Insect resistance. Russian wheat aphid (RWA)
Dn2, Dn4
Contributed by Junhua Peng, Scott Haley, and Nora Lapitan
Dn4 gene
Two microsatellite markers Xgwm106 and Xgwm337 flanking Dn4 with map distances of 7.4 and 12.9 cM, respectively, are useful for breeding (N. Lapitan unpublished, 4, 5). The progenies with the presences of the two markers are resistant with high probability (99.0%).
a1. Primers for Xgwm106Left primer: 5'-CTG TTC TTG CGT GGC ATT AA-3' Right primer: 5'-AAT AAG GAC ACA ATT GGG ATG G-3'
a2. Primers for Xgwm337
Left primer: 5'-CCT CTT CCT CCC TCA CTT AGC-3' Right primer: 5'-TGC TAA CTG GCC TTT GCC-3'
b. PCR reaction mix:
| Reagent | 1X |
| 1mM dNTPs | 5.0µl |
| 10x PCR buffer | 2.5µl |
| 25mM MgCl2 | 1.5µl |
| 10µM L-primer | 1.25µl |
| 10µM R-primer | 1.25µl |
| 10U Taq polymerase | 0.1µl |
| ddH2O | 8.4µl |
| 20ng Template DNA | 5.0µl |
| Total | 25.0µl |
c. PCR conditions
- Denaturing: 94°C 3 min
- 45 cycles of:
- 94°C 1 min
- 60°C 1 min (for Xgwm106) or 55°C (for Xgwm337)
- 72°C 2 min
- Extension: 72°C 10 min
d. Separation of PCR products
Xgwm106 is a dominant marker and can be visualized in normal 1.2% agarose
gels. Xgwm337
is a co-dominant marker with two co-migrating bands. The PCR products can
be separated in 3.0% agarose
gel or in 7% polyacrylamide gel.
e. Expected products
