Identification Of Molecular Markers Linked To Common Leaf Spot Disease Resistance Gene Of Alfalfa

We verify ISSR, AFLP and SRAP markers, which selected by predecessors. Markers with high accuracy and applicability will be selected. We build technology system to identify the resistance to common leaf spot disease, which can also screen genetic resources, that will increase the detection efficiency. The results of this study will not only provide high reliability marker-site to genetic maps and genes cloning, but also lay the foundation for Common Leaf Spot Disease Resistance gene mapping and cloning. The SCAR and CAPS markers can be used to identify and marker-assisted select of molecular that linked to gene conferring to the common leaf spot of alfalfa, but also increase the efficiency of resistant breeding of common leaf spot of alfalfa.1. In this research, four alfalfa cultivars with different genetic background were used as the expermental materials, we screen resistant and susceptible plants after inoculation. The order of the resistan to disease of the four alfalfa cultivars are: VS>VX>VZ>VL.The materials selected from totality were used for further analysis. 10 high resistant plants whose disease index ranged from 2.11 to 2.19, and another 10 susceptible plants whose disease index were ranged from 63.75 to 75.77.2. We verify the applicability of markers selected by ISSR, AFLP and SRAP. The experimental results demonstrate that susceptible marker ISSR22S450, resistant markers SRAPM3E3_R169 and ISSR20_R750 with high applicabilityn can be used for marker-assisted selection (MAS), which coincidence rate were from 75% to 85%. Susceptible markers ISSR6_S640 and AFLP38_S264, resistant markers AFLP1_R206, AFLP16_R185 and ISSR834_R450 were middle applicability, which were needed to be re-verificated by more materials. The susceptible markers ISSR11_S750 and ISSR22_S640 were poor applicability, that were not the reliability index for common leaf spot detection. The results of this study will not only lay the foundation for SCAR markers tansformed, but also provide high reliability marker-site to genetic maps and genes cloning.3. 10 resistance plants were chosen to set up resistance DNA bulk, another susceptible 10 plants were chosen to set up susceptible DNA bulk. We established the SCAR-PCR reaction conditions in alfalfa by studying the main parameters. Results showes the concentration of 6 components in 20μL reaction mixture are as follow: 2μL 10×PCR Buffer, 0.2μM dNTP, 0.25μM SCAR primers, 20 ng genomic DNA, 0.8 U Taq DNA Polymerase, ddH2O 9.24μL. The suitable PCR procedure are one cycle denaturing at 94℃for 5 min, that is followed by 36 cycles with denaturing at 94℃for 30 s, annealing 30 s, extending at 72℃for 1 min, a final extension at 72℃for 5 min, and then keep the temperature at 25℃.4. 53 pairs of SCAR primers were designed based on the markers with high and middle applicability we verified. Only 7 (11.48%) of The SCAR markers designed, ALSR834S-4, ALSR20-1, ALSS22A-1, ALSS22A-2, AFLP-M1P1-7, AFLP-M2P8-8 and AFLP-M5P6-2, were tansformed successfully. 5. The polymorphism is disappeared when SRAP markers marker converted to SCAR markers. After three SRAP-SCAR PCR products were digged, purified, cloned, sequenced and compared by DNAMAN, the results showed that the resistant sequence has the site of restriction endonuclease TaqⅠ, but the susceptible sequence don't have, therefore, we successfully. converted three SCAR-SRAP markers into CAPS markers by restriction endonuclease TaqⅠ.