AFLP Analysis For The Genetic Diversity Of Some Chrysanthemum Species

Chrysanthemum (Dendranthema X grandiflorum (Ramat) Kitam) originated fromChina, and it is one of the ten traditional famous flowers in China and the four famouscutting flowers in the world. There are lots of cultivars and variations among thiscultivated flower. It is estimated that there are more than 3,000 cultivars in China. Theprotection and identification of chrysanthemum cultivars are important but difficult.AFLP (amplified fragment length polymorphism) is a new DNA molecular markertechnique with high efficiency, promptitude, stability and accuracy. It has been widelyused in classification, cultivar identification, genetic mapping, breeding, location oftarget genein animals and plants. In order to provide scientific basis for theidentification, conservation and utilization of chrysanthemum germplasm, AFLPtechnique system suitable for chrysanthemum species was established. Thefingerprinting of 26 chrysanthemum materials were constructed, and the geneticdiversity of these materials was analysed.In this article, the totals DNA of Chrysanthemum were extracted from fresh leafusing an improved method of CTAB. The modified extraction buffer contained 100mmol/L Tris-HC1 (pH 8.0);20 mmol/LEDTA, 1.4mol/L NaCI, 2% CTAB, 2% PVP₄₀,and 50 mmo/Lβ-Mercaptoethanal(added before isolation). RNA was removed from thesolution with RNase, and DNA was purified by phenol/chloroform,chloroform/isoamyl alcohol. High quality DNA templates were isolated for AFLPanalysis. With this understanding the clear pictures of AFLP were obtained. Then anAFLP system fitting for Chrysanthemum genomic DNA analysis was asestablished.At the same time, AFLP marker technique was used to study the genetic diversityof 26 chrysanthemum species. Seven pairs of primers are chosen from 64 pair ofprimers with AFLP analysis, and the clear polymorphic fingerprints were obtainedwhich showed that it is feasible for using these primers in chrysanthemum speciesAFLP analysis. Using 7 primer combinations that were able to discriminate betweengenotypes could produce 385 AFLP-bands, and 285 of that is AFLP polymorphbands(74.03%), with an average of 40.7 bands produced per primer pair. The resultsshowed a high level of genetic diversity of Chrysanthemum and species level.On the other hand, UPGMA clustering method was carried out by one clusteranalysis softwares—NTSYS 2.0 package for the cluster analysis of AFLP data.Clustering of UPGMA analysis indicates that the Dice's coefficient of the 26chrysanthemum species ranges from 0.61-0.85. The genetic relationships among theseare relatively close. 26 Varieties could be taxonomied into five groups at 0.75similarity coefficients for NTSYS2.0.The result is the same of the traditional taxonomyhaply.The current Study indicated that AFLP had high repetition and rich polymorphism, and should be a reliable DNA fingerprinting technology, AFLP bandsusing 7 primer combinations. It can be used to their identification. So it is practical togroup chrysanthemum species and analyse genetic diversity with AFLPs.