Studies Of Genetic Marker-assisted-selection For Resistance To Brown Blot In Alfalfa

Alfalfa, one of herbages, can not be lacked in the development of farming and stockraising. But in our country, the study of Brown Blot [Pseudopeziza medicaginis (Lib.) Sace. in alfalfa got bogged down in the survey of field diseases. ? In orderto offer the theoretical basis, primary materials and DNA extracting methodsfor the breeding of alfalfa varieties resistant to Brown Blot, and promote eventually the formation and development of alfalfa industry, individuals of 5 alfalfa varietieswere studied with genetic markers. These 5 experiment varieties include 2 foreignalfalfa varieties--Iroquios, Salansi and 3 domestic alfalfa varieties桽hahe, Shawanand Jingyang, while the technologies of isozyme markers and RAPD markerswere used to analyze individual plants, different in the resistance to Brown Blot,after individuals of every variety were mixed. The experiment was carried out at theResearch Institute of Animal Husbandry, CAAS from March 2000 to January 2001. The results of experiment show as follow: (1)After inoculation, specific isoenzyme bands between resistant and susceptible plants of 4 varieties of alfalfa, that were isoenzyme markers linked to the generesistant of Brown Blot, were found at the given periods. One isoenzyme markerwas found in the Salansrs PPO zymogram, and in the zymogram of POD, differentisoenzyme markers were detected in the Salansi, Shawan, Shahe and Jingyang, and for SOD at last. one isoenzyme band disappeared in Iroquios and Shahe. (2)The determination of the isozyme activities was not directly relative betweenthe screening of individual plants resistant to Brown Blot and it only demonstrated in quantity that activities of 3 isoenzymes in resistant and susceptible plantswere different remarkably. But from the trend of the isozyme activities, the production and the action of enzymes were not at the same time, that is, the revulsivedefense reaction was detonated by proteins existing in the cells, and this was decided by the physiologic function of isoen.zymes. (3)Through modifying and simplifying 3 methods of genome DNA extraction, N,-CTAB, an improved method, was obtained, and it is more suitable for extraction ofalfalfa's DNA. In the study, alfalfa's DNA were extracted with 4 methods, including CTAB, SDS, N2-SDS, N2-CTAB, and then their outcomes were compared according to the UV absorption conditions, electrophoretic analysis and RAPD 3 4 analysis, and at last N2-CTAB was determined as the best way. In the novel way, a step, freezing leaf tissues with liquid N-,, was added, and while another, resolving RNA with RNase A, omitted. These changes not only led to high output of DNA and clear and high polymorphous products but also simplified DNA extraction steps. By now, this method has not been reported. (4) By analyzing 7 factors?amplified outcomes, the ideal RAPD system and the condition were determined. RAPD was strict with the reaction system, and the amplified outcome would be influenced by any changes of the reaction system. Therefore important factors, such as template DNA, M&...