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• W2589373389 abstract "Genomic in situ hybridization (GISH) was used to understand the process of interspecific tulip hybrid diploid (2n = 2x = 24), triploid (2n = 3x = 36) and tetraploid (2n = 4x = 48) cultivar formation. Simultaneous hybridization of differentially labelled genomic probes enabled chromosomes derived from T. gesneriana and T. fosteriana to be distinguished in all cultivars tested. Diploid cultivar ‘Shirayukihime’ contained one set of chromosomes from T. gesneriana and one set from T. fosteriana. In triploid cultivars ‘Come Back’, ‘Diplomate’, ‘Pink Impression’ and ‘Oxford’, 24 chromosomes were derived from T. gesneriana and 12 chromosomes originated from T. fosteriana. The genome of tetraploid cultivar ‘Ollioules’ comprised 36 chromosomes from T. gesneriana and 12 chromosomes from T. fosteriana. In all tulip hybrids, GISH signals were localized predominately in telomeric/subtelomeric and intercalary regions of T. gesneriana chromosomes, whereas T. fosteriana chromosomes were painted more evenly along the chromosome length. In triploid and tetraploid cultivars, cross-hybridization to putative rRNA gene sites was observed at telomeric and intercalary locations of some chromosomes, although additional experiments are needed to identify what kind of repetitive DNA families are arranged as nested elements interspersed in the tulip genome. INTRODUCTION Tulip is one of the most important ornamental crops. Every year many new cultivars are introduced to the market, some of which have been made by interspecific hybridization between genotypes of cultivated tulips (Tulipa gesneriana) and other Tulipa species (Van Eijk et al., 1991; Van Creij et al., 1997). Crosses between T. gesneriana and T. fosteriana are especially successful (Van Eijk et al., 1991; Van Creij et al., 1997), most of which are triploid (2n=36), whereas a few are diploid (2n=24) and tetraploid (2n=48). Vigorous traits such as large flowers, sturdy stems and large plant size are exhibited primarily by the triploid forms. Because of their distinct traits, these vigorous forms were given a classification of their own in the tulip classification list (Killingback, 1990), and are referred to as Darwin Hybrid Tulips. Because most of the diploid forms obtained from crosses between T. gesneriana and T. fosteriana do not show vigorous growth, they are classified in other horicultural groups; for example, ‘Shirayukihime’, is registered under Single Late Tulips. From these crosses, over 50 cultivars of Darwin Hybrid Tulips have been developed so far; most are triploid, one cultivar ‘Spring Song’ is a diploid, and a few are tetraploids (e.g., cultivar ‘Ollioules’ (Van Scheepen, 1996; Okazaki et al., 2005)). The triploid Darwin Hybrid Tulips have spontaneously resulted from unreduced gametes. By studying karyotype in tulips, Marasek et al. (2006) demonstrated that the Darwin Hybrid Tulips tested have two copies of the T. gesneriana genome and one copy of the T. fosteriana genome, indicating that T. gesneriana has supplied the diploid gamete. However, identification of the genome constitution of Darwin Hybrid Tulips using GISH has thus far been limited to the triploid cultivar ‘Yellow Dover’. For most Darwin Hybrid Tulip cultivars, little is known about their genome consitution, especially for the tetraploid form. In addition, it is unknown whether homologous recombination has occurred in polyploid genotypes between T. gesneriana and T. fosteriana chromosomes. 133 Proc. XX Intl. Eucarpia Symp. (Sect. Ornamentals) on Breeding for Beauty (Vol II) Eds. A. Mercuri and T. Schiva Acta Hort. 743, ISHS 2007 The objective of the present study was to use GISH to determine the genome composition in diploid (2n = 2x = 24), triploid (2n = 3x = 36) and tetraploid (2n = 4x = 48) tulip cultivars, which were obtained from interspecific crosses between T. gesneriana and T. fosteriana. MATERIALS AND METHODS Plant Materials The following tulip genotypes were used in this study: diploid cultivar ‘Shirayukihime’ (2n = 2x = 24), which was obtained from the interspecific hybridization between T. gesneriana and T. fosteriana (Kimura, 2002) and belongs to the classification group Single Late Tulips; triploid cultivars ‘Come Back’, ‘Diplomate’, ‘Pink Impression’ and ‘Oxford’ (2n = 3x = 36) (Darwin Hybrid Tulips); and tetraploid cultivar ‘Ollioules’ (Darwin Hybrid Tulips) (2n = 4x = 48). Cultivars were classified according to Van Scheepen (1996). Bulbs were obtained from the Toyama Bulb Growers Association, Japan. Chromosome Preparation Root tips were pre-treated with 0.05% colchicine at 18°C for 2.5 h and fixed in 3:1 (v/v) absolute ethanol:glacial acetic acid and stored at −20C. Root tips were washed in enzyme buffer (0.01 M sodium citrate, pH 4.8) for 20 min prior to digestion in an enzyme mixture containing 2% (w/v) cellulase Onozuka-RS (Yakult Honsha Co., LTD. Japan) and 1% (w/v) pectolyase Y-23 (Kyowa Chemical Products Co., LTD. Japan) for 2.5 h at 37oC. Meristems were squashed in a drop of 45% acetic acid, cover slips were removed by freezing on dry ice and preparations were dehydrated in absolute ethanol and air-dried. DNA Probes and In Situ Hybridization Total genomic DNA from ‘Red Emperor’ (T. fosteriana; 2n = 2x = 24) and ‘Queen of Night’ (T. gesneriana; 2n = 2x = 24) was extracted from the leaves using the standard cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). DNA was sonicated to yield 1–10-kb fragments and labelled by nick translation with either tetramethyl-rhodamine-5-dUTP (T. fosteriana) or digoxigenin-11-dUTP (T. gesneriana) (Roche). Autoclaved T. sacstatila DNA (100–500 bp) was used as blocking DNA in 30–75× excess of the probes. The method for in situ hybridization and probe detection followed Hasterok et al. (2001) with minor modifications. Slides were pre-treated with RNase A (100 μg/ml) for 1 h at 37oC, then washed in 2× SSC, post-fixed in 1% formaldehyde in PBS buffer for 10 min, washed again in 2× SSC and dehydrated in absolute ethanol. The hybridization mixture consisted of 50% deionized formamide, 10% dextran sulphate, 2× SSC, 1% SDS and probe DNA (200 ng of each per slide). The hybridization mixture was denatured at 75oC for 10 min and chilled on ice. Chromosome preparations and pre-denatured probes were denatured at 70oC for 10 min. Hybridization was carried out overnight in a humid chamber at 37oC followed by stringent washes in 2× SSC for 15 min at room temperature and in 0.1× SSC for 30 min at 42oC. The digoxigenated probe was immunodetected using the standard protocol with FITC-conjugated anti-digoxigenin (Roche). The chromosomes were counterstained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma) in Vectashield (Vector Laboratories). Images of fluorescently stained chromosomes were acquired using a digital camera attached to a microscope (BX51N, Olympus, Japan) with an appropriate filter and then processed using software (DP Manager, Olympus, Japan). RESULTS AND DISCUSSION Distribution of GISH Signals Simultaneous application of differentially labelled ‘Queen of Night’ (T. gesneriana) (green) and ‘Red Emperor’ (T. fosteriana) (red) total genomic DNA probes" @default.
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• W2589373389 title "GISH ANALYSIS OF HYBRIDS PRODUCED BY INTERSPECIFIC HYBRIDIZATION BETWEEN TULIPA GESNERIANA AND T. FOSTERIANA" @default.
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