parental lines. Lane M is a 100 bp DNA molecular-weight ladder and the figures on the left represent the molecular weight in bp.

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

References

1. Phenotypic assessment and mapped markers for H31, a new wheat gene conferring resistance to Hessian fly (Diptera: Cecidomyiidae). Williams CE, Collier N, Sardesai CC, Ohm HW, Cambron SE. In: Theoretical and Applied Genetics, 2003, 107:1516-1523. DOI:10.1007/s00122-003-1393-y

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