HORTSCIENCE 34(2):341–345. 1999. (RAPD) markers (Williams et al., 1990) have been widely used for determining variety and parentage identification, construction of high- Rose Germplasm Analysis with RAPD density genetic maps, targeted mapping for specific traits with bulk segregant analysis or Markers near-isogenic lines, and studying genetic di- versity and phylogenetic relationships (He et C.H. Jan1 and D.H. Byrne2 al., 1992; Hu and Quiros, 1991; Kresovich et Department of Horticultural Sciences, Texas A&M University, College Station, al., 1992; Tingey et al., 1993; Welsh et al., 1991). This method uses genomic DNA as a TX 77843-2133 template, is rapid, does not employ radioiso- J. Manhart2 and H. Wilson2 topes, and only requires nanograms of DNA per reaction (Williams et al., 1990). Department of Biology, Texas A&M University, College Station, TX 77843-3258 The objective of this study was to use Additional index words. Rosa, phenetics, classification RAPD markers to classify 36 rose species using a phenetic analysis for interpretation. Abstract. The genus Rosa consists of more than 100 species classified into four subgenera, Eurosa, Platyrhodon, Hesperhodos, and Hulthemia, and distributed widely throughout the Materials and Methods northern hemisphere. The subgenus Eurosa includes 11 sections. The other subgenera are monotypic. One hundred and nineteen accessions and 213 markers of 36 rose species that Plant materials. One hundred and nineteen include eight sections of the subgenus Eurosa and one species each from the subgenera rose genotypes representing 36 species were Hesperhodos and Platyrhodon were used to calculate a similarity matrix, which was surveyed. One to 11 species (mainly diploid) clustered with the unweighted pair group method using arithmetic means (UPGMA). The were selected to represent eight of 11 sections RAPD markers distinguished between all the rose accessions, and species grouped into within the subgenus Eurosa and one species their respective sections. Therefore, classification of Rosa using RAPD data generally each from the subgenera Platyrhodon and supports traditional classification. The Asian rose sections (Laevigatae, Banksianae, Hesperhodos (Table 1). Young leaves were Bracteatae, Pimpinellifoliae, Chinenses, and Synstylae) were consistently separated from collected from the greenhouse, screenhouse, the primarily North American sections (Cassiorhodon and Carolinae). The Cassiorhodon and field, put into labeled envelopes, and stored and Carolinae sections were grouped together with the subgenera Hesperhodos and in an ice chest for transport to the laboratory. Platyrhodon. Both subgenera separated out at the same level as sections within the In the laboratory the leaves were stored at –20 subgenus Eurosa, suggesting that they are more appropriately classified as sections within °C in a freezer until their DNA was extracted. the subgenus Eurosa. Sections Cassiorhodon and Carolinae overlapped, and are probably DNA isolation. A minipreparation proto- best grouped as one section as previously suggested. col utilizing a cationic hexadecyl trimethyl ammonium bromide (CTAB) method modi- fied from Doyle and Doyle (1987) and Pater- Roses are one of the most important flower Erlanson, 1934; Flory, 1950; Lewis, 1957a, son et al. (1993) was used. Modifications were and nursery crops throughout the world. About 1957b, 1958, 1970; Rehder, 1940). Because designed to counter the high level of second- 60 million rose plants for garden use are propa- each species of the genus Rosa has a wide and ary compounds found in rose leaves. These gated in the United States (Streeper, 1990) and overlapping range of morphological varia- compounds degrade DNA, and inhibit subse- $300 million is generated by the cut flower tions that are influenced by environmental quent enzyme digests and PCR reactions. The market annually (U.S. International Trade conditions, classification based on morphol- modifications included the use of 2-mercapto- Commission, 1995). The demand for greater ogy alone is not adequate (Lewis, 1957b). ethanol as an antioxidant, and further purifica- disease resistance has led to the use of more Chemotaxonomic studies of roses (Mikanagi tion through phenol extraction and gel filtra- wild rose species in breeding and the develop- et al., 1993, 1994; Okuda et al., 1992; Ray- tion. ment of unique interspecific derived germ- mond et al., 1995) based on the wide range of Young leaf tissue (50–70 mg) was ground plasm, such as amphidiploids (Byrne et al., variant polyphenolic compounds have been with liquid nitrogen in a 1.5-mL microfuge 1996). Unfortunately, this work is limited by reported. Isozyme markers have also been tube. The powder was then mixed with 1 mL the dearth of information available about the used for rose identification (Kim and Byrne, 4X CTAB solution and 2.5 µL 2-mercaptoet- genetic relationships among rose species. 1996; Kuhns and Fretz, 1978a, 1978b; Lee and hanol. The homogenate was incubated in a 65 The genus Rosa consists of more than 100 Kim, 1982; Walker and Werner, 1997; Yoneda °C water bath for 1–2 h with periodic gentle species, the exact number varying with the et al., 1993) and classification (Kim, 1994; vortexing, and the DNA was extracted twice classification system (Allen, 1973; Rehder, Kim and Byrne, 1994). However, the small with 24 chloroform : 1 isoamyl alcohol (CIA) 1940). Their wide geographic distribution, number of consistently resolvable loci (Kim, and twice with 25 phenol : 24 chloroform : 1 polyploidy, and frequent interspecific hybrid- 1994; Kim and Byrne, 1994) limits the utility isoamyl alcohol. The final pellet was dis- ization, together with human involvement, of isozyme markers. Direct DNA-based diag- solved in 150 µL TE buffer (10 mM Tris-HCl, makes this genus diverse and difficult to clas- nostic assays are considered powerful and pH 7.5, 0.1 mM EDTA), and further purified by sify. Traditional identification and classifica- reliable tools for genetic analysis because the gel filtration with 5% Sephadex G-50 gel tion of plants are based on morphological number of scorable loci is greater, and expres- column constructed from a 1.5-mL microcen- characteristics (Allen, 1969, 1973; Bean, 1970; sion is similar in all tissues (Hubbard et al., trifuge tube. DNA concentration was deter- 1992; Torres et al., 1993). DNA markers, such mined by 0.8% agarose gel electrophoresis in as restriction fragment length polymorphisms TE buffer (Sambrook et al., 1989) and com- (RFLPs) (Hubbard et al., 1992), random am- parison of band intensities with lambda DNA Received for publication 16 Nov. 1997. Accepted plified polymorphic DNA (RAPD) (Gallego standards. All DNA samples were diluted to for publication 11 Aug. 1998. Research conducted and Martinez, 1996; Torres et al., 1993; Walker 0.25 ng·µL–1 before use. in partial fulfillment of a Master of Science degree and Werner, 1997), and mini- and microsatel- RAPD assay. The ten 10-base-long arbi- by Chih-Hui Jan. The cost of publishing this paper lite probes (Vainstein and Ben-Meir, 1994) trary primers (Operon Technologies, Alameda, was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must have been used for rose identification, but only Calif.) that gave the most reproducible and be hereby marked advertisement solely to indicate recently has preliminary taxonomic research polymorphic patterns were selected from 80 this fact. been done with RAPD analysis (Debener et primers (Kit E, F, G, H). Those used were E14, 1Graduate student. al., 1996; Millan et al., 1996). E19, F06, F14, G11, G19, H06, H12, H15, and 2Professor. Random amplified polymorphic DNA H19. Amplification reactions were performed HORTSCIENCE, VOL. 34(2), APRIL 1999 341 BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES Table 1. Rose materials used in RAPD study. The rose has a small genome (up to 0.825 pg per haploid genome) (Dickson et al., 1992) Number of Subgenus Sectionz Rosa species accessions and a high level of polymorphism. The high Eurosa L. Chinenses Sér. R. chinensis Jacq. 8 number of bands per primer may also be re- R. ×odorata (Andr.) Sweet. 1 lated to the direct selection of the primers with Banksianae Lindl R. banksiae Ait. 9 more polymorphic bands. The size of markers R. ×fortuniana Lindl. 1 ranged from 260 (E19) to 2300 bp (G11) (Fig. Laevigatae Thory. R. laevigata Michx. 3 1). Bracteatae Thory. R. bracteata Wendl. 5 Relationships between the sections and R. clinophylla Thory 3 subgenera. The phenogram (Fig. 2) places the Synstylae DC R. brunonii Lindl. 5 roses into two divisions. The second division R. filipes Rehd. & Wils. 1 included only the section Bracteatae. The first R. gentiliana Rehd. & Wils. 1 R. luciae Franch. & Rochebr. 2 division is divided into two groups. Group 1 R. moschata Herrm. 5 includes two subgenera, Hesperhodos (group R. mulliganii Bouleng. 2 A) and Platyrhodon (group B), as well as two R. multiflora Thunb. 15 sections of the subgenus Eurosa, Cassiorhodon R. setigera Michx. 6 (group C1 to C4) and Carolinae (group C5 and R. wichuraiana Crép. 5 D). Group 2 includes five sections of the Pimpinellifoliae DC R. hugonis Hemsl. & Wils. 2 subgenus Eurosa, the sections Banksianae R. omeiensis Rolfe 3 (group E), Synstylae (group F), Chinenses R. primula Bouleng 5 (group G), Pimpinellifoliae (group H), and R. xanthina Lindl. 1 R. sericea Lindl. 3 Laevigatae (group I). Cassiorhodon Dumort The section Bracteatae (2nd division) is (Cinnamomeae DC) R. arkansana Porter.y 1 considered to be allied with Banksianae and R. blanda Ait. 2 Laevigatae due to free and caducous stipules R. californica Cham. & Schechy 2 (Rehder, 1940), although it is easily distin- R. majalis Herrm. 2 guished by its deeply incised stipules, large R. ×paulii Rehd. 1 inflorescence bracts, and woolly receptacle R. rugosa Thunb. 19 (Bean, 1970). Both the isozyme (Kim, 1994) R. woodsii Lindl. 4 and RAPD analyses indicate that these three R. forrestiana Bouleng. 1 R. davurica Pallas 1 sections are distantly related, although the R. macrophylla Lindl. 1 analyses differ from each other in the grouping Carolinae Crép.