Introduction
Lilium tigrinum is distributed nation-wide because of its vigorous growth, its resistance to Fusarium, and its bulbil formation. It is generally known to include both diploid (2n = 2x = 24) and triploid (2n = 3x = 36), making it a polyploid complex species (Hwang et al. 2011; Noda 1978, 1986).
Flavell et al. (1974) mentioned that the genome sizes of angiosperm are different from amount of repetitive DNA contents and not from gene numbers. For example, fungi have low DNA content with 10 ~ 20% of repeated sequences, while plants with high DNA contents have over 50% of repeats. In eukaryotes, various types of repetitive DNA families, including several classes of tandem repeated sequences (such as highly repetitive satellite DNA and minisatellite or microsatellite sequence), transposons, retrotransposons (moderately repetitive sequences), and rRNA genes (moderately repetitive sequences) are scattered in the genome (Sun et al. 1999). The various types of highly to moderately repetitive DNA elements were reported in plant, such as del2 in Lilium spp. (Leeton and Smyth 1993), Tourist in cereals (Bureau and Wessler 1994), and KpnI in Pennisetum spp. (Ingham et al. 1993).
The mobile DNAs termed as retrotransposons existed in the repetitive DNA that forms the large genome in plants (Kumar and Bennetzen 1999). Retrotransposons, which generally constitute the major class of transposable elements in eukaryote, are exist abundant forms as long terminal repeats (LTR) or non-LTR retrotranposons in plant genome (Bennetzen 1996; Grandbastien 1992; Kumar 1996). The repeat sequences occupy a large portion of the plant genome consisting of 30.5% in Brassica (Lim et al. 2005), 50% in rice, 50% in Sorghum (Kim et al. 2005), 78% in maize, 83% in wheat, 92% in rye, and 95% in onion (Flavell et al. 1974).
The Lilium species composed a huge and extensive genome size with haploid DNA content (1C value) varying from 13,400 (L. amabile Palib.) to 46,900 Mbp (L. canadense) (Zonneveld et al. 2005). Lilium species were assumed to have large amounts of repetitive DNA, however, until now, only few highly repetitive DNA sequences, such as del2 (Leeton and Smyth 1993), Bam family sequences (Sentry and Smyth 1985), long terminal repeats (Sentry and Smyth 1989), and Ty1-copia-like retrotransposon (Lee et al. 2013) have been characterized.
Cot analysis, first developed by Britten and Kohne in 1968, is a technique based on the DNA re-naturation kinetics. Cot value is affected by DNA concentration in moles per liter (C0), re-association reaction time in seconds (t), and cation concentration in the buffer. Cot-analysis can produce a series of DNA samples, such as highly repetitive, moderately repetitive, and single/low copy DNA. Through this analysis, estimation of the size and the amount of repetitive or single/ low copy sequences in the genome can be made possible (Peterson et al. 2002). Cot analysis was applied to the FISH karyotype analysis in several Brassica species (Hwang et al. 2009; Wei et al. 2005, 2007). Recently developed Cot-based cloning and sequencing (CBCS) method combining with Cot analysis can be used to identify the unique sequences. CBCS has been applied to analyze highly repetitive sequences in banana (Høibová et al. 2004), Sorghum (Peterson et al. 2002), and maize (Yuan et al. 2003).
The FISH with Cot-1 DNA probe in the present study allowed the characterization of L. tigrinum chromosomes based on the distribution patterns of the repetitive DNAs.
Materials and Methods
Genomic DNA extraction
The extraction of genomic DNA was performed following the CTAB method (Doyle 1991). Briefly, young leaf was grinded in pre-cooled mortar and pestle with liquid nitrogen. Leaf powder was mixed with preheated (65°C) DNA extraction buffer [100 mM Tris-HCl, 20 mM Na2 ethylenediaminetetraacetic acid (EDTA), 1.4 M NaCl and 2% cetyltrimethyl-ammonium bromide (CTAB) with 0.2% (v/v) β-mercaptoethanol (pH 8.0)] and incubated at 65°C for 1 hr. The solutions were mixed with chloroform-isoamylalcohol (v/v, 24 : 1). The samples were centrifuge at 13,000 rpm for 15 min at room temperature. Supernatant was transferred to a new tube and mixed with 0.7 volumes of cold isopropanol by gently inverting. To obtain the DNA pellet, the mixture was centrifuged at 13,000 rpm for 10 min. The pellets were washed with 700 μL of 70% ethanol and dissolved in distilled water.
Genomic DNA shearing, Cot-1 DNA isolation
Genomic DNA shearing and Cot-1 isolation was performed using the method described by Zwick et al. (1997). The genomic DNA was diluted to a concentration of 500 mg • L–1 in 0.3 mol • L–1 NaCl and then samples were autoclaved at 121°C for 10 min to make DNA fragments about 100-1000 bp. Re-annealing reaction was performed using the following Cot- 1 DNA time calculation formula:
Cot = DNA Concentration (moleL–1) × re-annealing time in sec (Ts)
Ts is the re-annealing time measured with following formula:
Ts = 1 / DNA Conc.
Co = (0.500 g • L–1) / (339 g/mol, an average molecular weight for a deoxynucleotide monophosphate) = 1.47 × 10–3 mol • L–1
and therefore:
DNA was re-associated at 62.4°C for calculated reannealing time. Following the time allotted for re-annealing, remove the tube, add the calculated amount of 10x S1 buffer, and mix thoroughly. Add the S1 nuclease and again mix the solution thoroughly, but gently, immediately place the tube in the 23°C water bath for 30 min.
Chromosome preparation, Probe labeling, and FISH analysis
Diploid (2n = 2x = 24) L. tigrinum, collected by Prof. Jong Hwa Kim, Kangwon National Universtiy, was used in this study. Chromosome preparations and Fluorescence in situ hybridization were performed according to Lim et al. (2005). Freshly growing root tips were collected and pretreated with ábromonaphtalene at 20°C for 3 hr, and were fixed in Acetoethanol (Acetic acid : Ethanol=1 : 3, v/v) at room temperature for at least 2 hr. The materials were stored in 70% ethanol solution at –20°C before use. For the chromosome preparations, the root tips were treated with an enzyme mixture (0.3% pectolyase, 0.3% cellulase, 0.3% cytohelicase in 150 mM citrate buffer) at 37°C for 1.5 hr. The root tips were squashed in a drop of 60% acetic acid and then air-dried.
Slides were pre-treated with RNase A (100 μL • mL–1) in 2x SSC for 1 hr at 37°C, and then washed in 2x SSC for three times. Post-fixation was performed in 4% para-formaldehyde solution for 10 min. The Cot-1 DNAs were directly labeled with digoxygenin-11-dUTP by nick translation (Roche, Germany). The hybridization mixture (50% deionized formamide, 10% dextran sulfate, 2x SSC, and 20 μL • mL–1 of probe DNA) was denatured at 70°C for 10min. The slides were denatured at 80°C for 5 min, followed by incubation overnight at 37°C in a humid chamber. After hybridization, the slides were washed in 0.1x SSC at 42°C for 30min and then digoxygenin was detected using FITC conjugated anti-digoxygenin antibodies (Roche, Germany). The chromosomes were then counterstained with 2 μL • mL-1 of 4’, 6-diamidino-2-phenylindole (DAPI) in Vectashield (Vecta laboratories Inc., USA). The chromosomes were observed with Nikon BX 61 fluorescent microscope. Images were captured using Genus image analysis workstation software (Applied Imaging Corporation, Genus version 3.8 program).
Results and Discussion
In the present study, we isolated Cot-1 (highly repetitive) DNA from diploid L. tigrinum (2n = 2x = 24). These repeat fractions were hybridized in the somatic metaphase chromosomes of a diploid L. tigrinum by FISH technique.
Genomic DNA fragmentation
Genomic DNA samples were adjusted to a concentration of 500 mg • L–1 for the experiments to isolate Cot-1. The preparation and shearing of genomic DNA and the isolation of Cot-1 result is shown in Fig. 1. Two aliquots of genomic DNA were autoclaved for 7 min, with all fragments within the desired size range of 100-2000 bp, mainly in 300 to 1000 bp. The isolated Cot-1 showed a size range of approximately 100-1000 bp.
Distribution of Cot-1 signals on diploid L. tigrinum
Flavell et al. (1974) cited that repetitive DNAs that represents more than 100 copies in the genome occupied approximately between 50 ~ 95% in higher plant. Genome size of Lilium species (1C = 13,400 ~ 46,900 Mbp) is remarkably larger than that of tomato (1C = 960 Mbp) (Arumuganathan and Earle 1991) and Brassica (1C = 520 Mbp) (Hwang et al. 2009). In addition, the largest chromosome length was observed to be 30.72 mm in chromosome #1 of L. tigrinum (Hwang et al. 2011), not exceeding 3 mm compared in tomato (Brasileiro-Vidal et al. 2009) and 5 mm in Brassica species (Hwang et al. 2009; Lim et al. 2005). Based on extremely large genome size and chromosome length results in Lilium species, L. tigrinum is also expected to possess a large amount of repetitive sequences.
Cot analysis is one of the powerful tools for examining repetitive DNA in the eukaryote genome. Until now, Cotanalysis was adapted to the Cot-based cloning and FISH karyotype analysis. Cot-based cloning was used to analyze highly repetitive sequences in wheat (Lamoureux et al. 2005; Šimková et al. 2007), in Amaranthus (Sun et al. 1999), and in maize (Yuan et al. 2003). In addition, FISH karyotype using Cot-DNA as probes were conducted to the tomato (Chang et al. 2008) and several Brassica species (Hwang et al. 2009; Wei et al. 2005, 2007).
The FISH with Cot-DNA results are shown in Fig. 2 and 3. Cot-1 was hybridized on overall chromosomes; however, signal intensity was discriminated region by region as decreased intensity were irregularly distributed in between the signals. The regions with less or no Cot-1 hybridization are expected to be euchromatin or low/single copy sequences regions.
Cot-1 signal was weakly hybridized in 1) NOR regions of chromosome #1, #2, and #7 (Fig 3B, red arrow); 2) centromere and pericentromere regions of all chromosomes; 3) DAPI band region in chromosome #9 (Fig 3B, white arrow). 4) In the short arm of chromosome #7, #8, #9, #10, #11, and #12, Cot-1 signal was especially weak or partly observed in the interstitial region (Fig 3B, yellow arrow). It seems that short arm of chromosome #7, #8, #9, #10, #11, and #12 are mostly euchromatin or single/low copy region.
In general, NOR, centromere region and DAPI band were known to comprise the highly repetitive DNAs in plant taxa (Ingle et al. 1975; Lim et al. 2005; Macgregor and Kezer 1971; Trask 1999; Yasmineh and Yunis 1971). Cot-1 DNA was established to consist of highly and moderately repetitive DNA sequences including 45S rDNA and 5S rDNA, as well as several tandem repetitive sequences including centromeric and telomeric repeats (Chang et al. 2008; Chen et al. 2010; Wei et al. 2007). Previous reports of FISH karyotyping in B. oleracea and B. napus stated that Cot-1 DNA predominantly located in NOR and pericentromeric regions (Wei et al. 2005, 2007). Chang et al. (2008) reported the Cot-1 signals in tomato to be situated on the NOR and macrosatellite regions whereas weakly detected in the distal and pericentromere region. Relatively small sized genome plant, such as tomato and Brassica, showed that repetitive sequences concentrated on NORs, centromeric or pericentromeric, and telomeric regions. Interestingly, Cot-1 DNAs were sporadically distributed all over the chromosomes of L. tigrinum, except for pericentromeric region. As well as, in the chromosome #1, #2, #3, #6, and #10, Cot-1 signal was co-localized with DAPI bands but displayed the weak intensity in DAPI band region on chromosome #8 of L. tigrinum. The AT-rich heterochromatin region of chromosome 8 stained by DAPI that were observed to have a weak Cot-1 DNA signal could somehow be implied that the certain repeats in that particular region could be a low copy even though it’s an AT-rich. Joseph et al. (1990) reported that Cot-1 fractions isolated from L. henryi and L. longiflorum abundantly consist of del sequences and 5S rDNA. In our FISH study, Cot-1 DNA was strongly hybridized with 5S rDNA position in the chromosome #3, as well as sequencing data from microdissected chromosome DNA library (Hwang et al. 2015) showed that 57% of total sequences matched with retrotransposons with constituting Del-1 retrotransposon.
In this present study, we established Cot 1-DNA distribution L. tigrinum somatic metaphase chromosomes. FISH with Cot-1 DNA result showed that highly-, moderately- and single/low repetitive sequences are distributed in the all over the L. tigrinum chromosomes. Cot-analysis with FISH technique can make accessible to solve the question of how large genome size in the Lilium species.
