Scientia Horticulturae 144 (2012) 153–160 Contents lists available at SciVerse ScienceDirect Scientia Horticulturae journa l homepage: www.elsevier.com/locate/scihorti Confirmation of hybrid origin of Cyrtanthus based on the sequence analysis of internal transcribed spacer a b c d d,∗ Ae Kyung Lee , Jeong Hong , Gary R. Bauchan , Se Hee Park , Young Hee Joung a Department of Environmental Horticulture, College of Bio-Resources Science, Dankook University, Cheonan, Chungnam, South Korea b Department of Horticulture, Division of Environmental & Life Science, Seoul Women’s University, Nowon-Ku, South Korea c USDA, ARS, BARC-East, Electron and Confocal Microscope Unit, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA d School of Biological Science & Technology, Chonnam National University, Gwangju, South Korea a r t i c l e i n f o a b s t r a c t Article history: The objectives of this study were to create interspecific hybrids between Cyrtanthus elatus and C. san- Received 31 May 2012 guineus and to confirm the hybrid origin of the progeny based on morphological characters and using Received in revised form 4 July 2012 molecular markers. The tip of the leaves, the shape and size of cells, and stomata distribution in abaxial Accepted 5 July 2012 and adaxial surface of leaf epidermis were analyzed by low temperature scanning electron microscope (LT-SEM). Molecular markers generated from random amplification polymorphic DNA (RAPD) and single Keywords: nucleotide polymorphisms (SNPs) analysis of internal transcribed spacer 1 and 2 of rRNA gene (ITS 1, 2) Amaryllidaceae were also analyzed to confirm hybrid status. Putative C. elatus × C. sanguineus hybrid A 3 resembled C. Interspecific hybrids elatus and C. sanguineus ‘Pringle Bay’ (PB) × ‘Fred Meyer’ (FM) B 23 and was similar to C. sanguineus at ITS 1 flowering. However, the hybrid origin of hybrid A 3 could not be confirmed based on the morphological 2 region Low temperature-scanning electron characteristics of flowers, the tip of the leaves, and cell structure and surface images in both abaxial and microscopy adaxial images of LT-SEM. Based on RAPD markers, putative hybrid A 3 and other putative hybrids that Random amplification polymorphic DNA did not flower (A 4, A 5, A 13 and D 39, D 40, and D 42) can be considered as a hybrid based on the presence Single nucleotide polymorphisms of both species-specific bands for parental taxa. SNP markers of ITS 1, 2 region also revealed that other putative hybrids of A and D that did not flower during the evaluation can be interspecific hybrids of C. elatus and C. sanguineus. Therefore, the use of SNP markers of ITS 1, 2 is considered a more accurate tool to characterize hybrid origin than morphological characteristics and RAPD markers. © 2012 Elsevier B.V. All rights reserved. 1. Introduction A new hybrid with a flower shape intermediate between C. elatus and C. sanguineus or similar to C. elatus Jacq. Traub with relatively The genus Cyrtanthus Aiton (Amaryllidaceae) is native to South easy flowering as observed in C. sanguineus would be desirable for Africa with waxy tubular white, orange, and red flowers formed as pot plant use. Therefore, it would be ideal to combine the free clusters at the tip of scape (Koopowitz, 1986; Reid and Dyer, 1984). flowering characters inherited from C. sanguineus and multiple Cyrtanthus elatus (Jacq.) Traub, C. makenii Baker, and C. sanguineus flowers (4–5 flowers) and strong scape from C. elatus by inter- Lindl. Walp. among about 50 species in the genus, have been culti- specific hybridization. Over the years, self- and cross-pollination vated for many years. Induction of flowering of C. makenii following of these two species did not yield viable seeds, perhaps due to some chilling treatment received outdoors before moving into a self-incompatibility in these species. In 2002, when controlled pol- ◦ 20 C greenhouse was reported (Mori et al., 1990). Cyrtanthus san- linations between C. sanguineus and C. elatus were made, seeds were ◦ guineus flowered following 6 weeks of 5 C treatment and C. ‘Orange obtained from C. elatus × C. sanguineus. Floral development has not Gem’ × C. eucallus R. A. Dyer can be induced to flower after water been investigated in many Cyrtanthus species, except the report ◦ stress (Lee, personal observation). However, controlled flowering that flower bud abortion occurred at 22 C in C. elatus (Van Nes and of C. elatus and C. sanguineus for year-round production with con- vonk Noordegraaf, 1977). trolled cultural practices such as temperature, photoperiod, and When two hybrid plants flowered in 2011, one putative inter- plant growth regulators has not been reported. specific hybrid A 3 from C. elatus × C. sanguineus resembled flowers of C. elatus and one intraspecific hybrid B 23 from C. sanguineus ‘Pringle Bay’ (PB) × ‘Fred Meyer’ (FM) was similar to C. sanguineus. Based on the morphological characteristics of leaves during cul- ∗ ture and of flowers, it was not possible to determine definitely Corresponding author. Tel.: +82 62 530 5202; fax: +82 62 530 2199. E-mail addresses: [email protected], [email protected] (Y.H. Joung). whether this putative hybrid A 3 was of a hybrid origin although 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.07.010 154 A.K. Lee et al. / Scientia Horticulturae 144 (2012) 153–160 Table 1 Species and cross pollination, designation and lane numbers, and the presence of species specific bands, and also morphological characters of tips and mid-ribs in mature leaves. a b c Species/hybrids Designation (Fig. 4) Lane numbers in Fig. 4 Morphological characteristics Tip Vein Female Male Female Male C. elatus CE 1, 42 1, 42 1, 42 C. sanguineus CS 24 24 24 × C. elatus C. sanguineus A 2–15 3, 5 2 2, 5 D 39–41 39, 40 39 C. sanguineus PB × FM B 16–23 16–23 16–23 C. sanguineus FM × PB C 25–36 34 25–34, 36 25–34, 36 C. herrei CH 43 a C. sanguineus from Fred Meyer (FM) and Pringle Bay (PM). b Designation of cross pollination and lane number in Fig. 4. c Morphological characteristics of sample number 1, 3, and 5, for example, resemble C. elatus and of sample number of 17–23, for example, resemble C. sanguineus. hybrids showed mosaic morphological patterns either close to after anthesis. Clonal selections of C. sanguineous, obtained from each parental species, or intermediate with extreme characters Waldo Essen, Pringle Bay, South Africa (PB) and Fred Meyer, Cali- (Rieseberg and Ellstrand, 1993). fornia, USA (FM) were also pollinated; C. sanguineus PB × FM and C. Morphological characters are often used to establish that spe- sanguineus FM × PB. Seeds were sown in the greenhouse in Metro cific plants have a hybrid origin. Characters such as the shape of Mix 200 and germinated in 35 days in 2006. After germination, the tip of the leaf in Tsuga (Roh et al., 2007), can be used to iden- seedlings were grown in pots in a greenhouse for 3 years. Four- tify the species. However, the shapes of the apices of needles from teen and 3 seedlings were obtained from C. elatus × C. sanguineus young plants could not be used as a possible key character to iden- capsule A and D, respectively, 8 seeds from C. sanguineus PB × FM tify T. dumosa (D. Don) Eichler in Engler & Prantl. and T. chinensis (capsule B), and 14 seeds from C. sanguineus FM × PB (capsule C). var. forrestii (Downie) Silva. The shape of the epidermis cells was Bulbs were harvested (April, 2009) and were stored dry in a paper used to separate taxa in the genus Lilium (Lee, 1989). When the bag for 18 months (Table 1). Bulbs were planted in October, 2010 ◦ ◦ anatomy of leaf cross sections of the mid-rib in the genus Lilium and cultivated in a greenhouse maintained at 21–24 C/16–18 C, was investigated, L. hansonii Leich. had no apparent raised mid-rib day/night. One bulb was planted per 12.5 cm pot, except C. elatus and L. tsingtauense Gilg. had a raised mid-rib. However, no differ- and C. herrei (Leighton) R.A. Dyer which was planted in 15 cm and ences were apparent on the surface image and undulation of cell 21 cm pots, respectively. shape between these two species (Lee, 1989). Molecular markers generated by random amplification poly- 2.2. Identification of hybrid origin based on the morphological morphic DNA (RAPD) and Single nucleotide polymorphisms (SNPs) characteristics of the nuclear ribosomal DNA (nrDNA) and chloroplast DNA (cpDNA) have been used to verify and determine whether seedlings At anthesis, leaves from putative interspecific C. elatus × C. san- are of hybrid origin in Pulsatilla tongkangensis (Lee et al., 2010), guineus hybrid A 3 (putative hybrid A 3) resembling the female Arisaema (Lee et al., 2011), and many other plants (Roh et al., 2007, parent C. elatus, and intraspecific C. sanguineus PB × FM hybrid B 23 2008). Single nucleotide polymorphisms (SNPs) and DNA sequence (hybrid B 23) (Fig. 1), and parental taxa were collected during the variations occurring with a single nucleotide (Brooks, 1999) have third week of April, 2011. The abaxial and adaxial surfaces between been useful to study the genetic variation and hybridization among the mid-rib and leaf margin at the mid-point of mature leaves were species, and sequence analysis of cpDNA, and nuclear internal observed using low temperature scanning electron microscopy as transcribed spacers (ITS) were used in Anemone and related gen- described by Roh et al. (2012). The distribution of stomata from both era (Ehrendorfer and Samuel, 2001; Roh et al., 2007; Schuettpelz × leaf surfaces was observed from the images at 100 and the number et al., 2002).