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Insect resistance. Hessian fly.

Gene H25

Protocol contributed by Ed Souza (esouza@uidaho.edu), Justin Wheeler (jwheeler@uidaho.edu) and Mary Guttieri (mgttri@uidaho.edu)


The H25 gene is derived from rye and conditions resistance to most Hessian fly populations in North America. The hard red winter (HRW) variety KS92WGRC20, developed by the USDA-ARS Manhattan KS, has a rye translocation that carries H25 on wheat chromosome 4A. Other HRW wheat varieties have the rye segment containing H25 translocated to different wheat chromosomes (1). IDO584 is an alternative source of H25 in a spring background. For more information, see the "available germplasm" section on the main page.

Two microsatellite loci located outside the rye translocation but close to it are Xgwm397-4A (7 to 9 cM) and Xgwm610-4A (5 to 6 cM). Since there is limited recombination between any of these loci and the H25 gene, only one marker is enough for MAS.

Microsatellite marker Xgwm610

Primers

    WMS610-L   5’- CTG CCT TCT CCA TGG TTT GT -3’  
 	WMS610-R   5’- AAT GGC CAA AGG TTA TGA AGG -3’ 

The WMS610-L primer was labeled with IRDye 700 for detection in a LI-COR model 4200 sequencer (LI-COR Inc., Lincoln, NE).

PCR conditions

  • Denaturing step: 94°C, 5 min
  • 34 cycles of: [94°C 30 sec, 60°C 30 sec, 72°C 45 sec]
  • Terminal cycle: [94°C 30 sec, 60°C 30 sec, 72°C 5 min]

Products were stored at 4°C.

Sample preparation for gel evaluation

In a new plate, add 1 µl of PCR product to 16 µl of water. Then to each sample add 3 µl of 700 loading dye. Denature plate at 94 °C for 3 min.

Gel evaluation

Load 1 µl sample onto 3% polyacrylamide Licor imaging gel. Run at for 2 hours and 30 min.

Expected products

Segregation of codominant marker Xgwm610 in an F2 population of IDO599/ IDO584. S: homozygous susceptible; R: homozygous resistant; H: heterozygous.

Microsatellite marker Xgwm397

Primers

 WMS397-L  5'- TGT CAT GGA TTA TTT GGT CGG -3' 
 WMS397-R  5'- CTG CAC TCT CGG TAT ACC AGC -3'  

The WMS397-L primer was labeled with IRDye 700 for detection in a LI-COR model 4200 sequencer (LI-COR Inc., Lincoln, NE).

PCR conditions

  • Denaturing step: 95°C, 2 min
  • 35 cycles of: [95°C 1 min, 55°C 1 min, 72°C 1 min]
  • Terminal cycle: [95°C 1 min, 55°C 1 min, 72°C 5 min]

Products were stored at 4°C.

Expected products

PCR reactions were denatured and electrophoresed as described by the manufacturer using a 6% acrylamide gel in a LI-COR model 4200 sequencer.

Denaturing polyacrylamide gel electrophoresis of amplification products

The 168 to 170 bp product of Xgwm397 used as a marker for H25 originates from 'Amigo' wheat chromosome 4A. The Xgwm397 allele from Amigo is closely linked to a translocated chromosome segment from 'Balbo' rye carrying the H25 resistance gene.

References

  1. Amigo (- H25)
  2. Octillo durum (- H25)
  3. IDO415 (- H25)
  4. IDO470 (- H25)
  5. IDO586 (+ H25)
  6. WGRC20 (+ H25)
  7. WGRC41 durum (+ H25)
  8. Suwon 92 (- H25)
  9. TAM W-104 (- H25)
  10. TAM W-106 (- H25)
  11. Balbo rye (+ H25)

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

References

1. Registration of KS92WGRC17, KS92WGRC18, KS92WGRC19, and KS92WGRC20 winter wheat germplasms resistant to Hessian fly. Sebesta EE, Hatchett JH, Friebe B, Gill BS, Cox TS, Sears RG. In: Crop Science, 1997, 37(2):635.

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