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Insect resistance. Hessian fly.

Gene H9

Contributed by Herbert Ohm and Lingrang Kong

Hessian fly resistance gene H9 was recently re-located on chromosome arm 1AS (1, 2).

SCAR marker SOPO05909

This PCR marker was developed by Kong et al. (1). SOPO05909 maps at a distance of 2.8 cM from H9

Primers

SOPO05-F  5’- CCC AGT CAC TCA TAT GCT ACC TAT -3’  
SOPO05-R  5’- CCG AGT TGA TAT GCA CGA TG -3’ 
 	

PCR reaction mix

  • H2O: 11 µl
  • 10× PCR Buffer: 2.5 µl
  • dNTPs (2 mM): 2.5 µl
  • MgCl2 (25 mM): 1.8 µl
  • Forward primer (2.5 µM): 2.5 µl
  • Reverse primer (2.5 µM): 2.5 µl
  • Taq: 0.2 µl
  • DNA: 2 µl (~50-100 ng)

Total reaction volume: 25 µl

PCR conditions

  • 35 cycles [ 94°C for 30 sec; 56°C for 45 sec; 72°C for 60 sec]
SCAR-H9 marker analysis in an F2 population. The band (909 bp) is present in resistant plants that are homozygous or heterozygous for resistance gene H9. Br- resistant bulk, Bs- susceptible bulk, Pr- resistant parent, Ps- susceptible parent, R- resistant F2 plants, S- susceptible F2 plants

Microsatellite markers

Liu et al. (2) determined that microsatellite loci Xbarc263 and Xcfa2153 flank H9 and H10 genes within a range of 0.3-0.5 cM. H11 is linked to flanking markers Xcfa2153 and Xbarc263 at 0.3 cM and 1.7 cM, respectively. The germplasm used as sources of Hessian fly resistance genes for this study were, Iris (H9), Ella (H9, ref. 10), Elva (H9 and H10), Joy (H10), Karen (H11).

Locus Xbarc263-1A

Primers
BARC263-L   5'- GGA AGC GCG TCA GCA CTA GGC AAC -3' 
BARC263-R   5'- GGC TTC TAG GTG CTG CGG CTT TTG TC -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, 5 min
  • Amplification step (35 cycles)
    • 94°C 40 sec
    • 55°C 40 sec
    • 72°C 60 sec
  • Extension step: 72°C 10 min
Expected products

The PCR products were separated on 3% agarose gels. The band sizes for different parents are shown in the table below.

Locus Xcfa2153-1A

Primers
CFD132-F:  5'- TTG TGC ATG ATG GCT TCA AT -3'
CFD132-R:  5'- CCA ATC CTA ATG ATC CGC 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 (30 cycles)
    • 94°C 30 sec
    • 60°C 30 sec
    • 72°C 30 sec
  • Extension step: 72°C 10 min
Expected products

The PCR products were separated on 3% agarose gels. The band sizes for different parents are shown in the table below.


    allele sizes
marker distance resistant parent susceptible parents
Xbarc263 0.3 cM to H9
0.3 cM to H10
1.7 cm to H11
  H9: 220-bp
H10: 220
H11: 210
210-bp or null
Xcfa2153 0.5 cM to H9
0.5 cM to H10
0.3 cM to H11
  H9: 180
H10: 180
H11: 195
210-bp or null

RAPD marker

For the H9 locus two RAPD markers are available H9-1 and H9-2. The first requires two primers (OPA09 and OPA17), while for the second only one primer is used (OPO05).

Primers

H9-1: OPA09: 5'- GGG TAA CGC C -3' ;  OPA17: '5'-GAC CGC TTG T -3'

H9-2: OPO05: 5'- CCC AGT CAC T -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. Molecular mapping determines that Hessian fly resistance gene H9 is located on chromosome 1A of wheat. Kong L, Ohm HW, Cambron SE, Williams CE. In: Plant Breeding, 2005, 124, 525-531. DOI:10.1111/j.1439-0523.2005.01139.x

2. H9, H10, and H11 compose a cluster of Hessian fly-resistance genes in the distal gene-rich region of wheat chromosome 1AS. Liu XM, Fritz AK, Reese JC, Wilde GE, Gill BS, Chen MS. In: Theoretical and Applied Genetics, 2005, 110:1473-1480. DOI:10.1007/s00122-005-1982-z

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