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. 2013 May 10;112(1):123–134. doi: 10.1093/aob/mct103

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

Rabeya Begum 1, Falk Zakrzewski 2, Gerhard Menzel 2, Beatrice Weber 2, Sheikh Shamimul Alam 1, Thomas Schmidt 2,*
PMCID: PMC3690992  PMID: 23666888

Abstract

Background and Aims

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification.

Methods

A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling.

Key Results

Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species.

Conclusions

The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Keywords: Corchorus olitorius, Corchorus capsularis, jute, karyotype, satellite DNA, FISH, physical mapping, DNA methylation, immunolabelling

INTRODUCTION

Jute is a dicotyledonous plant belonging to the genus Corchorus of the family Malvaceae (Sinha et al., 2011). At the end of the 18th century, it was identified as an alternative fibre crop to Cannabis sativa L., the European hemp (Ghosh, 1983). Today, jute is the world's second most cultivated fibre crop next to cotton, and mostly distributed in the tropical and sub-tropical regions of Africa, America, Australia and Asia (Kundu, 1951). India and Bangladesh rank as the top two countries for jute production. Although >100 different species of the genus Corchorus have been classified, only two species, namely Corchorus capsularis L. (White jute) and Corchorus olitorius L. (Tossa jute), are widely cultivated (Sarker et al., 2007). Both C. capsularis and C. olitorius differ in growth and branching habit, and characteristics relating to leaf, flower, fruit, seed and bast fibre properties, and photosensitivity. As a self-pollinated crop, jute contains only limited genetic variability with respect to adaptability to agronomic environments, fibre quality and yield, as well as susceptibility to diseases and pests (Basu et al., 2003).

Despite the agronomic importance of jute, only a few molecular data are available (Alam et al., 2010; Ahmed et al., 2011; Sharmin et al., 2011). These studies were mostly focused on the estimation of the genetic diversity of cultivated jute accessions and wild jute species. Molecular markers were mainly based on simple and intersimple sequence repeat (SSR, ISSR) polymorphisms and RAPD (random amplified polymorphic DNA), and showed the genetic diversity of jute species (Mir et al., 2008). Moreover, a genetic linkage map with very low marker density has been constructed which consists of six linkage groups containing 55 SSR markers (Das et al., 2012). Concerning the repetitive DNA of the jute genome, only the heterogeneity of reverse transcriptase gene domains of long terminal repeat (LTR) retrotransposons has been investigated (Ahmed et al., 2011).

Recently, the genome size of a wide range of jute accessions has been analysed by flow cytometry, revealing a plasticity of the nuclear genome and sizes ranging from 430 to 460 Mbp in C. olitorius and from 390 to 396 Mbp in C. capsularis (Benor et al., 2011).

Jute cultivars are diploid (2n = 2x = 14) while wild Corchorus species show higher ploidy levels; however, the chromosomes of Corchorus species are only poorly characterized and their unequivocal discrimination is not possible. Reports about chromosome identification by fluorochrome staining are ambiguous (Alam and Rahman, 2000).

Although the jute genome sizes are only approximately three times the size of the Arabidopsis thaliana genome and similar in size to rice, there are only very few reports about the presence and structure of the repetitive DNA (Huq et al., 2009; Alam et al., 2010; Ahmed et al., 2011). The repetitive DNA, with repeating motifs from 2 bp to >10 kb, constitutes the main fraction of plant genomes and can account for 85 % of the nuclear DNA (Schnable et al., 2009). While satellite DNA is highly amplified and arranged in long monotonous arrays, dispersed repetitive DNA sequences are scattered throughout the genome, interspersed with other sequences and distributed over all or most chromosomes. The majority of dispersed DNA sequences originate from transposable DNA sequences, in particular from retrotransposons (Bennetzen, 2009).

High-throughput sequencing technologies provide a valuable resource for the bioinformatic identification of repetitive DNA families, but only for genomes which were sequenced and are publicly available. Patterns of the long-range organization of repeats cannot be resolved by current sequencing technologies, but can be unequivocally determined by fluorescent in situ hybridization (FISH). FISH is a powerful approach to study chromosome and genome structure, and the organization and distribution of repetitive DNA families. Moreover, the method enables comparative physical mapping of repeated DNA families in related species.

Here, we describe the characterization of a major satellite DNA family and retrotransposon sequences which are present in the genome of both jute cultivars. The abundance, genomic organization and diversity of these repeat families were analysed by sequencing and comparative Southern hybridization. The physical mapping of the chromosome-specific satellite arrays and partial LTR retrotransposons in combination with 18S–5·8S–25S rRNA genes and 5S rRNA genes enabled the discrimination of all chromosomes in both species and provides FISH karyotypes for C. olitorius and C. capsularis.

MATERIALS AND METHODS

Plant material and genomic DNA extraction

Seeds of the cultivated jute species Corchorus capsularis ‘CVL-1’ (2n = 2x = 14) and C. olitorius ‘O-4’ (2n = 2x = 14) were kindly provided by the Bangladesh Jute Research Institute (Dhaka, Bangladesh). Seedlings were grown under greenhouse conditions. Genomic DNA was isolated from young leaves using the CTAB (cetyltrimethylammonium bromide) standard protocol (Saghai-Maroof et al., 1984).

Generation of Corchorus-specific plasmid libraries and clone sequencing

Genomic DNA of C. olitorius and C. capsularis was digested with the restriction endonuclease AluI and separated by agarose gel electrophoresis. DNA fragments corresponding to a size range from 100 to 500 bp were purified from the gel using the QIAquick gel extraction kit (Qiagen) and ligated in the SmaI site of the pUC18 vector. After transformation, clones of each species were collected and stored in 384-well plates. The plasmid libraries were transferred to Hybond-N+ nylon membrane (GE Healthcare) and hybridized with radioactively labelled genomic DNA from both Corchorus species. Plasmid clones containing repetitive sequences were identified by the signal strength and further analysed.

Sequence analyses

Plasmid clones were sequenced on the CEQ 8000 capillary sequencer (Beckman) using M13 universal primers. Raw sequence data were analysed with Geneious software. Sequence alignments were generated by Geneious 6 (http://www.geneious.com/), using the MUSCLE algorithm (Edgar, 2004). The diversity of satellite repeats was analysed by the maximum parsimony algorithm using the MEGA software version 5 (Tamura et al., 2011).

PCR amplification and cloning

Monomers and multimers of the satellite DNA family CoSat I were amplified from 100 ng of template DNA from C. olitorius and C. capsularis using the primer combination forward 5′-TAGTTAGGCCATAAACAATGG-3′ and reverse 5′-TCATTTTGGTGAGTTAGTCC-3′. Polymerase chain reactions were performed using GoTaq DNA polymerase (Promega). Standard PCR conditions were 94 °C for 2 min, followed by 30 cycles of 94 °C for 20 s, annealing for 30 s at 54 °C, 72 °C for 20 s and a final incubation at 72 °C for 5 min. After gel electrophoresis, PCR fragments were purified with the QIAquick gel extraction kit (Qiagen) and ligated into the pGEM-T vector (Promega). Plasmid clones were sequenced on the CEQ 8000 capillary sequencer.

Chromosome preparation

Primary roots were collected from seedlings after germination on wet filter paper, incubated in 2 mm 8-hydroxyquinoline for 2 h at room temperature, fixed in fixative solution (ethanol:glacial acetic acid = 3:1) for 15 min at 4 °C, and stored in 70 % ethanol at 4 °C for use. Fixed roots were washed in enzyme buffer (10 mm citric acid–sodium citrate, pH 4·6) and macerated at 37 °C for 25 min in an enzyme mixture containing 1·25 % (w/v) pectinase from Aspergillus niger (Sigma), 1·25 % (w/v) cellulase Onozuka R 10 (Serva) and 1·25 % (w/v) pectolyase from Aspergillus japonicus (Sigma). The suspension was dropped onto pre-cleaned glass slides as described by Schwarzacher and Heslop-Harrison (2000).

Labelling of FISH probes

For physical mapping of rDNA sites, the probe pZR18S (accession no. HE578879) containing a 5066 bp fragment of the sugar beet 18S–5·8S–25S rRNA gene labelled with digoxigenin-11-dUTP by nick translation and the probe pXV1 (Schmidt et al., 1994) for the 5S rRNA gene labelled with biotin-11-dUTP by PCR were used. The telomeric probe pLT11 was labelled with digoxigenin-11-dUTP by nick translation. The satellite probe CoSat I was labelled with digoxigenin-11-dUTP, and the retrotransposon sequences CoRetro I and CoRetro II were labelled with digoxigenin-11-dUTP and biotin-11-dUTP by PCR, respectively. For karyotyping, the following labelling was used: the probe pZR18S was labelled with DY-647-dUTP and the probe pXV1 was labelled with DY-415-dUTP (Dyomics, http://www.dyomics.com). The satellite probe CoSat I was labelled with biotin-11-dUTP and the retrotransposon sequence CoRetro I was labelled with digoxigenin-11-dUTP. Polymerase chain reaction labelling was performed with standard M13 primers using the following program: 5 min 95 °C, 35 cycles of (1 min 95 °C, 40 s 56 °C, 1 min 72 °C), 10 min 72 °C. Probes were purified from unincorporated nucleotides by precipitation and resuspended in water.

Fluorescent in situ hybridization

Fluorescent in situ hybridization was performed according to Heslop-Harrison et al. (1991). Chromosome spreads were pre-treated with 100 µg mL−1 RNase A in 2× SSC for 1 h at 37 °C and washed twice in 2× SSC. After incubation with 10 µg mL−1 pepsin in 0·01 mm HCl for 20 min at 37 °C, preparations were stabilized in freshly de-polymerized 4 % (w/v) paraformaldehyde in water for 10 min, dehydrated in a graded ethanol series and air dried. In total, 30 µL of the hybridization mixture containing 50 % formamide in 2× SSC (stringency 76 %), 7·5–10 % dextran sulfate, 0·2 % SDS, 300 ng of sheared salmon sperm DNA and 50–150 ng of the labelled FISH probes were denatured at 70 °C for 10 min and applied per slide. After application of the hybridization mixture, the slides were covered with plastic coverslips, denatured at 70 °C for 5 min, cooled down stepwise to 37 °C in an in situ omnislide thermal cycler (Thermo Electron) and hybridized overnight at 37 °C in a humid chamber. Stringent post-hybridization washes were performed at 42 °C in 20 % formamide/0·1× SSC (stringency 85 %). Biotin-labelled probes were detected by streptavidin coupled to Cy3. Digoxigenin-labelled probes were detected with anti-digoxigenin coupled to fluorescein isothiocyanate (FITC; Roche, http://www.roche-applied-science.com). Probes labelled with the fluorochromes DY-415 and DY-647 were detected directly. 4′,6-Diamidino-2-phenylindole (DAPI) in antifade solution (citifluor AF1) was used to counterstain chromosomes.

Examination of slides was carried out using a Zeiss Axioplan2 fluorescence microscope equipped with filters 09 (FITC), 15 (Cy3) and 01 (DAPI), and filter sets F36-710 (emission at 467 nm) and F36-760 (emission at 672 nm, AHF: http://www.ahf.de) for the direct detection of the dyes DY-415 and DY-647, respectively.

Images were acquired with the Applied Spectral Imaging v. 3.3 software, coupled with the high-resolution CCD camera ASI BV300-20A, and arranged using Adobe Photoshop v. 7.0 software after contrast optimization.

Immunolabelling

For detection of cytosine methylation, slides with prometaphase and metaphase nuclei were prepared according to the FISH procedure. Chromosome spreads were pre-treated with 100 µg mL−1 RNase A in 2× SSC for 1 h at 37 °C and washed twice in 2× SSC. After incubation with 10 µg mL−1 pepsin in 0·01 mm HCl for 20 min at 37 °C, the slides were washed three times in 1× phosphate-buffered saline (PBS) buffer for 5 min at room temperature followed by fixing in 4 % paraformaldehyde and washing three times in 1× PBS buffer. The chromosomes were denatured in 70 % formamide (in 2× SSC) at 80 °C for 3 min and dehydrated immediately, first using 70 % ethanol and then 100 % ethanol for 3 min. After incubation in 200 µL of blocking solution (4 % bovine serum albumin, 0·1 % Tween-20 in 1× PBS), slides were incubated with 50 µL of the primary antibody against 5-methylcytosine (Calbiochem: anti-5mC 162 33 D3; diluted 1:250) at 37 °C overnight. Detection of the 5mC antibody was carried out with 50 µL of secondary antibody (Invitrogen: antimouse-Alexa Fluor 488 A-11001) in antibody solution diluted 1:250 at 37 °C for 1 h. After washing three times in 1× PBS, DAPI in antifade solution (citifluor AF1) was used to counterstain chromosomes. The examination of slides and image acquisition were performed as described above.

Southern hybridization

Aliquots of genomic DNA were digested with several restriction endonucleases, separated in 1·1 % agarose gels and transferred onto positively charged nylon membranes (Hybond-N+, GE Healthcare) using alkaline transfer. Southern hybridizations using 32P-labelled probes were performed according to standard protocols (Sambrook et al., 1989). Filters were hybridized at 60 °C overnight and washed at 60 °C in 2× SSC/0·1 % SDS and 1× SSC/0·1 % SDS for 10 min each. Signals were detected by autoradiography.

RESULTS

Identification and classification of repetitive sequences from Corchorus species

In order to identify repetitive DNA sequences in Corchorus species, genomic DNA of C. capsularis and C. olitorius was digested with the restriction endonuclease AluI and separated by gel electrophoresis. From both species, restriction fragments in the size range from 100 to 500 bp were cloned, and plasmid clones were collected in 384-well plates and comparatively hybridized with genomic Corchorus DNA in a species-specific manner. In total, 59 clones from C. capsularis and 30 C. olitorius clones, respectively, showing strong hybridization signals, were sequenced. By nucleotide and protein homology searches in the EMBL database as well as local sequence clustering, the 89 jute sequences could be classified. A total of 53 clones represent repetitive sequence families, 18 clones showed similarity to genomic Corchorus DNA such as SSR markers, chloroplast and mitochondrial DNA and coding sequences, while 18 clones showed no significant similarity to known sequences. Out of these 53 clones, two and three clones contained fragments of retrotransposons and ribosomal genes, respectively. The remaining clones of both jute species harboured highly similar sequences of varying size including three C. olitorius and two C. capsularis plasmid clones with multimers of a 154 bp repeating unit. These sequences can be regarded as satellite DNA which is present in both Corchorus species and is designated CoSat I.

In order to study the diversity within the CoSat I family over a wider range, 18 CoSat I repeats from C. olitorius and 22 repeats from C. capsularis were isolated by PCR. For further analysis, multimers were separated into individual monomers. The characteristic features of the CoSat I satellite are given in Table 1. The dendrogram derived from the sequence alignment of all full-length CoSat I monomers from C. olitorius (CoSat I 1–22) and C. capsularis (CoSat I 23–46) resulted in the formation of two species-specific CoSat I clades (Fig. 1A). The consensus sequence of both clades forms subfamilies with 92 % similarity due to diagnostic nucleotide changes (Fig. 1B).

Table 1.

Characteristic features of the CoSat I satellite DNA in Corchorus species

Species Analysed monomers Consensus size (bp) Identity (%) G/C content (%) Accession numbers
C. olitorius 22 154 79–100 34·9 HE962036–HE962057
C. capsularis 24 154 64–100 36·6 HE962058–HE962081
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Fig. 1.

Fig. 1.

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(A) Neighbor–joining analysis of CoSat I repeats based on the nucleotide alignment of 46 monomers reveals two species-specific CoSat I clades. The genetic distance of the sequences is given by the scale bar. (B) Consensus nucleotide sequences of subfamilies of the satellite CoSat I in C. olitorius and C. capsularis. The consensus sequences are based on the alignment of 22 monomers of C. olitorius and 24 monomers of C. capsularis. Differences in nucleotide composition between subfamilies are indicated by black boxes. The recognition sites of the restriction endonucleases HaeIII and AluI are underlined.

Two clones revealed significant similarities to known sequences of Ty3-gypsy retrotransposons, and therefore were named CoRetro I and CoRetro II. The fragments of CoRetro I and CoRetro II are 360 and 330 bp long, respectively, and Fig. 2 shows partial amino acid similarities to three plant Ty3-gypsy retrotransposons such as CRM2 (Zea mays), LjRE2 (Lotus japonicus) and Beetle1 (Patellifolia procumbens). The retrotransposon CoRetro I contains the conserved integrase motif GPF and a chromodomain downstream of the polypurine tract extended into the 3′ LTR, whereas CoRetro II contains a protease sequence. For clarity, CoRetro I and CoRetro II have been aligned with a hypothetical retrotransposon; however, it cannot be concluded that both fragments belong to the same retrotransposon copy or family (Fig. 2).

Fig. 2.

Fig. 2.

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The partial retrotransposon sequences CoRetro I and CoRetro II show similarities to the coding region of centromeric Ty3-gypsy retrotransposons such as CRM2 (Z. mays), LjRE2 (L. japonicus) and Beetle1 (P. procumbens). CoRetro I contains sequences of a retrotransposon integrase which is characterized by the presence of a chromodomain situated downstream of the polypurine tract within the 3' LTR. The CoRetro II probe comprises a retrotransposon protease sequence. Conserved amino acid residues are shaded in black.

Genomic organization of repetitive sequences in both Corchorus species

To investigate the organization and the abundance of the three repeat families in Corchorus genomes, genomic DNA of C. olitorius and C. capsularis was digested with the restriction enzymes RsaI, HaeIII and AluI, respectively, blotted, and hybridized with CoSat I, CoRetro I and CoRetro II probes (Fig. 3A–C).

Fig. 3.

Fig. 3.

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Comparative Southern hybridization of genomic DNA of C. olitorius (lanes 1–3) and C. capsularis (lanes 4–6) using probes for the DNA satellite family CoSat I (A) and the retrotransposons CoRetro I (B) and CoRetro II (C). The genomic DNA was restricted with RsaI (lanes 1, 4), HaeIII (lanes 2, 5) and AluI (lanes 3, 6). The molecular size marker is given on the left.

After hybridization with the clone CoSat I-10, a typical ladder-like banding pattern was observed in both species (Fig. 3A, lanes 1–6). RsaI-digested DNA showed signals of CoSat I multimers and strong hybridization of higher molecular weight DNA, suggesting that a large fraction of the satellite DNAs lack the RsaI sites (Fig. 3A, lanes 1 and 4). HaeIII and AluI restriction sites are more conserved, resulting in oligomers in both species' satellites (Fig. 3A, lanes 2, 3, 5, 6). Interestingly, in C. olitorius smaller monomers were observed, resulting from digestion of a conserved internal HaeIII in many monomers (Fig. 3A, lane 2). This internal HaeIII site is often diverged in C. capsularis satellite monomers (Supplementary Data Fig. S1).

The hybridization patterns of CoRetro I and CoRetro II showed different signal strength and band sizes in both species (Fig. 3B, C, lanes 1–6). The partial retrotransposon sequences CoRetro I and CoRetro II hybridized to multiple conserved fragments along with considerable background hybridization, indicating a dispersed genomic organization. The conserved fragments of CoRetro I and CoRetro II differ between both jute species, while the abundance is similar in C. olitorius and C. capsularis (Fig. 3B, C). The stronger hybridization signals of CoRetro I showed that this retrotransposon belongs to a family consisting of a higher number of copies (Fig. 3B).

Physical mapping of repetitive sequences along Corchorus chromosomes

Fluorescent in situ hybridization was applied to localize the tandemly arranged 18S–5·8S–25S rRNA and 5S rRNA genes on C. olitorius and C. capsularis chromosomes using heterologous probes from sugar beet (Beta vulgaris).

In both C. olitorius and C. capsularis, the 18S–5·8S–25S rRNA genes are located in the terminal position of one chromosome pair (Fig. 4A, C, green fluorescence). Similarly, two signals of 5S rRNA were observed in both C. olitorius and C. capsularis (Fig. 4A, C, red fluorescence). However, the chromosomal position of the 5S rRNA genes was different in both species. In C. olitorius, the 5S rRNA gene signals were detected in the interstitial regions of two homologous chromosomes (Fig. 4A, red fluorescence). In C. capsularis, 5S rRNA gene arrays were located in the pericentromeric regions of one pair of chromosomes (Fig. 4C, red fluorescence). The fluorescent in situ hybridization with the telomeric probe pLT11 showed that cultivated jute species possess Arabidopsis-like telomeric sequences at all chromosome termini (Fig. 4B, D, green fluorescence). Moreover, it was notable that very faint intercalary telomeric repeats were found in both species.

Fig. 4.

Fig. 4.

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Fluorescent in situ hybridization of interphase and metaphase chromosomes of C. olitorius and C. capsularis. DAPI-stained DNA (blue fluorescence) shows the morphology of the chromosomes. (A and C) Signals show the position of the 18S–5·8S–25S rRNA genes (green fluorescence) and 5S rRNA genes (red fluorescence) in C. olitorius and C. capsularis, respectively. (B and D) Localization of the telomeric probe pLT11 (green fluorescence) on chromosomes of C. olitorius and C. capsularis, respectively. (E and F) Chromosomal position of CoSat I repeats (green fluorescence) in C. olitorius and C. capsularis, respectively. In (F), arrows indicate two moderate CoSat I repeat signals. (G) Interphase nuclei of C. capsularis show that most but not all DAPI-positive chromocentres co-localize with the satellite signals (green signals); chromocentres without satellite signals are marked by arrows. (H) Immunolabelling with an antibody against 5-methylcytosine (green fluorescence) reveals that regions showing depletion in DNA methylation (examples marked by arrows) correspond to large CoSat I satellite arrays (red signals). (I and J) Green signals show the localization of CoRetro I on chromosomes of C. olitorius and C. capsularis, respectively. (K and L) Chromosomal localization of CoRetro II in C. olitorius and C. capsularis (red), respectively.

Fluorescent in situ hybridization has also been applied to compare the chromosomal distribution pattern of the three repetitive sequences CoSat I, CoRetro I and CoRetro II on metaphase chromosomes of C. capsularis and C. olitorius.

Hybridization with the full-length satellite monomer clone CoSat I-10 revealed signals at brightly stained DAPI-positive centromeric and pericentromeric heterochromatic blocks in both jute species. All chromosomes of C. olitorius and C. capsularis possess the CoSat I; however, the size of the satellite arrays varied enormously between chromosomes. In particular, six chromosomes of C. olitorius revealed very strong signals with high intensity, whereas four showed less intense and hence moderate hybridization (Fig. 4E). The remaining four chromosomes showed very faint signals in the pericentromeric regions of C. olitorius chromosomes (Fig. 4E). In C. capsularis, four chromosomes showed strong signals, while moderate intensity was observed on two chromsomes (Fig. 4F, arrows) and the remaining chromosomes showed only weak signals. After hybridization on interphase nuclei, most but not all DAPI-positive chromocentres co-localize with the satellite signals (Fig. 4G, arrows). Some of these heterochromatic regions may contain only a small number of CoSat I or other, as yet unknown, repetitive sequences (Fig. 4G, arrows).

Fluorescent in situ hybridization using the retrotransposon-derived sequences CoRetro I and CoRetro II as probes showed signals on all mitotic metaphase chromosomes of both jute cultivars (Fig. 4I–L). The retrotransposon CoRetro I showed a localization predominantly in the centromeric and pericentromeric regions on all chromosomes of both jute species (Fig. 4I–J, green fluorescence). However, some peculiarities have been observed. While in C. olitorius the signals are moderate to strong and confined to centromeric and pericentromeric regions (Fig. 4I, green fluorescence), in C. capsularis the signals are more diffuse, with two chromosomes revealing strong signals (Fig. 4J, green fluorescence).

The retrotransposon CoRetro II showed less pronounced and weaker signals in the centromeric regions of both species (Fig. 4K, L, red fluorescence).

DNA methylation on jute chromosomes

To determine the level of DNA methylation along C. olitorius chromosomes, an antibody against 5-methylcytosine was used for immunolabelling. Signals were dispersed along late prometaphase chromosomes and depletion in centromeric regions was observed (Fig. 4H, green fluorescence). The subsequent FISH with CoSat I-10 showed that these depleted regions correspond to satellite arrays (Fig. 4H, arrows). This hypomethylation of cytosines is clearly detectable in large satellite arrays but less pronounced in chromosomes carrying smaller arrays.

A reference FISH karyotype of jute species

Staining techniques do not enable the discrimination of jute chromosomes, and the unambiguous identification of each chromosome in a full mitotic complement has not been performed so far.

A set of repetitive probes was used for FISH to develop the molecular–cytogenetic karyotype of jute species. The probes were pZR18S (partial 18S–5·8S–25S rRNA gene), pXV1 (5S rRNA gene), the satellite DNA monomer CoSat I-10 and the partial retroelement CoRetro I. The probes CoSat I-10 and CoRetro I were labelled with biotin-11-dUTP and digoxigenin-11-dUTP, respectively, and used in the first FISH experiment. The probes for the 18S–5·8S–25S rRNA and 5S rRNA genes were labelled with DY-647-dUTP and DY-415-dUTP, respectively, and used in rehybridizations. The hybridization strengths and patterns were unique for each repeat and specific for individual chromosome pairs, and supported by the occurrence of brightly DAPI-stained regions (Fig. 5). To arrange the chromosomes of C. olitorius (Fig. 5A) and C. capsularis (Fig. 5C), we used the signal strength of the major repeat CoSat I as a quantitative reference, classifying the signals from very strong, strong, weak and faint. To indicate the position and strength of the signals, we converted the hybridization patterns into schematic karyograms for both species (Fig. 5B, D). Chromosome pair 1 and chromosome pair 2 carried very strong CoSat I signals in both species. Nevertheless, a strong cytogenetic marker was found on chromosome 2 that is able to differentiate both jute species. In C. olitorius, chromosome pair 2 possesses a very strong signal of the tandemly repeated 18S–5·8S–25S rRNA genes at the terminal region of one arm which is not present in C. capsularis. In C. capsularis, this chromosome can be recognized by a strong DAPI band in the centromere. In both species, chromosome pair 3 showed weak CoSat I signals in the centromeric region; however, C. capsularis revealed a strong signal of the 18S–5·8S–25S rDNA at the terminal region of one arm. Chromosome pair 4 showed a strong CoSat I signal in centromeric regions of both species. In both species, chromosome pair 5 showed a faint CoSat I and strong CoRetro I signal. A very strong CoSat I array was found in the centromeric region of the chromosome pair 6 in C. olitorius, while in C. capsularis a weak satellite signal was observed. Chromosome pair 7 possesses a strong 5S rDNA signal on the short arm of both species.

Fig. 5.

Fig. 5.

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FISH karyotypes and schematic karyograms of C. olitorius (A and B) and C. capsularis (C and D). DAPI-stained DNA (blue fluorescence) shows the morphology of the chromosomes. Chromosomes were numbered and arranged according to descending size. Chromosomes were probed with satellite CoSat I (red fluorescence), retrotransposon sequence CoRetro I (green fluorescence), 18S–5·8S–25S rRNA (magenta fluorescence) and 5S rRNA (orange fluorescence). The scale bar is 5 µm. Chromosomes in the schematic karyogram display the positions of the probes used for identification, but are not to scale.

DISCUSSION

Highly repetitive DNA sequences of jute genomes

We have isolated a major satellite DNA family, named CoSat I, and fragments of the Ty3-gypsy families CoRetro I and CoRetro II from the jute species C. olitorius and C. capsularis and analysed their molecular structure, genomic organization and chromosomal localization.

Several strategies can be applied to identify repetitive sequences, such as cloning of c0t1-DNA, isolation of restriction satellites or identification during genome sequence annotation. Because the genome sequence of jute is not publically available and restriction analyses did not reveal conserved satellite fragments, we have applied shotgun cloning of restriction fragments produced by the frequently cutting enzyme AluI, which has a balanced AT/GC recognition site, avoiding ambiguities regarding the GC content of fragments. Nevertheless, the CoSat I family is AT rich which is characteristic for many plant satellite DNAs (Schmidt et al., 1991). Repetitive DNA sequences are often strongly methylated at cytosines in different sequence contexts (Zakrzewski et al., 2011). However, hypomethylation of centromeric repeats has been demonstrated by immunostaining in Pennisetum glaucum, A. thaliana and B. vulgaris (Kamm et al., 1994; Chan et al., 2005; Zakrzewski et al., 2011). Similarly, the long CoSat I arrays in the jute centromeres are also hypomethylated, as observed after immunostaining using antibodies against 5-methylcytosine DNA (Fig. 4H), which is also consistent with the high AT and low GC content. The low level of cytosine methylation of the CoSat I satellite can be explained by continuous deamination of 5-methylcytosine. The deamination of 5-methylcytosine results in thymine and is the most common single nucleotide mutation (Hendrich et al., 1999). Within the CoSat I repeats from C. olitorius there are 12 cytosine positions where the transition to thymine have been found in a sub-set of monomers (Supplementary Data Fig. S1). Therefore, we propose that cytosine deamination during CoSat I satellite evolution leads to the high AT content and is fixed by homogenization. Another reason may be the hypomethylation of cytosines in relation to their sequence context. Bisulfite sequencing of centromeric satellite monomers in B. vulgaris revealed that cytosines in the frequently occurring CHH sequence contexts are only weakly methylated, which consequently leads to a DNA hypomethylation of the centromere (Zakrzewski et al., 2011). Similarly, a CHH hypomethylation of CoSat I could also be responsible for the low methylation in jute centromeres. Nevertheless, it is likely that the satellite DNA in jute is organized as heterochromatin (strong DAPI staining in Fig. 4E, F) involving also chromatin modifications such as mono- and dimethylation of Lys9 in histone 3. Together with the biased base composition, sequence divergence and monomer length, the CoSat I satellite is similar to many plant satellite DNA families (Schmidt et al., 1991; Kamm et al., 1994; Hemleben et al., 2007). Another typical feature of satellite DNA is the formation of subfamilies which we observed by comparison of monomers of C. olitorius and C. capsularis. They form two species-specific subfamilies which most probably evolved during species radiation within the genus Corchorus. The most prominent nucleotide change regards an internal HaeIII recognition site which is diverged in 70 % of the C. capsularis satellite fragments. In contrast, this site is largely conserved in C. olitorius, resulting in the detection of an additional 77 bp fragment in Southern experiments (Fig. 3A, lane 2).

The evolutionary diversification and amplification of satellite DNA families results in the formation of species-specific satellite DNA (Ma et al., 2007; Cohen et al., 2008). Although the existence or absence of satellite families can support the interpretation of species relationships, any taxonomic conclusion based on molecular data of repetitive DNA alone needs careful reconsideration and should be verified on a large sample of accessions. Approximately 215 species, subspecies, varieties and landraces have been reported in the genus Corchorus (Global Biodiversity Information Facility 2008: http://www.gbif.org) often with different ploidy levels. It would be interesting to investigate landraces and cultivars of different geographical origin or distantly related Corchorus species in the future.

In jute, a recent study by Ahmed et al. (2011) reported the diversity of the reverse transcriptase gene domains of jute LTR retrotransposons. These fragments show a clustering, indicating the existence of distinct families. Ahmed et al. (2011) estimated that 31 000 Ty1-copia and 3600 Ty3-gypsy retrotransposons exist in the genome. However, these numbers are rough estimations only because of the limited specificity of the degenerate primers used in this study, the unknown size of full-length retrotransposons and the incorrect genome size of 1250 and 1000 Mbp for C. olitorius and C. capsularis, respectively (Edmonds, 1990; Palve et al., 2003). Therefore, it is likely that the number of LTR retrotransposons is much higher. Genome sequencing by next-generation technologies has shown that >80 % of the barley genome consists of mobile DNA sequences, the vast majority of these sequences belonging to LTR retrotransposons (Mayer et al., 2012). In maize, 75 % of the genome is made up exclusively by LTR retrotransposons which can be assigned to >400 diverse families (Schnable et al., 2009).

In our study, we identified partial sequences of two different Ty3-gypsy retrotransposons designated CoRetro I and CoRetro II and consisting of an integrase motif and a chromodomain downstream of the polypurine tract and a protease motif, respectively. CoRetro I and CoRetro II showed a relatively high conservation in their putative amino acid sequence with CRM2 (Zea mays), LjRE2 (Lotus japonicus) and Beetle1 (P. procumbens) (Mroczek and Dawe, 2003; Holligan et al., 2006; Weber and Schmidt, 2009). In particular, the integrase sequence of CoRetro I with a chromodomain extending into the 3' LTR represents a conserved feature identified in plant centromeric retrotransposons (Langdon et al., 2000; Nagaki and Murata, 2005; Weber and Schmidt, 2009; Wollrab et al., 2012; Weber et al., 2013). Also, the centromeric and pericentromeric localization of CoRetro I and CoRetro II is typical for chromoviruses, as shown in different plant families (Neumann et al., 2011).

The FISH karyotype of jute

Karyotypes and cytogenetic maps have been developed for most major crop species such as all cereals, soybean and potato (Pedersen and Langridge, 1997; Brown et al., 1999; Dong et al., 2000; Findley et al., 2010). Regarding molecular cytogenetic studies the jute species C. olitorius and C. capsularis are neglected crops, although they represent the major cash crop of Bangladesh.

Jute chromosomes are relatively small, and the length of the chromosomes varies between 1·5 and 3·5 µm (Sinha et al., 2011). The chromosomes are mostly metacentric to sub-metacentric and of similar morphology; hence, discrimination of chromosomes has not been readily possible so far. Several attempts to identify jute chromosomes have been undertaken in the past decades, e.g. by determination of chromosome lengths and arm ratios (Samad et al., 1993; Morakinyo and Baderinwa, 1997) and fluorescent banding methods (Alam and Rahman, 2000). However, only two chromosomes could be identified by fluorochrome staining using CMA (chromomycin-A3) and DAPI (Alam and Rahman, 2000).

We used four repetitive probes consisting of two major sequence families and ribosomal genes in multicolour-FISH and rehybridization to develop karyotypes of jute. Genes for the 5S rRNA and 18S–5·8.S–25S rRNA are evolutionary extremely conserved and often used as heterologous probes to mark a sub-set of chromosomes (Galasso et al., 2001; Han et al., 2008). The in situ hybridization using sugar beet ribosomal gene probes provided landmarks for chromosomes 7, and 2 and 3 in C. olitorius and C. capsularis, respectively. The telomeric sequence pLT11 detected pairs of signal doublets mostly at the ends of all chromosomes, but no polymorphism between chromosomes as in other plant species such as conifers has been observed (Schmidt et al., 2000).

The variation of the CoSat I array size enabled the discrimination of chromosome sub-sets, which was important for the establishment of the jute karyotype. Tandemly repeated DNA sequences have been used in numerous studies to identify plant chromosomes (Castilho and Heslop-Harrison, 1995; Dong et al., 2000; Kato et al., 2005). In many plants, the centromeric and pericentromeric regions are enriched with satellite DNA, for example in Crucifer species (Grellet et al., 1986; Maluszynska and Heslop-Harrison, 1991; Harrison and Heslop-Harrison, 1995; Kamm et al., 1995), in Vignia unguiculata (Galasso et al., 1995) and in P. glaucum (Kamm et al., 1994). Centromeric satellite DNA is, together with Ty3-gypsy-like elements, of particular functional importance, because of its role in active plant centromeres. Therefore, these tandem repeat families are often present on all centromeres of a species (Zakrzewski et al., 2013) and their potential to discriminate chromosomes is limited.

Satellite families also occuring in intercalary regions or in the close vicinity of the telomere (Sharma and Raina, 2005) have been reported from cereal species (Brandes et al., 1995; Vershinin et al., 1995), and have also been found in Allium cepa (Barnes et al., 1985; Pich et al., 1996) and potato (Dong et al., 2000). Apparently, non-centromeric satellite DNA tends to show more variation in distribution, number and size of sites, resulting in increased chromosome specificity.

The FISH-based karyotypes for jute provide a resource for unequivocal discrimination of chromosomes and correlation of physical chromosomes with the genome sequences. Knowledge of the major sequence classes, their diversity and chromosomal localization is also important for jute breeding. Improvements in yield, stress tolerance and quality are important aims in jute breeding and might be achieved by combining the agronomic traits of C. olitorius and C. capsularis; however, the hybrid progeny suffer from genomic instability (Haque, 1987). The results of this study might be helpful to elucidate whether processes such as chromosome rearrangements or differences in methylation are involved in this instability.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of Figure S1: alignment of CoSat I satellite repeats for C. olitorius (CoSat I 1–22) and C. capsularis (CoSat I 23–46).

Supplementary Data
supp_112_1_123__index.html (1.1KB, html)

ACKNOWLEDGEMENTS

R.B. acknowledges a Georg Forster fellowship of the Alexander von Humboldt Foundation. R.B. also thanks the BJRI (Bangladesh Jute Research Institute) for providing cultivated jute seeds. We are grateful to I. Walter, K. Seibt and N. Fliegner for excellent technical assistance.

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