Introduction

Plant chromosomes contain a number of complex repetitive sequences, including retroelements, which influence their genome sizes and chromosome evolution [3, 11, 12, 14, 15]. Fluorescence in situ hybridization (FISH) is frequently used to visualize chromosomal distribution of these repetitive elements in plants [2, 7, 9, 13, 24] as a powerful tool for comparative genomics at the microscopic level. In addition, FISH using bacterial artificial chromosome (BAC) probes (BAC FISH) is a useful cytogenetic technique for physical mapping and chromosome marker screening. Because each plant BAC clone usually carries repetitive sequences, random BAC FISH without competitive DNA can visualize complex chromosome organization [18, 20].

The family Orchidaceae is a highly evolved and largest angiosperm family having enormous number of species. Their hybrids often retain its enchanting and splendid flowers. The economically important orchid hybrids have a big share in cut-flower industries worldwide. The genome and transcriptome data of some Orchidaceae plants have been reported recently [1, 4, 16, 22, 23, 25]. Therefore, molecular breeding of orchids has begun vigorously in Asia. Many Asian researchers are interested in the orchid molecular cytogenetics, because their chromosome number is not so variable and inter-species hybrids are easily obtained. Molecular cytogenetic techniques are undoubtedly valuable tools to elucidate genome organization in orchids; BAC-FISH analysis has important contribution in chromosome-based comparisons.

We are interested in the intergeneric hybrid of orchid, eg. Neostylis Lou Sneary, which is an economically valuable orchid hybrid of Neofinetia falcata (2n = 38) and Rhynchostylis coelestis (2n = 38). These two parental species are both aerophyte native to East Asia, which possess beautiful flowers (Fig. 1). Especially, N. falcata is historically famous in Japan and called as “Furan.” In this study, to elucidate the genomic and chromosomal organization of the highly crossable N. falcata and R. coelestis, we constructed BAC libraries of these plants, and performed BAC-FISH analysis. These BAC libraries can be further used as important tools for molecular cytogenetics in the orchid hybrids.

Fig. 1
figure 1

Flowers of two orchid plants, Neofinetia falcata (a) and Rhynchostylis coelestis (b)

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Materials and methods

Plant materials

Neofinetia falcata (2n = 38) and Rhynchostylis coelestis (2n = 38) were grown at room temperature. Young leaves and roots were used to isolate high-molecular-weight (HMW) DNA and chromosomal preparation, respectively.

Chromosomal preparation

Root tips were pretreated with 2 mM 8-hydroxyquinoline for 5 h at 14 °C. The pretreated root tips were fixed in ethanol/glacial acetic acid (3:1) for several days, and then squashed in 45 % acetic acid on glass slides as described by Mukai et al. [8].

Construction of BAC libraries

BAC libraries were constructed according to the conventional method as described before [5, 10, 18, 20, 21]. HMW DNA embedded in agarose plugs was prepared by the typical rapid procedure [17, 20], and was partially digested with HindIII (Takara) at 37 °C for 12 or 20 min. Partially-digested DNA was then subjected to pulsed field gel electrophoresis (PFGE) using a CHEF mapper (Bio-Rad) with a 1 % low melting point agarose gel (SeaPlaque GTG, FMC) in 0.5× TBE buffer with the conditions of 6 V/cm, constant linearly ramped pulse time of 90 s for 4 h at 14 °C, followed by 6 V/cm, constant linearly ramped pulse time of 6 s for 12 h at 14 °C. The target sizes of DNA fragments were released from the selected fractions (around 150 kb) of the gel with ß-Agarase I (New England Biolabs), and ligated into the 8.2-kb HindIII-digested CopyControl™ pCC1BAC™ vector (Epicentre) with T4 ligase (Promega). Subsequently, the ligated DNA was electroporated into Escherichia coli strain ElectroMAX DH10B (Invitrogen) using a Gene Pulser II (Bio-Rad) under the conditions of 1.25 or 1.5 kV, 25 μF, and 100 Ω. Recombinants were clearly distinguished from nonrecombinants on an LB agar plate containing 12.5 mg/l chloramphenicol, X-gal and IPTG after 20–24 h of incubation at 37 °C. Insert size of BAC DNA was determined by NotI digestion followed by PFGE.

Probe labeling and BAC-FISH analysis

Randomly selected BAC colonies were inoculated into 3 ml of LB with 12.5 mg/l chloramphenicol, and shaken at 37 °C overnight. BAC DNA purified by the standard alkali method was dissolved in 20 μl of TE, and the 4-μl aliquot was labelled with digoxigenin-11-dUTP or biotin-16-dUTP using a nick translation kit (Roche Diagnostics). For the FISH analysis, chromosomal DNA was denatured in 70 % formamide-2×SSC for 2 min at 69 °C and dehydrated in an ethanol series at -20 °C. The 10 μl of hybridization mixture (50 % formamide, 10 % dextran sulfate, 50 μg of blocking DNA, and 2×SSC, with 2.5 μl of the labelled BAC DNA), which had been denatured for 10 min at 100 °C and cool on ice for 10 min, was applied to each slide. Overnight hybridization was done in a moist chamber at 37 °C. After hybridization, the slides were washed in 2×SSC at room temperature for 5 min, 50 % formamide-2×SSC at 37 °C for 15 min, 2×SSC at room temperature for 15 min, 1×SSC at room temperature for 15 min, and 4×SSC at room temperature for 5 min. Biotin and digoxigenin were simultaneously detected with fluorescein isothiocyanate (FITC)-conjugated avidin (Roche Diagnostics) and rhodamine-conjugated sheep anti-digoxigenin Fab fragment (Roche Diagnostics), respectively. Slides were incubated in 2 μg/ml FITC-conjugated avidin and 2 μg/ml rhodamine-conjugated antidigoxigenin in detection buffer consisting of 4×SSC-1 % BSA for 1 h at 37 °C. After incubation, the slides were washed in 4×SSC for 10 min, 0.1 % Triton X-100 in 4×SSC for 10 min, 4×SSC for 10 min, and 2×SSC for 5 min, all at room temperature. Slides were mounted in a fluorescence antifade solution (1.25 % DABCO, 90 % glycerol). DAPI was used as chromosome DNA counterstaining in the antifade solution at 200 ng/ml. Each fluorescent signal on the slide was captured with an Axioskop fluorescence microscope (Zeiss) coupled to a cooled CCD camera (Hamamatsu Photonics, model 4880). Images were pseudo-colored and merged using Photoshop software (Adobe).

Results and discussion

The BAC libraries of N. falcata and R. coelestis were constructed from HindIII partially digested DNA by using the 8.2-kb pCC1BAC vector. In total, 21,000 and 10,600 clones were selected as glycerol stocks for N. falcata and R. coelestis BAC libraries, respectively. For the glycerol stocks, we used the 100-clone pooled procedure [19], which can be used for polymerase chain reaction (PCR) screening. Average inset sizes of N. falcata and R. coelestis BAC clones were 74.6 and 50.8 kb, respectively. Because the most abundant range of insert sizes was from 40 to 60 kb (Fig. 2), we concluded that the BAC clones were sufficient for FISH probes. Total insert sizes were calculated as 1.57 and 0.54 Gb in N. falcata and R. coelestis, respectively. Leitch et al. [6] estimated the DNA amounts corresponding to the 1C value of Neofinetia and Rhynchostylis as 2.4 and 2.6 pg, which can be converted to 2.35 and 2.54 Gb of genome sizes (using 1 pg = 978 Mb), respectively. Accordingly, our BAC libraries of N. falcata and R. coelestis estimate 0.67 and 0.21 genome coverage, respectively.

Fig. 2
figure 2

The insert size of the two orchid BAC libraries. a Distribution of the insert size in 125 BAC clones in Neofinetia falcata. b Representative results of PFGE analysis of the NotI-digested BAC clones in Rhynchostylis coelestis. Each insert DNA fragment migrated more slowly than the common pCC1BAC vector band (Vector)

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Since we have previously reported that localization of repetitive DNA can be detected by BAC-FISH analysis without adding competitive DNA [18, 20], the same strategy was applied to the two orchid species. In N. falcata, 106 BAC clones were applied to the BAC-FISH analysis (Table 1). Almost half of the BAC clones showed dispersed repetitive signals on the chromosomes; this result was more similar to asparagus-type than onion-type of the BAC-FISH patterns [20]. It might be possible to speculate that 32 clones showing no signal might contain unique sequences, because high-quality chromosomal preparation in orchid was quite difficult to obtain higher resolution signals by the BAC-FISH analysis. We found comparatively high proportion (ca. 20 %) of clones showing proximal and distal BAC FISH signals, suggesting that chromosomal region specific localization of repetitive sequences is rather common in N. falcata.

Table 1 BAC FISH results of randomly selected clones in Neofinetia falcata
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Figure 3 showed the representative results of the BAC FISH analysis. Two different BAC signals of N. falcata were simultaneously detected on the N. falcata chromosomes (Fig. 3a), showing that BACs contain respective repetitive sequence(s) preferentially distributed to proximal and distal regions of all chromosomes. Similarly, signals from one BAC clone of R. coelestis were appeared on the distal regions of the most chromosomes (Fig. 3b). The distribution patterns of repetitive sequences are also known in other plants (eg. asparagus, onion, and wheat) [18, 20].

Fig. 3
figure 3

Representative BAC FISH results in Neofinetia falcata (a) and Rhynchostylis coelestis (b). a FISH signals from two BAC clones, C-2 (66 kb) and A-53 (65 kb), were simultaneously detected in proximal (red) and distal (green) regions of N. falcata chromosomes, respectively. b Distal repetitive signals (red) from a B-15 BAC clone (125 kb) were detected on R. coelestis chromosomes (blue). The Bars represent 10 μm

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In the present study, we constructed BAC libraries of N. falcata and R. coelestis, and BAC-FISH revealed the chromosomal distribution of the orchid repetitive sequences. A previous study reported that centromeric BAC clones were obtained from a tobacco BAC library whose cloning site was HindIII [10]. Because our orchid BAC libraries were also used the HindIII digestion for cloning, we will try to isolate the centromeric DNAs in Neofinetia and Rhynchostylis, which are useful for comparative cytogenetic studies in their hybrids.