Study On Genetic Diversity By Morphological And ISSR Markers Of Bermudagrass Resources In South China

Bermudagrass (Cynodon Richard) is a perennial grass with abundant variation, distributing widely in the world. It is one of the most important warm-season turfgrass and it is a merit legume as well. There is abundant wild bermudagrass germplasm with great protential of utilization in South China.After a whole investigation in South China,118 collections were selected as experimental materials to evaluate the genetic diversity on morphological and DNA molecular levels for the first time. The results provide the theoretic and practical scientific data for learning about genetic relationships of bermudagrass accessions, effectively protecting or utilizing wild bermudagrass germplasm and constructing core germplasm pool. The research results showed that:16 morphological characteristics of 118 bermudagrass materials collected from the South China were described, including leaf length and width of erect shoot, height of erect shoot, leaf length and width of creeping shoot, internode length and diameter, inflorescence length, branch number of inflorescence, spikelet number, spikelet color, anther color, stigma color, stem color, leaf color and leaf hair. The results indicated that stem color had biggest variation which coefficient of variation was 67.38%; Stigma color had bigger variation and its coefficient of variation was 55.54%; The lowest variation was branch number of inflorescence which coefficient of variation was only 11.85%. Height of erect shoot, spikelet color, anther color and leaf hair had high variation and the coefficient of variation were over 40%.A simple correlation analysis of morphological characteristics showed that there were 56 couples index which were significant correlation with each other. Cluster analysis of 118 bermudagrass materials according to 16 morphological characteristics indicated that they were grouped into nine types at 1.40 or five types at 1.54.DNA of bermudagrass were extracted successfully by using improved CTAB method, the DNA detection result by sepharose and ultraviolet spectrophotometer showed that the DNA could be used in ISSR amplify for the amplified results were steady, clear, good repeatability and multiple polymorphic bands.In this study orthogonal design was used to optimize ISSR amplification system on bermudagrass materials in two factors (template DNA and primers) at four levels. The optimal primers for ISSR-PCR reaction and their annealing temperatures were proposed by PCR. And a suitable ISSR reaction system was established, namely 15uL reaction system containing 60 ng template DNA,10μmol/L primer,7.5 uL 2×Easy Taq PCR Super Mix and 3.5uL ddH2O.The optimized ISSR-PCR system and 14 screened ISSR primers were used for ISSR amplify, the result showed that 210 DNA fragments with an average of 15 DNA bands per primer were amplified from 118 samples to give 210 fragments that were polymorphic, the average percentage of polymorphic bands was 100%. The genetic similarity computed by NTSYS among all accessions ranged from 0.4714 to 0.9714, indicating a very rich genetic diversity among the tested accessions. Through UPGMA cluster based on NTSYS genetic similarity, the 118 accessions were divided into five groups when the similarity coefficient stayed at Dice's coefficient of 0.73.