Abiotic Stress and Agronomic Traits. Dwarfing Genes
Microsatellite marker Xgwm261 for Rht8 (3, 8-10).
Korzun et al. (8) mapped Rht8 on the short arm of chromosome 2D and determined that microsatellite locus Xgwm261 was located at 0.6 cM distal to Rht8.
Primer sequencesWMS261-F 5'- CTC CCT GTA CGC CTA AGG C -3' WMS261-R 5'- CTC GCG CTA CTA GCC ATT G -3'Final concentrations of the reagents used in the PCR amplification (8)
- 50-100 ng template DNA.
- 250 nM of each primer
- 1 unit of Taq DNA polymerase
- 0.2 mM of each dNTP
- 1.5 mM MgCl2
Total volume: 25 µl reaction
PCR conditions- Denaturing step: 94°C, 3 min
- Amplification step (45 cycles)
- 94°C, 60 sec
- 55°C, 60 sec
- 72°C, 120 sec
- Extension step:72°C, 10 min
Expected products
Korzun et al. (8), Worland et al (9, 10) and Ahmad and Sorrellls (3) analyzing different wheat lines found that the dwarfing allele of Rht8 is linked to a 192-bp size allele for Xgwm261. Cultivars with the wild type allele have alleles of several sizes:165-bp, 174-bp, 180-bp, 198-bp, 200-bp and 204-bp. Ellis et al. (11) found several cultivars derived from Norin 10 in which the 192bp allele is not linked to Rht8.
Conditions presented here should be consider only as a starting point of the PCR optimization for individual laboratories.