Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.23 No.4 pp.250-254

Chromosome Characterization of Lilium tigrinum based on Cot DNA Analysis

Yoon-Jung Hwang*
Chromosome Research Institute, Department of Life Science, Sahmyook University, Seoul 139-742, Korea
Corresponding author: Yoon-Jung Hwang Tel: +82-2-3399-1718
October 19, 2015 November 4, 2015 November 4, 2015


The genus Lilium has a huge nuclear genome size (approximately 13,400 to 46,900 Mbp), implying that Lilium genome is composed of a larger amount of repetitive sequences. To understand the organization of plant genome is required to observe repetitive DNA sequences found in the Lilium chromosome. In this study, Cot DNA analysis was introduced, in which repeated DNAs were assessed. The Cot analysis revealed that Cot-1 DNAs were a target region that contained highly and moderately repetitive sequences. In addition, Cot-1 DNA as a probe in fluorescence in situ hybridization (FISH) was used to detect somatic chromosomes at the metaphase stage of diploid (2x) Lilium tigrinum. The FISH analysis showed that bright fluorescent signals on the Cot-1 DNA were sporadically distributed in all over the L. tigrinum-chromosomes. However, relatively weak signals were displayed in nucleolarorganizing regions (NORs) of chromosome #1, #2, and #7 as follows: centromere and peri-centromere regions of all chromosomes; distinct DAPI band region in long arm of chromosome #8; and short arm of chromosome #7, #8, #9, #10, #11, and #12. In conclusion, the random Cot-1 DNA distribution pattern has proven that L. tigrinum genome is composed of dispersed repetitive DNAs.


    Rural Development Administration
    No. PJ010448


    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)

    Cot−1=1=mol L −1 xTs

    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:

    T=1/ 1.47 × 10 −3 =680.27sec

    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.



    Genomic DNA shearing was performed with autoclaving for 7 min to make 100 to 1000 bp DNA fragment length.


    Application to FISH analysis with Cot-1 (green fluorescence) probe to the somatic metaphase chromosome of diploid L. tigrinum. White, red, and yellow arrows indicate centromere, NOR, and DAPI band positions, respectively.


    Chromosomal distribution of Cot-1 DNAs in diploid L. tigrinum. Chromosomes were counterstained with 4’, 6’-Diamidino-2-phenylindole (A) and hybridized with Cot-1 signals (B, Green fluorescence). Red, white, and yellow arrows indicate weakly detected signals of centromere, DAPI band, and short arm chromosome positions, respectively. Bars = 10 μm.



    1. Arumuganathan K , Earle ED (1991) Estimation of nuclear DNA content of plants by flow cytometry , Plant Mol Biol Rep, Vol.9 ; pp.208-218
    2. Bennetzen JL (1996) The contributions of retroelements to plant genome organization, function and evolution , Trends Microbiol, Vol.4 ; pp.347-353
    3. Brasileiro-Vidal AC , Melo-Oliveira MB , Carvalheira GMG , Guerra M (2009) Different chromatin fractions of tomato (solanum lycopersicum L.) and related species , Micron, Vol.40 ; pp.851-859
    4. Britten RJ , Kohne DE (1968) Repeated sequences in DNA , Science, Vol.161 ; pp.529-540
    5. Bureau TE , Wessler SR (1994) Mobile inverted-repeat elements of the Tourist family are associated with the genes of many cereal grasses , Proc Natl Acad Sci USA, Vol.91 ; pp.1411-1415
    6. Chang SB , Yang TJ , Datema E , Van Vugt J , Vosman B , Kuipers A , Meznikova M , Szinay D , Lankhorst RK , Jacobsen E , De Jong JH (2008) FISH mapping and molecular organization of the major repetitive sequences of tomato , Chromosome Res, Vol.16 ; pp.919-933
    7. Chen Y , Guan L , Liu H , Li G , Qin R (2010) A new protocol for extraction of C0t-1 DNA from rice , Afr J Biotechnol, Vol.9 ; pp.4482-4485
    8. Doyle JJ Hewitt GM (1991) DNA protocols for plants-CTAB total DNA isolation , Molecular technique in taxonomy, Springer, ; pp.283-293
    9. Flavell RB , Bennett MD , Smith JB , Smith BD (1974) Genome size and the proportion of repeated nucleotide sequence DNA in plants , Biochem Genet, Vol.12 ; pp.257-269
    10. Grandbastien MA (1992) Retroelements in higher-plants , Trends Genet, Vol.8 ; pp.103-108
    11. Høibová E , Macas J , Neumann P , Doležel J (2004) Isolation of highly repetitive DNA sequences in Musa acuminate using reassociation kinetics , In. Abstr. 1st, Int Congr Musa,
    12. Hwang YJ , Kim HH , Kwon SJ , Yang TJ , Ko HC , Park BS , Chung JD , Lim KB (2009) Karyotype analysis of three Brassica species using five different repetitive DNA markers by fluorescence in situ hybridization , Korean J Hort Sci Technol, Vol.27 ; pp.456-463
    13. Hwang YJ , Kim HH , Kim JB , Lim KB (2011) Karyotype analysis of Lilium tigrinum by FISH , Hort Environ Biotechnol, Vol.52 ; pp.292-297
    14. Hwang YJ , Lim KB (2011) Development of microdissection and chromosome specific genomic library in Lilium tigrinum , Genes and Genomics, Vol.33 ; pp.451-455
    15. Hwang YJ , Yang TJ , Kim HH , Younis A , Lim KB (2015) Random PCR of Micro-dissected chromosome amplified predominantly repeated DNA in Lilium tigrinum , Int J Agric Biol, Vol.17 ; pp.169-174
    16. Ingham LD , Hanna WW , Baier JW , Hannah LC (1993) Origin of the main class of repetitive DNA within selected Pennisetum species , Mol Gen Genet, Vol.238 ; pp.350-356
    17. Ingle J , Timmis JN , Sinclair J (1975) The relationship between satellite deoxyribonucleic acid, ribosomal ribonucleic acid gene redundancy and genome size in plants , Plant Physiol, Vol.55 ; pp.496-501
    18. Joseph JL , Sentry JW , Smyth DR (1990) Interspecies distribution of abundant DNA sequences in Lilium , J Mol Evol, Vol.30 ; pp.146-154
    19. Kim JS , Islam-Faridi MN , Klein PE , Stelly DM , Price HJ , Klein RR , Mullet JE (2005) Comprehensive molecular cytogenetic analysis of Sorghum genome architecture: Distribution of euchromatin, heterochromatin, genes and recombination in comparison to Rice , Genetics, Vol.171 ; pp.1963-1976
    20. Kumar A (1996) The adventures of the Ty-1-copia group of retrotransposons in plants , Trends Genet, Vol.12 ; pp.41-43
    21. Kumar A , Bennetzen JL (1999) Plant retrotransposons , Ann Rev Genet, Vol.34 ; pp.479-532
    22. Lamoureux D , Peterson DG , Li W , Fellers JP , Gill BS (2005) The efficacy of Cot based gene enrichment in wheat (Triticum aestivum L.) , Genome, Vol.48 ; pp.1120-1126
    23. Lee SI , Park KC , Son JH , Hwang YJ , Lim KB , Song YS , Kim JH , Kim NS (2013) Isolation and characterization of novel Ty1-copia-like retrotransposon form lily , Genome, Vol.56 ; pp.1-9
    24. Leeton PRJ , Smyth DR (1993) An abundant LINE-like element amplified in the genome of Lilium speciosum , Mol Gen Genet, Vol.237 ; pp.97-104
    25. Lim KB , De Jong JH , Yang TJ , Park JY , Kwon SJ , Kim JS , Lim MH , Kim JA , Jin M , Jin YM , Kim SH , Lim YP , Bang JW , Kim HI , Park BS (2005) Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa , Mol Cells, Vol.19 ; pp.436-444
    26. Macgregor HC , Kezer J (1971) The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus , Chromosoma, Vol.33 ; pp.167-182
    27. Noda S (1978) Chromosomes of diploid and triploid forms found in the natural populations of tiger lily in Isushima , Bot Mag Tokyo, Vol.91 ; pp.279-283
    28. Noda S (1986) Cytogenetics behavior, chromosome differentiations, and geographic distribution in Lilium lancifolium (Liliaceae) , Plant Species Biol, Vol.1 ; pp.69-78
    29. Peterson DG , Schulze SR , Sciara EB , Lee SA , Bowers JE , Nagal A , Jiang N , Tibbitts DC , Wessler SR , Paterson AH (2002) Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery , Genome Res, Vol.12 ; pp.795-807
    30. Sentry JW , Smyth DR (1985) A family of repeated sequences dispersed through the genome of Lilium henryi , Chromosoma, Vol.92 ; pp.149-155
    31. Sentry JW , Smyth DR (1989) An element with long terminal repeats and its variant arrangements in the genome of Lilium henryi , Mol Gen Genet, Vol.215 ; pp.349-354
    32. Šimková H , Janda J , H ibová E , Šsfá J , Doležel J (2007) Cotbased cloning and sequencing of the short arm of wheat chromosome 1B , Plant Soil Environ, Vol.53 ; pp.437-441
    33. Song NH (1987) Analysis of C-banded karyotypes and chromosomal relationships of Lilium species , PhD thesis. Kyungpook National University. Korea,
    34. Sun M , Chen H , Leung FC (1999) Low-Cot DNA sequences for fingerprinting analysis of germplasm diversity and relationships in Amaranthus , Theor Appl Genet, Vol.99 ; pp.464-472
    35. Trask B Birren B , Green ED , Heiter P , Klapholz S , Riethman H , Roskams J (1999) Florescence in situ hybridization , Genome analysis: A laboratory manual vol: 4 Mapping genomes, Cold spring Harber Laboratory, Cold Spring Harbor, ; pp.391-404
    36. Wei WH , Zhao WP , Wang LJ , Chen B , Li YC , Song YC (2005) Karyotyping of Brassica napus L. based on Cot-1 DNA banding by fluorescence in situ hybridization , Journal of Integrative Plant Biology, Vol.47 ; pp.1479-1484
    37. Wei WH , Zhang SF , Wang LJ , Chen B , Wu XM , Song YC (2007) Karyotyping of Brassica oleracea L. based on Cot-1 and ribosomal DNAs , Botanical Studies, Vol.48 ; pp.225-261
    38. Yuan Y , SanMiguel PJ , Bennetzen JL (2003) High-Cot sequence analysis of the maize genome , Plant J, Vol.34 ; pp.249-255
    39. Yasmineh WG , Yunis JJ (1971) Satellite DNA in calf heterochromatin , Exp Cell Res, Vol.64 ; pp.41-48
    40. Zwick MS , Hanson RE , McKnight TD , Islam-Faridi MH , Stelly DM , Wing RA , Price HJ (1997) A rapid procedure for the isolation of Cot-1 DNA from plants , Genome, Vol.40 ; pp.138-142
    41. Zonneveld BJM , Leitch IJ , Bennett MD (2005) First nuclear DNA amounts in more than 300 angiosperms , Ann Bot, Vol.96 ; pp.229-244