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Insect resistance. Hessian fly.

Genes H26 and H32

Contributed by Steven Xu, Guotai Yu, Xiwen Cai, Christie Williams, and Marion Harris

The gene H26 for Hessian fly [Mayetiola destructor (Say)] resistance was found in Aegilops tauschii and introgressed into common wheat (1) and synthetic hexaploid wheat (2, 3). Currently H26 is one of the most effective genes to control populations of Hessian fly that are virulent against several other sources of resistance. Hessian fly populations have been increasing over the last years, and within the US heavily infested wheat fields have been observed more frequently in Texas, Oklahoma, and Kansas (4). Among the 22 resistance genes tested on these fly populations, H26 was one of the few genes that were consistently effective in all of them, providing protection to more than 80% of the plants. In particular, H26 was one of only six genes that were effective against the highly virulent Kay-OK-GH-06 population found at Kay County, Oklahoma. Wang et al. (3) mapped H26 to the deletion bin 3DL3-0.81-1.00 on chromosome 3D, and later Yu et al. (5) developed two EST-based STS markers, Xrwgs11 and Xrwgs12, that map 1 cM distal to the H26 locus. Another marker Xrwgs10, which was designed based on Brachypodium genomic sequence, is 3.2 cM proximal to H26.

The gene H32, another gene for Hessian fly resistance from Ae. tauschii (6), confers resistance to highly virulent Hessian fly populations and biotype L (6). It was transferred into a synthetic wheat line, W7984, which is one of the parents of the ITMI mapping population. The gene was mapped to the deletion bin 3DL3-0.81-1.00 of chromosome 3D (6). Comparative mapping analysis indicated that H26 and H32 are either different Hessian fly-resistance alleles at the same gene locus or two different, but tightly linked H genes. The three STS markers Xrwgs10, Xrwgs11 and Xrwgs12 closely-linked to H26 were also linked to H32 in ITMI population (7). Xrwgs10 and Xrwgs12 flanked the H32 locus with a genetic distance of 0.5 cM and Xrwgs11 co-segregated with H32. Three markers were validated in 13 bread wheat cultivars and an advanced breeding line. The H26 and H32 resistance sources had the same band for each of three markers. The bands of the H26 and H32 resistance sources at three marker loci were different from those in all 13 susceptible bread wheat genotypes (7), demonstrating that the three markers are suitable for marker-assisted selection in breeding for Hessian fly-resistance in wheat.

STS markers

Three STS markers, Xrwgs10, Xrwgs11 and Xrwgs12 (5, 7), are presented here. They are tightly-linked to H26/H32 (Figure. 1).

3DL maps
Figure 1. Partial genetic maps of 3DL harboring H26 in the synthetic wheat population and H32 in the ITMI population.

Primers

Xrwgs10

    rwgs10-F   5'- CCT AAC TGA GGT CCC ACC AA -3' 
    rwgs10-R   5'- GCA AAG GAC TTG ATG CCT GT -3' 

Xrwgs11

   rwgs11-F   5'- GGA GAG TCG CAG GAT CCA -3' 
   rwgs11-R   5'- TCT CTG CCC AGT CCA ACT TT -3' 

Xrwgs12

   rwgs12-F   5'- CGT ATC GGC GAC AAG GTA AT -3' 
   rwgs12-R   5'- ACT GGA AGA AGC CCC AGT CT -3' 
PCR conditions for three STS markers

DNA was amplified as follows in a 9800-fast thermal cycler (Applied Biosystems, Foster, CA).

Segment I

  • Denaturing step: 4 min at 94°C
  • 10 cycles of:
    • 45 sec at 94°C
    • 45 sec at from 60°C to 55°C decreased by 0.5°C per cycle
    • 45 sec at 72°C

Segment II

  • Denaturing step: 45 sec at 94°C
  • 35 cycles of:
    • 45 sec at 94°C
    • 45 sec at 55°C
    • 45 sec at 72°C
  • Extension step: 7 min at 72°C
Final concentrations of the products used in the PCR amplifications (12 µl total volume):
  • 1X buffer (Qiagen);
  • 1.5-2.0 mM MgCl2
  • 100 µM of each nucleotide
  • 0.5 µM of the forward and reverse primers
  • 1.0 Unit of Taq DNA polymerase (Qiagen)
  • 10ng/µl template DNA
Expected products

PCR products were separated on a 6% poly-acrylamide gel in TBE buffer. Images were taken after staining with GelRed (Biotium, Inc. Hayward, CA).

Xrwgs10
Figure 2. Gel image for marker Xrwgs10. L: DNA Ladder; 1: SW8 (H26donor); 2: Newton (recurrent parent); 3 - 10: Newton/SW8/2/Newton BC2F1 plants (3 - 6 and 9 - 10: homozygous plants; 7 and 8: heterozygous).

Xrwgs11
Figure 3. Gel image for marker Xrwgs11. L: DNA Ladder; 1: KS92WGRC26 (H26 donor); 2: Newton (recurrent parent); 3 - 10: Newton/KS92WGRC26/2/Newton BC2F1 plants (3 and 10: homozygous plants; 4 - 9: heterozygous).

Xrwgs12
Figure 4. Gel image for marker Xrwgs12. L: DNA Ladder; 1: SW8 (H26 donor); 2: Newton (recurrent parent); 3 - 10: Newton/SW8/2/Newton BC2F1 plants (6 and 8 - 10: homozygous plants; 3 - 5 and 7: heterozygous).

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

References

1. Hessian fly resistance gene H26 transferred from Triticum tauschii to common wheat. Cox TS, Hatchett JH. In: Crop Science, 1994, 34:958–960. [link]

2. Registration of two synthetic hexaploid wheat germplasms resistant to Hessian fly. Xu SS, Cai X, Wang T, Harris MO, Friesen TL. In: Crop Science, 2006, 46:1401–140. DOI:10.2135/cropsci2005.06-0137

3. Genetic characterization and molecular mapping of Hessian fly resistance genes derived from Aegilops tauschii in synthetic wheat. Wang T, Xu SS, Harris MO, Hu J, Liu L, Cai X. In: Theoretical and Applied Genetics, 2006, 113:611–618. DOI: 10.1007/s00122-006-0325-z.

4. Virulence analysis of Hessian fly populations from Texas, Oklahoma, and Kansas. Chen MS, Echegaray E, Whitworth RJ, Wang H, Sloderbeck PE, Knutson A, Giles KL, Royer TA. In: Journal of Economic Entomology, 2009, 102:774-780. DOI:10.1603/029.102.0239.

5. Saturation and comparative mapping of genomic region harboring Hessian fly resistance gene H26 in wheat. Yu GT, Cai X, Harris MO, Gu YQ, Luo MC, Xu SS. In: Theoretical and Applied Genetics, 2009, 118:1589-1599. DOI:10.1007/s00122-009-1006-5

6. Identification and mapping of H32, a new wheat gene conferring resistance to Hessian fly. Sardesai N, Nemacheck JA, Subramanyam S, Williams CE. In: Theoretical and Applied Genetics, 2005, 111:1167–1173. DOI:10.1007/s00122-005-0048-6

7. Identification, Development and Validation of Molecular Markers closely linked to Hessian Fly-resistance Gene H32 in Wheat. Yu GT, Williams CE, Harris MO, Cai X, Mergoum M, Xu SS. In: Crop Science, 2010, In press.

 

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