HORTSCIENCE, VOL. 31(5), SEPTEMBER 1996 858 BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES HORTSCIENCE 31(5):858–861. 1996. Received for publication 16 Feb. 1995. Accepted for publication 22 Mar. 1996. This study was under- taken with funding assistance from G. and E. Cook of Helidon and the Rural Industries Research and Development Corp. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper there- fore must be hereby marked advertisement solely to indicate this fact. Distinction of Ozothamnus diosmifolius (Vent.) DC Genotypes Using RAPD H.L. Ko and R.J. Henry Department of Primary Industries, Queensland Agricultural Biotechnology Centre, Gehrmann Laboratories, The University of Queensland, Queensland 4072, Australia P.R. Beal, J.A. Moisander, and K.A. Fisher Department of Primary Industries, Redlands Research Station, P.O. Box 327, Cleveland, Queensland 4163, Australia Additional index words. riceflower, genotype, selection Abstract. An assessment was made to determine the suitability of RAPD analysis for identification of the Australian wildflower Ozothamnus diosmifolius (Vent.) DC [syn. Helichrysum diosmifolium (Vent.) Sweet] cultivars and lines. Of 19 arbitrary primer sequences tested, 16 revealed a high degree of polymorphism between the six most important genotypes with commercial significance, producing a total of 166 markers, of which 70% were polymorphic. Several primers (such as OPD-03 and OPM-07) were able to distinguish all tested genotypes from one another, showing an intracultivar consistency. These results indicate that RAPD analysis is a useful tool for establishing genetic diversity in this species as well as assisting in commercial protection of plant breeders’ rights. netic linkage maps) have been applied to re- lated species of the Asteraceae family for determination and comparison of genetic di- versity (Brunel, 1994; Fritsch et al., 1993; Lawson et al., 1994; Rieseberg et al., 1993; Wolff and Peters-Van Rijn, 1993; Wolff et al., 1993). These previous studies using RAPD- PCR analysis indicated that this technology is a potentially useful tool for genotype identifi- cation within this family. In our study, RAPD analysis was under- taken on six of the currently most important O. diosmifolius cultivars and lines, of which two are commercialized and protected by PVR. The usefulness of this identification method for this species was explored and established. Materials and Methods Plant material. All plant material origi- nated in Australia. Two commercial cultivars (‘Cook’s Tall Pink’ and ‘Cook’s Snow White’) and two lines under development (‘Cook’s Salmon’ and ‘Cook’s Pom Pom’) were pro- vided for testing by Graham and Esther Cook of Helidon, Queensland, who pioneered com- mercial riceflower growing (personal com- munication). Two lines (40b3 and 54.4), de- veloped by the Queensland Department of Primary Industries’ (QDPI) Redlands Research Station in Cleveland, Australia, were also in- cluded. Selected vegetative and floral charac- teristics of these lines are listed in Table 1. These lines, all identified as O. diosmifolius, were originally collected from diverse wild populations in eastern parts of Queensland and New South Wales. DNA extraction. DNA for PCR was ex- tracted from the youngest leaves according to Graham et al. (1994). Concentrations of the genomic DNA were determined spectropho- tometrically and the quality of the extracted DNA was assessed on 0.7% (w/v) agarose gels. PCR. PCR conditions were optimized by titrating the concentrations of MgCl 2 , Taq DNA polymerase, and template to give the optimum resolution of amplification products. Riceflower is a native Australian wild- flower (Fig. 1). It has been grown commer- cially in Australia since 1987 and marketed as a cut flower (a filler); most of it is destined for the overseas export market. This species has been introduced to intensive commercial hor- ticulture by selection of wild seedlings for vegetative propagation. The best clonal lines were identified after comparative evaluations over 3 or more years in field or laboratory trials, then further propagated for commercial cut flower production. Selection within the species has been successful in isolating forms that differ in flower color (white and a range of pinks) and flowering season (Beal, 1994; Lacey, S., personal communication). Vegeta- tive characteristics of this woody shrub spe- cies are difficult to distinguish with confi- dence. It is only at the flowering stage that the diverse flower shades become evident. Other properties, such as early flowering, cannot be detected morphologically. The short flower- ing period, 2 to 5 days of commercial accept- ability for most cultivars, requires the use of many cultivars to achieve continuity of sup- ply. With plant variety rights (PVR) being sought by growers, an early method of charac- terization and identification of various geno- types is essential. Polymerase chain reaction [PCR (Saiki et al., 1988)] analysis using arbitrary oligonucle- otide primers in random amplified polymor- phic DNA [RAPD (Williams et al., 1990)] and other systems (e.g., microsatellites and ge- Table 1. Description of Ozothamnus diosmifolius cultivars and lines included in the experiments. Characteristics Line/cultivar Vegetative Floral Cook’s Salmon z Tall shrub to 2 m height, bushy, Domed corymbs, closely packed, rounded shape, branches straight, round to oval, medium-sized salmon medium-green leaves. to pink buds with white tips, some uneven flowering, main flush in spring. Cook’s Tall Pink z Tall shrub >2.5 m height, upright, Loose, flat-topped corymbs, oval to open, inverted cone shape, mature pointed, large dark pink buds with bush has many long, very thin white base, isolated early flowers, straight branches, dark green leaves. main flush in spring. Cook’s Snow White z Tall shrub >2.5 m height, bushy, Flat-topped corymbs, round and young plants have circle of light, very small white buds, flowering secondary growth at base, branches in spring. spread from base before growing upright and straight, dark blue-green leaves. Cook’s Pom Pom Shrub to 1.5 m height, bushy, Highly domed corymbs closely rounded, medium-green leaves. packed, large, round to oval pink buds with white base, earliest (≈1 week earlier than Cook’s Tall Pink) flowering line currently identified. QDPI line 40b3 Tall shrub to 2.5 m height, with Domed corymbs, large, oval to upright growth, medium-green pointed pink buds with white base, leaves. latest (late spring, ≈2–3 weeks later than Cook’s Tall Pink) flowering line currently identified. QDPI line 54.4 Small rounded shrub, 1–1.5 m Domed corymbs, small, round white height, closely branched medium- buds, flowering ≈2 weeks later green leaves. than Cook’s Tall Pink. z From Cook and Law (1993).