Disease resistance. Leaf Rust Resistance.
Lr50
Contributed by Gina Brown-Guedira and Suhkwinder Singh
Lr50 is flanked by to microsatellite markers Xgwm382 (6.7 cM) and Xgdm87 (9.4 cM) on wheat chromosome arm 2B long.
Primers for GWM382 (2)WMS382-F 5'- GTC AGA TAA CGC CGT CCA AT -3' WMS382-R 5'- CTA CGT GCA CCA CCA TTT TG -3'Primers for GDM87 (1)
GDM87-left 5'- AAT AAT GTG GCA GAC AGT CTT GG -3' GDM87-right 5'- CCA AGC CCC AAT CTC TCT CT -3'
Components of PCR reaction
Final volume: 25µl reactions containing,
- 50 ng genomic DNA
- 12.5 pmol each primer
- 0.2 mM each dNTP
- 0.625 U Taq
- 1x PCR buffer
- 2 mM MgCl2
- Denaturing step: 4 min at 94°C
- Amplification step: (30 cycles)
- 30 s at 94°C
- 30 s at 60°C
- 30 s at 72°C
- Extension step: 5 min at 72°C.
PCR products are resolved on 2.3% agarose gels (GeneMate GenePure HiRes Agarose - ISC BioExpress Cat# E-3115-500). Some susceptible cultivars have microsatellite bands of similar mobility as the resistant germplasm. Therefore, a preliminary screening is necessary to test the existence of polymorphism between the donor and the selected recurrent parents. For example, see the bands produced by line TAM 107 for Xgdm87 in panel B.
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| Molecular markers linked to the Lr50 locus in KS96WGRC36. DNA fragment amplified by primer pairs GWM382 (A) and GDM87 (B) from genomic DNA of the leaf rust resistant T. armeniacum accession TA870, resistant wheat germplasm line KS96WGRC36, and susceptible wheat cultivars TAM 107 and Wichita. Allelism studies determined that leaf rust resistant line U3193 (TAM 107*4/TA874) also has Lr50 transferred from accession TA874 of T. armeniacum. Arrows indicate sizes of polymorphic fragments. |
Conditions presented here should be considered only as a starting point of the PCR optimization for individual laboratories.