Study On Poiygenetic Relationship Of Narcissus By Molecular Makers

Polygenetic relationship among different types and resources of Narcissus was studied by RAPD and ISSR. And special marker in French type of Narcissus by RAPD was transferred to SCAR, which is more iterative and stable to identify this type materials.1. DNA concentration extracted from Narcissus by 2% CTAB little method was higher than that from other plants by this method or mass method. This indicated that tender leaf tissue with little cellulose was the direct cause of high deriving of Narcissus DNA. Inclusion in the leaf of Narcissus had little effect on DNA exacting. High quality DNA could be gained by 2% CTAB little method without supplementing any additives.2 .Four levels were assigned in all factors of RAPD for orthogonal analysis. In the results, 6 systems showed that the proper assembly of respective factor and concentration affected amplification result, not the concentration of certain factor. Primers used in RAPD were random, and their base sequence differed greatly, so their combination with other factors in the system caused different optimal reaction system.3. Classification of Narcissus by RAPD was basically coincident to that of British royal horticulture academy.58 materials was clustered into 10 types. Clove type, big-cupped subcorolla type, bugled subcorolla type and polyphyll type clustered respectively. Among them, clove type was more original, while the other three were closer with average similarity coefficient of 0.610. The change of flower color probably could be caused by wider genetic variation. Geographical location also could result in different variation in French type, with near location varying similarly. Relationship between French and clove type, big-cupped subcorolla type, bugled subcorolla type, polyphyll type was far with average similarity coefficient of 0.493 and they belong to different species respectively. Relationship between escape type and types above was farthest; with average similarity coefficient of 0.229 and escape type belong to another species. Further, French and escape type clustered at the top of the dendrogram, and they may be the later type of evolution. In evolution process, flower organ of Narcissus evolved from simple to complex, appearing as subcorolla changing from little, big to petalled.4. Four levels of all the factors were assigned for orthogonal analysis in ISSR system of Narcissus. Amplification effect of ISSR was controlled by concentration combination of different factors in the system too. Primers of 4 base repeat, especially (AGTG)4 and (GACA)4 showed better amplification ability. High annealing temperature was used at the beginning of the ISSR procedure, while lower temperature was selected in later cycle. Such system had benefits for clear and polymorphic results.5. Classification result of ISSR was accordant to that of RAPD. Result of ISSR was rough due to the little number of ISSR primers. However, results of 6 ISSR primers weresimilar to that of 14 RAPD primers, which indicated ISSR had prominent efficiency and polymorphism.6 .In RAPD, primer S488 got a special band S488-631 in 17 French type materials. After sequence analysis, the band could be transferred to SCAR. That significantly raised the specificity and stability of the marker. This marker could be conveniently and rapidly used in identifying French type.