A COMPARISON OF GARDENIA AUGUSTA CULTIVARS USING ISOZYMES AND RAPD MARKERS

Abstract

Numerous cultivars of the florist gardenia [Gardenia augusta (L) Merrill. = G. jasminoides J. Ellis] exist but there is confusion over naming as a result of different sources that apply different names to their materials. Two studies were conducted with (some) of the same plant materials to determine whether clear distinctions could be made. Comparisons were also made to other Gardenia species available to us. The isozyme study compared polymorphisms in seven enzyme systems in buffered leaf extracts using starch gel electrophoresis. Total genomic DNA was extracted recently matured leaves using the DNeasy Mini Kit (QIAGEN, Inc., Valencia, CA, USA) extraction protocol. Nine random 10-mer RAPD primers (Operon Technologies, Alameda, CA, USA) were selected for analyses. Gels were digitally imaged and analyzed to compile a presence/absence matrix. For the isozyme study, phosphoglucomutase (PGM), phos-phoglucoisomerase (PGI), and uridine diphosphoglucopyrophosphorylase(UDPP) were the only successful enzyme systems to yield clear interpretations for a total of 5 loci, and dendrograms based on Jaccard’s or simple matching coefficients of similarity were very comparable. However, the three enzyme systems were not sufficient to uniquely fingerprint all of the G. augusta accessions. The RAPD analysis using the nine 10-mer primers showed well-defined (bootstrap values >80) groups: the G. augusta, with subgroupings and the G. brighamii and species. Both isozyme and RAPD analyses separated or matched cultivars of G. augusta as well as G. brighamii and other Gardenia species. Coupled with leaf and floral measurements and morphological descriptions, it is possible to distinguish among commercial florist gardenias in a decisive manner. INTRODUCTION The florist gardenia (Gardenia augusta = G. jasminoides; family Rubiaceae) is a woody shrub native to east Asia and widely cultivated as a garden ornamental in tropical regions and temperate greenhouses (Staples and Herbst, 2005). The fragrant flowers are used as decorations and in corsages by florists. Many named double-flowered cultivars are available in the nursery trade, but names have been lost as plants have been traded among nurseries and homeowners. Brief descriptions of many cultivars can be found in the literature (Watkins, 1950; Everett, 1981; Huxley, 1992; Ruppert and Bradshaw, 1993) or plant patents (e.g., PP 93, 1934; PP 364, 1940; PP 622, 1944; PP 654, 1945), but these often do not provide sufficient information to distinguish among similar-appearing cultivars. Additionally, gardenia is known to mutate and more than two dozen clones have arisen as bud sports (Watkins, 1950), although new cultivars have also been selected from hybrid seedling populations (Milbocker, 1994). Although morphological traits and isozyme marker analysis have been used to distinguish cultivars and/or hybrids, both systems have limitations, the first due to environmental effects and the latter to selection of markers. Both isozyme and randomly amplified polymorphic DNA (RAPD) markers have been useful in determining genetic relationships among closely related species. Isozyme electrophoresis is chosen for its relative simplicity and because it provides direct visualization of gene products (Brewer Proc. XXVII IHC-S5 Ornamentals, Now! Ed.-in-Chief: R.A. Criley Acta Hort. 766, ISHS 2008 462 and Sing, 1970) and potentially can provide a unique fingerprint for each genetically distinct clone (Lebot et al., 1991). RAPD analysis requires minimal preliminary work and is quick and robust (Demeke et al., 1992; Molnar et al., 2000) and has become widely used for routine cultivar identification and genetic diversity of many plants. RAPD analysis has been used for genetic diversity of wild gardenia populations in Korea (Huh