Insect resistance. Russian wheat aphid (RWA)
Dn2, Dn4
Contributed by Junhua Peng, Scott Haley, and Nora Lapitan
Dn2 gene
Four markers including two microsatellites (2, 4) and two RAPD-derived SCAR (3) are available. The linkage distances between Dn2 and the four markers are in the range of 2.8-3.3 cM. So, the reliability of selection based on these markers is >95%.
a1. Primers for microsatellite Xgwm437
WMS437-F: 5'- GAT CAA GAC TTT TGT ATC TCT C -3'
WMS437-R: 5'- GAT GTC CAA CAG TTA GCT TA -3'
a2. Primers for microsatellite Xgwm111
WMS111-F: 5'- TCT GTA GGC TCT CTC CGA CTG -3'
WMS111-R: 5'- ACC TGA TCA GAT CCC ACT CG -3'
a3. Primers for SCAR marker B10880
B10880-1: 5'- CTG CTG GGA CGA AGC GTT TGA C -3'
B10880-2: 5'- CTG CTG GGA CCC GAT GAA TTG T -3'
a4. Primers for SCAR marker N1400
N1400-1: 5'- CTC ACG TTG GGA GCC ATT GAC G -3'
N1400-2: 5'- CTC ACG TTG GCA TCA GGG ATA A -3'
b. PCR reaction mix
For microsatellite markers, the PCR recipe is the same as described above for Dn4 gene. The recipe for SCAR marker is the following (3):
| Reagent | 1X |
| 1mM dNTPs | 3.75 µl |
| 10x PCR buffer | 2.5 µl |
| 25mM MgCl2 | 2.0 µl |
| 10µM L-primer | 1.0 µl |
| 10µM R-primer | 1.0 µl |
| 10U Taq polymerase | 0.1 µl |
| ddH2O | 13.65 µl |
| 20ng Template DNA | 1.0 µl |
| Total | 25.0µl |
c. PCR conditions
The PCR conditions for microsatellite markers are the similar as described for Dn4 gene except the annealing temperatures, 50°C for Xgwm437, and 55°C for Xgwm106.The PCR conditions for SCAR markers is the following (3):
- Denaturing: 94°C 3 min
- 35 cycles of:
- 94°C 20 s
- 62°C 30 s
- 72°C 45 s
- with 1 s increase in each cycle
- Extension: 72°C 5 min
d. Separation of PCR products
Due to the small band size and multiple bands, the PCR products of both Xgwm111 and Xgwm437 need to be separated in 10% polyacrylamide gel or sequence gel. The SCAR markers can be visualized in 2% agrose gel.
e. Expected products
