Development Of A RAPD Molecular Marker For The Leaf Rust Resistance Gene Lr38 In Wheat

The random amplified polymorphic DNA (RA PD) analysis was carried out in Thatcher, near-isogenic lines carrying twenty-one different genes conferring resistance against leaf rust and seven different varieties. The main results were as follows: 1. The optimized RAPD reaction system was established. In a total volume of 25 LI I, containing 10mM KCI, 8mM(NH4)2S04, 10mM Tris-HCI(pH- 9.0), NP- 40, 2.0mM Mg2~, 200 Li M dNTP, 45ng primer, 20ng template DNA, I.5U Taq DNA polymerase. Amplification performed in a AmpGene DNA Thermal Cycler 4800 was programmed for 5 mm at 94C, 40 cycles of 30s at 94t, 60s at 37t, 90s at 72℃, and a final extention at 72C for 8 mm after the last cycle. 2. One polymorphic RAPD marker (S33-800) linked to leaf rust resistance gene Lr38 was screened. All of the 160 random primers screened for RAPD analysis gave clear amplification products, 4 of them amplified the polymorphic DNA in the NILs Thatcher and Lr38/6°hatcher when screened first time. Further investigation demonstrated that primers S30 and S33 showed the same discriminating results in more than five replications. The polymorphic bands were named as S30-200, S33-800, respectively. Using these two primers to detect a resistant material with Lr38 and other susceptible material without Lr38, it was shown that only S33-800 could be amplified in the resistant material and absent in all of the susceptible materials. This demonstrated that the marker S33-800 was linked to resistant gene Lr38. Further research will be focused on RAPD analysis of F2 population using primer S33 in order to evaluate the genetic distance between S33-800and Lr38 resistant gene. 45 3. The 800bp specific fragment was recoveried. The 800bp specific fragment, which was amplified in Lr38/&Thatcher with primer S33, was recoveried using phenol-chloroform extraction method, and then amplified with the above-mentioned primer S33. The amplification product was separated in 1.4% agarose gels and visualized by ethidium bromide staining. A clear band was gained and it implied that the primer and the non-specific products have been clean up.