| Alfalfa (Medicago sativa L.) is an important protein feed, which was called the king of forages. Common leaf spot disease caused by Pseudopeziza medicaginis (Lib.) Sacc.is the most common and destructive disease in alfalfa. The most economical and effective way to control the common leaf spot disease was the breeding and employment of disease resistant materials. In this study, three populations were used as the experimental materials—resistant and susceptible strains of five alfalfa varieties, moderate resistant×moderate resistant crosses and backcross population of (resistant×susceptible)×resistant. The object of our study was to obtain several molecular markers linked to the common leaf spot disease resistance genes in alfalfa, which would do help to the alfalfa marker-assisted selection system. And at the same time, we want to explor the inheritance law of the common leaf spot disease resistance in alfalfa.The research has led to the following results:1. By studying the main parameters we established the optimal ISSR-PCR reaction conditions in alfalfa. Results showes that the optimum concentration of seven components in 20μL reaction mixture is as follow: 15 ng genomic DNA, 0.2 mmol/L dNTP, 0.4μmol/L ISSR primer, 0.8 U Taq DNA Polymerase, 2μL 10×PCR Buffer, 1.5 mmol/L MgCl2, 2.5% deionized formamide. The suitable PCR procedure is one cycle denaturing at 94℃for 4 min; 11 cycles each involved denaturing at 94℃for 30 s, annealing at 62℃(62℃～58℃) for 45 s, with declining 1℃each cycle, extending at 72℃for 1 min 45 s, which is followed by 24 cycles each with denaturing at 94℃for 30 s, annealing at 52℃for 45 s, extending at 72℃for 1 min 45 s, and a final extension at 72℃for 5 min, then keep the temperature at 52℃.2. Resistant and susceptible strains of five alfalfa varieties (Iroquois, Saranac, Shawan, Shahe and Jingyang) were used to construct resistant and susceptible DNA pools. Four ISSR markers associated with the common leaf spot disease resistance genes were obtained. They were ISSR6-S640, ISSR22-S450, ISSR22-S660 and ISSR834-R450.3. Six highly resistant plants, six highly susceptible plants and four moderate resistant plants were selected by the disease inoculation and investigation. By mutual hybridization, six highly resistant×highly resistant crosses, six highly susceptible×highly susceptible crosses, twenty-one highly resistant×highly susceptible crosses and four moderate resistant×moderate resistant crosses were constructed. Then by inoculation and investigation, two highly resistant×highly resistant crosses, two highly susceptible×highly susceptible crosses, one highly resistant×highly susceptible crosses and one moderate resistant×moderate resistant crosses were selected to be further research materials or as original alfalfa materials of common leaf spot disease resistance breeding in future.4. Using moderate resistant×moderate resistant population, one SRAP marker named M3E3-R169 was obtained. It was linked to the alfalfa common leaf spot disease resistance gene and the genetic distance was 11.75 cM. By cloning and sequencing of this marker, we discovered that high inentity as much as 97% existed between the No 3855～4006 base sequence of alfalfa glutamine synthetase gene and the No 6～157 base sequence of this marker, which coverd the gene's No 11 exon and intron.5. Innoculation and investigation were applied to the two BC1 populations. The ratio of the resistant plants to the susceptible plants in the two populations were both in accordance with 3:1. And in the highly resistant×highly resistant population, most were resistant plants. So we inferred alfalfa resistance to the common leaf spot disease was inherited as a single dominant locus, accompanied with some minor genes with moderate effects.6. Using the BC1 population, three AFLPmarkers linked to the common leaf spot disease resistance genes were obtained. They were M1P1-R206, M2P8-R185 and M5P6-S264, with the genetic distance of 8.95 cM, 10.95 cM and 4.35 cM. By cloning and sequencing of the three markers, we found high identity existed between these markers and some clones of Medicago truncatuca L.The four molecular markers obtained in this study was linked to the common leaf spot disease resistance genes in alfalfa. They will provide evidence for the identification of alfalfa germplasm with common leaf spot disease resistance, and also provide basis for the construction of the marker-assisted selection system. It will improve the selection efficiency, shorten the breeding period. And on the other hand , the BC1 population created in this study can also be used to construst the linkage map of tetraploid alfalfa.
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