| Common Leaf Spot of Alfalfa (Medicago sativa L.) caused by Pseudopeziza medicaginis (Lib.) is the most common and serious disease occurred among varieties of Medicago. So far,breeding resistant varieties was generally acknowledged as the best method to prevent and control the disease. However, for the population of autotetraploid Alfalfa are high heterozygous, it is difficult to breed a new resistant variety. The traditional field investigation is a great work, which would take too much time; therefore, screening large number of resistant germplasm under that way is inefficient. The purpose of this research is to make use of molecular marker techniques such as ISSR (Inter-simple sequence repeat) and SCAR (Sequence-characterized amplified regions) to develop molecular markers linked to the resistant genes of common leaf spot in Alfalfa. Then by means of MAS, the efficiency of identification and selection will be increased, and also the breeding process will be accelereted.In this research, we use the F1 population of intermediate resistance×intermediate resistance as the material. The ISSR markers which closely linked with resistant gene of Common Leaf Spot were screened by techniques of Inter-simple sequence repeat (ISSR) and BSA. After that the ISSR markers were transformed into SCAR markers that can quickly and accurately identify the disease resistance of Common Leaf Spot of Alfalfa. The main results were as follows:1. The disease index of A×B cross showed normal distribution,the scope is 1.25～28.57, therefore,the materials could be used for further analysis. 12 plants whose disease index were less than 6.67 from F1 population were chosen to set up resistance DNA bulk,another 12 plants whose disease index were more than 18.4 were chosen to set up susceptible DNA bulk.2. After the 93 ISSR primers had been screened in resistance and susceptible DNA bulk, we found that there were 6 primers could amplify polymorphic bands .But single marker analysis showed that only the polymorphic bands generated by NO.11 and NO.20 primers closely linked with resistant QTL and susceptible QTL respectively. The two markers were renamed as 11-S750 and 20-R750. After that the 2 ISSR markers were verified by 25 plants apiece from 4×5,2×6,1×3 three highly resistant×highly resistant crosses and 25 plants apiece from 10×7,11×12 two highly susceptible×highly susceptible crosses, the result showed that the coincidence rate was 76.8% and 61.6% with chi-square of 39.38(p<0.05) and 9.74(p<0.05).3. After 11-S750 and 20-R750 were digged,purified,cloned,sequenced and compared through BLAST homologous analysis, the results showed that the sequences of these two markers were closely similar to part of the sequences of different chromosomes of Medicago truncatula and the similarity degree was about 80%, it means that at least there exsited two QTLs which related with common leaf spot. In addition,11-S750 maybe a part of sequence of serine/threonine protein kinase after comparative analysis of protein sequence.4. 11-S750 and 20-R750 markers were transformed into SCAR markers according to DNA sequence results. 20-R750 was transformed successfully into two dominant markers named SCAR20-R1 and SCAR20-R3. The coincidence rate of SCAR20-R1 and SCAR20-R3 was the same as the coincidence rate of 20 primers. The two SCAR markers would have important significance on increasing the efficiency of resistant breeding of the common leaf spot in Alfalfa.
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