Differentiation of bermudagrass (Cynodon spp.) genotypes by AFLP analyses

Bermudagrasses (Cynodon spp.) are major turfgrasses for home lawns, public parks, golf courses and sport fields, and are widely adapted to tropical an d warmer temperate climates. Morphological and physiological characteristic s are not sufficient to differentiate some bermudagrass genotypes because t he differences between them are often subtle and subject to environmental i nfluence. In this study, a DNA-typing technique, amplified fragment length polymorphism (AFLP), was used to differentiate bermudagrass genotypes and t o explore their genetic relationships. Twenty seven bermudagrass cultivars and introductions, mostly from the Coastal Plain Experiment Station in Tift on, Ga., were assayed by the radioactive (P-32) and the fluorescence-labele d AFLP methods. The AFLP technique produced enough polymorphism to differen tiate all 27 bermudagrass genotypes, even the closely related ones. An aver age of 48-74 bands in the 30-600-bp size range was detected by the P-32-lab eled AFLP method. The results indicated that most of the 14 primer combinat ions tested in this study could be used to distinguish bermudagrass genotyp es, and that some single primer-pairs could differentiate all 27 of them. T o test the reliability and reproducibility of the AFLP procedure, three DNA isolations (replications) of the 27 bermudagrass genotypes were assayed us ing five primer pairs. Only 0.6% of the bands were evaluated differently am ong the three replications. One replication of one genotype (which was most likely a planting contaminant) was grouped in an unexpected cluster using the Unweighted Pair Group Mean Average (UPGMA) method. A one- or two-band d ifference in scoring did not change the clustering of genotypes or the repl ications within genotypes. The 27 genotypes were grouped into three major c lusters, many of which were in agreement with known pedigrees. Trees constr ucted with different primer combinations using P-32- and fluorescence-label ling formed similar major groupings. The semi-automated fluorescence-based AFLP technique offered significant improvements on fragment sizing and data handling. It was also more accurate for detection and more efficient than the radioactive labelling method. This study shows that the AFLP technique is a reliable tool for differentiating bermudagrass genotypes and for deter mining genetic relationships among them.