Studies On The DNA Fingerprinting Of Prunus Mume Cultivar Resources Via AFLP Markers

Prunus mume have been long cultivated and appreciated by the Chinese people.The number of cultivars were so many and the morphology characters were abundant, so it was too difficulty to classify and identify only by using morphology methods.Also,there have no explicit researches on the origins and evolvement of P.mume cultivars, the complicate genetic relationships, the genetic variance and diversity among the cultivars. Based on the systematic investigation of conserved P. mume cultivars in Wuhan and Nanjing, P. mume cultivars were analyzed via AFLP markers with silver staining, DNA fingerprints were established and analyzed, the genetic relationships and variance among some cultivars were studied, the similarities and differences between the morphological taxonomy and the clustering results of molecular markers were discussed with using NTSYS 2.1 software. The main results were as follows:1. Based on the comprehensive investigation on the Mei Flower Research Center of China in Wuhan, China and Sun Yat-sen Mausoleum, Nanjing, the total numbers of P. mume cultivars were cleared up and 350 cultivars were registered and conserved including all of 3 branches, 5 group and 18 forms. The cultivars with typical, peculiar and differential characters were evaluated.2. The methods of genomic DNA extraction were optimized. The genomic DNA template of high quality and concentration which was suitable for P. mume was obtained. The optimized methods were fit for the dry leaves of P. mume. A simple and convenient operated method was provided for International P. mume Cultivar Registration (ICR) and new variety registration of the cultivars. On the base of research results before, the Sliver-staining AFLP system suitable for P. mume was further optimized. Clear and high diviesity of DNA fingerprints were obtained by optimized measures such as the time and the system of restriction-ligase reaction, the concentration of genomic DNA template, the system of double amplification, the process of electrophoresis and staining techniques in experiment.3. By using EcoRⅠ-MseⅠrestriction endonuclease combinations, the best 5 primer pairs with clear and high polymorphism bands were selected from 156 primer combinations which could be reliably analyzed more cultivars. 22 representational cultivars were analyzed via AFLP markers.Different similarity coefficients and Nei's distance coefficient were calculated.The results indicated that there has a higher interspecific genetic diversity, mainly between P. mume and P. mume var. bungo, P.×blireiana. Cluster analysis results showed that AFLP markers can effectively distinguish 3 groups and were generally consistent with the morphological taxonomy. The first and second class criterium in morphological taxonomy specific germplasm and branch gesture were also supported.The cultivars with obvious phylogenetic relationship clustered together revealed that the AFLP could be used to analyze the phylogenetic relationships.4. With using of 5 selected primer pairs and AFLP markers, DNA fingerprints of 163 samples were obtained.The primer E-ATA/M-CTG was taken as an example to calculate the similarity coefficient.The results show that AFLP clustering were approximately consistent with morphological taxonomy on group and branch partition. Single Flowered Form, Cinnabar Purple Form and ext in Upright Mei Group were clustered together. There were some genetic variances among the Upright Mei group:Pink Double Form might come from different origins and with complex evolution levels, the genetic variances among those cultivars were obvious;There were no distinct differences between Versicolor Form and Flavescens Form, there mightbe some phylogenetic relationships between these two forms. Pendulous Groups were clustered togther showed that branch gesture was an important ornamental character in Mei flowers.The objectives of this study are to effectively conserve and utilize the wild and the cultivar germplasm resources, to select and breed eminent cultivars, to systematicaly study the origions, the genetic relationships and diversity on species and cultivars. To provide molecular techincal supports for Molecular marker-assisted selection, the important genotype marking, the establishing of registred P. mume cultivars'standardized fingerprints.