DNA Amplification Fingerprinting and Hybridization Analysis of Centipedegrass

Abstract

DNA amplification fingerprinting (DAF) with arbitrary oligonucleotide primers provides germplasm analyses for cultivar identification and protection of proprietary rights, and constitutes an effective tool in marker‐assisted breeding and in the study of genetic variation within natural and domesticated populations. In this study, DAF was used to infer genetic relationships in centipedegrass [Eremochloa ophiuroides (Munro) Hack.]. An optimization of reaction components defined amplification conditions that reproducibly fingerprinted centipedegrass and other turfgrass species. Cultivars Tennessee Hardy, Tennessee Tuff, Oklawn, Centennial, and Tifton common were analyzed with 14 octamer primers. Four primers produced polymorphic patterns, two of which differentiated all cultivars. Phylogenetic analysis using parisomy (PAUP) distinctly separated cold‐tolerant Tennessee Hardy. Numerous monomorphic bands were obtained, suggesting that the centipedegrass cultivars are closely related and are from a common origin. Several DAF bands were isolated from silver‐stained polyacrylamide gels for use as hybridization probes. A monomorphic 200‐base pair (bp) band that hybridized to a single amplification product detected eight homologous regions in genomic centipedegrass DNA. When hybridized to DAF patterns obtained from other turfgrass species, such as bermudagrass [Cynodon dactylon (L) Pers. × Cynodon transvaalensis Burtt‐Davyl, rough bluegrass (Poa trivialis L.) and Zoysia (Zoysia japonica Steud.), the 200‐bp fragment, and a 175‐bp fragment generated with a different primer, resulted in smearing patterns. While these products are only present within one size amplicon in centipedegrass fingerprints, the smearing indicated that the putative sequence was present in multiple copies throughout the amplification profile, was abundant in the heterologous grass genomes, and may have represented a family of repetitive DNA sequences. Our results indicated that DAF can generate molecular markers that can be used reliably for DNA fingerprinting of important centipedegrass cultivars. This tool would be useful in determining genetic relationships and diversity, and in establishing possible origins of cultivars.