JXB Advance Access originally published online on February 27, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 847-854, April 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Genome remodelling in three modern S. officinarumxS. spontaneum sugarcane cultivars
6 November 2003; 22 December 2003
1 Department of Cell Biology and Genetics, University of Alcalá, 28871 Alcalá de Henares, Madrid, Spain
2 Instituto de Investigaciones de la Caña de Azúcar, Carretera a Central Martinez Prieto Km 2 1/2, Boyeros, Ciudad de La Habana, Cuba
3 Centro de Investigaciones Biológicas, CSIC, Velázquez, 144. E-28006 Madrid, Spain
* To whom correspondence should be addressed. Fax: +34 91 8854758. E-mail: angeles.cuadrado{at}uah.es
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Abstract |
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This study provides evidence that nuclear and chromosome remodelling has taken place in sugarcane, a vegetative crop with a complex genome derived from interspecific hybridizations between Saccharum officinarum and S. spontaneum. Detailed knowledge on the chromosomal compositions of the three clones analysed was acquired. (1) All hybrid cultivars were found to be aneuploid, affecting both parental genomes (having chromosomes in addition to full genomes), with chromosome numbers from 2n=102–106 in My5514 and up to 2n=113–117 in C236-51. (2) Comparative in situ hybridization showed that about 16% of these chromosomes are inherited from S. spontaneum and less than 5% are recombinant or translocated chromosomes containing sequences of both S. officinarum and S. spontaneum. (3) Differences between the observed DNA contents (estimated by flow cytometry) and those expected from the number of chromosomes, allowed the introgression of additional S. spontaneum or S. officinarum DNA pieces into the B42231 [GenBank] and C236-51 cultivars to be estimated. (4) Size heterogeneity between S. officinarum homologous chromosomes carrying the 18S–5.8S–25S and 5S ribosomal genes (identified by FISH with pTa71 and pTa794, respectively) confirms remodelling occurred by chromosomal interchange events, at least in these homologous chromosomes. (5) Simultaneous visualization of nucleoli and NORs showed that all 18S–5.8S–25S loci were potentially functional in the three clones, independent of their origin and size.
Key words: Flow cytometry, genome remodelling, in situ hybridization, intergenomic recombination, nucleolar competition, ribosomal genes, Saccharum spp.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Modern cultivated sugarcane varieties (Saccharum spp.) such as those analysed in this work, are derived from artificial interspecific hybridizations between S. officinarum (2n=80) and S. spontaneum (2n=40–128). These were performed 100 years ago using S. officinarum as the maternal partner (Pérez et al., 1997). This is a very short time in evolutionary terms, but long enough to allow genome remodelling, as this paper shows.
Sugarcane cultivars are clones that are propagated by stem cuttings. Vegetative propagation is well tolerated, in fact, sugar cane was introduced to America by Columbus in his second journey (1493), and was propagated in this way for the next 250 years (Grassl, 1974, 1977). Vegetative reproduction causes the meiotic filter to be bypassed and permits the accumulation of numerical and structural chromosome changes.
Because of the complexity of the sugarcane genome (which probably exceeds that of any important polyploid crop, and of which no diploid relatives are known), it has received relatively little attention from plant scientists. However, in recent years, molecular cytogenetic approaches and the use of molecular markers have led to significant advances in establishing the origin and genomic structure of sugarcane (Grivet and Arruda, 2001).
Genomic in situ hybridization is the method of choice for identifying parental chromosomes in interspecific hybrids, and for testing for the exchange of material between genomes (Schwarzacher et al., 1989; Cuadrado and Jouve, 1995). D’Hont et al. (1996) demonstrated the feasibility of using comparative genomic in situ hybridization to identify the parental genomes of sugarcane. Among the chromosomes of cultivar ‘R570’ (2n=107–115), about 10% were identified as originating from S. spontaneum and another 10% as recombinant chromosomes, demonstrating that exchanges have occurred between chromosomes derived from S. officinarum and S. spontaneum.
The numbers and locations of the ribosomal RNA genes in different clones of S. spontaneum and S. officinarum have been established. The nucleolar organizer regions (NORs) of S. officinarum are distal, while those of S. spontaneum are interstitial. The 5S rDNA loci are located at an interstitial position in both species (D’Hont et al., 1998; Ha et al., 1999; Acevedo et al., 2002). For both families, sites of various intensities have been detected, reflecting differences in the copy number of repeats. However, the observation of Acevedo et al. (2002) in the cultivar ‘Cristalina’ (2n=80), with its major as well as minor NOR sites, does not indicate suppression of their activity. The eight NORs found in this S. officinarum clone are all potentially functional.
The long-debated size of sugarcane’s basic chromosome set was established by the physical mapping of ribosomal RNA genes. S. officinarum and S. spontaneum have basic chromosome numbers of x=10 and x=8, respectively (D’Hont et al., 1998). As a consequence, two distinct chromosome organizations coexist in current hybrid cultivars.
The aim of the present work was to visualize any possible chromosomal rearrangements that might have taken place in three sugarcane cultivars during the adaptive process following interspecific hybridization. This was accomplished via: (i) the evaluation of the total DNA content of resting cells, (ii) the use of genomic in situ hybridization to differentiate between S. officinarum and S. spontaneum chromosomes, and (iii) the location of 18S–5.8S–25S and the 5S non-nucleolar ribosomal DNA by in situ hybridization in order to identify homologous chromosomes with these repetitive sequences.
An analysis of the number of NORs associated with nucleoli in interphase was also made in order to determine whether all 45S rDNA loci are equally active in the allopolyploids or whether nucleolar competition exists between the NORs of the parental species (amphiplasty).
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Materials and methods |
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Materials
Four Saccharum spp. cultivars: ‘Cristalina’, ‘My5514’, ‘B42231’, ‘C236-51’, supplied by the Cuban INICA (Instituto Nacional de Investigaciones de la Caña de Azúcar) were examined. ‘Cristalina’ is a typical S. officinarum and produces the highest sugar yield. However, its high susceptibility to sugar cane mosaic virus (SCMV) precludes its agronomic use. The ‘My5514’ cultivar is used commercially because of its high resistance to SCMV and fungal (Ustilago scitaminea Sydow) infections. The ‘B42231’ and ‘C236-51’ cultivars are not used commercially because of their susceptibility to the fungus U. scitaminea Sydow and to SCMV, respectively.
Culture
Sugarcane stem cuttings containing radical bands were cultured on wet filter paper and cotton in a Refritherm-5 (Struers) incubator at 30 °C/85% relative humidity in the dark. Samples of quiescent primordia were excised from the radical bands before soaking. Roots were also taken from plants cultivated in pots.
Sample preparation
Quiescent and proliferative roots were fixed in 4% paraformaldehyde in PBS pH 7.4 buffer in order to observe nucleoli and NORs in the same preparation. Roots were treated with 0.04% hydroxyquinoline for 3 h and then fixed for 72 h in ethanol:acetic acid (3:1, v:v).
The fixed materials were squashed onto clean microscope slides in a drop of 45% acetic acid following the method of Schwarzacher et al. (1989) with modifications. Before squashing, the roots were washed in water to remove the fixative. Root tips were then digested with an enzyme solution containing 2 ml of 2% cellulase (Onozuka R10, Yakult Honsha Co., Tokyo) and 20% liquid pectinase (from Aspergillus niger, Sigma Chemical Co., St Louis, Mo) for 50 min at 37 °C. The samples were washed again in the above buffer before freezing the slides to remove the cover. They were then air-dried.
DNA probes
The DNA probes used were: pTa71 (a 9 kb fragment from Triticum aestivum containing the 18S–5.8S–25S rDNA and intergenic spacers [Gerlach and Bedbrook, 1979]), and pTa794 (a 410 bp fragment, also from wheat, containing the 5S rDNA-gene repeated unit [Gerlach and Dyer, 1980]). For genomic in situ hybridization, DNA was isolated from S. spontaneum and S. officinarum according to Hoisington (1992). The probes were labelled with digoxigenin-11-dUTP, biotin-14-dUTP, or rhodamine-4-dUTP by nick translation (pTa71, S. spontaneum and S. officinarum genomic DNA) and PCR (pTa794). The digoxigenin and biotin were detected using anti-digoxigenin FICT conjugate (green), and anti-biotin Cy3 conjugate (red), respectively.
Fluorescence in situ hybridization
Preparations were incubated in 100 µg ml–1 DNase-free RNase in 2x saline sodium citrate (SSC) (0.03 M Na citrate and 0.3 M NaCl) for 1 h at 37 °C. The slides were then washed in 2x SSC for 5 min, post-fixed in freshly depolymerized 4% (w/v) paraformaldehyde in water for 10 min, washed in 2x SSC for 10 min, dehydrated in a graded ethanol series, and air-dried.
Preparations and probe denaturation, in situ hybridization, post-hybridization washing, and detection were all performed according to Cuadrado and Jouve (1994). For genomic in situ hybridization, 50 ng of genomic DNA form each species (differently labelled) were used.
Microscopic analyses were made using an epifluorescence Axiophot Zeiss system. Photographs were taken with Fuji Super G 400 ASA colour film. For figures, negatives were digitized and processed with Adobe Photoshop 5.0 software, using only those functions that applied equally to all pixels in the image.
Flow cytometry
Quiescent roots were fixed in 1% paraformaldehyde in TRIS buffer (10 mM TRIS, 10 mM Na2EDTA, 100 mM NaCl, pH 7.5). The samples were then washed in the same buffer and digested with an enzyme mixture (2% cellulase, 1% pectinase, 0.05% macerozyme (Serva, Heidelberg), and 0.4 M mannitol (Merck, Darmstadt). Nuclei were isolated from the quiescent roots by using an Ultra Turrax homogenizer in 500 µl lysis buffer (15 mM TRIS, 2 mM Na2EDTA, 80 mM KCl, 20 mM NaCl, 0.1% Triton X-100, pH 7.5).
The homogenate was filtered through a 30 µm nylon mesh and centrifuged at 600 g for 20 min at 4 °C. The pellet was resuspended in 500 µl of lysis buffer and 0.1 mg ml–1 propidium iodide and DNase-free ribonuclease A added (Serva, Heidelberg). Salmo trutta erythrocytes were used as a standard to evaluate the total DNA content of the four cultivars. Samples were analysed in an EPICS XL (Coulter, FL., USA) flow cytometer.
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Results |
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DNA content
The DNA content of the pre-replicative nuclei in quiescent root primordia gave an idea of the DNA content of all the chromosomes. Table 1 shows that DNA contents varied from a minimum of 6.0 pg in ‘Cristalina’ to a maximum of 9.9 pg in ‘C236-51’. Because the genome size of a somatic cell (2C) is approximately the same in S. officinarum (2n=80) and S. spontaneum (2n=80) (D’Hont and Glaszmann, 2001), the mean DNA content per unreplicated S. officinarum or S. spontaneum chromosome could be unequivocally estimated in ‘Cristalina’ by taking into account the chromosome number (2n=80, Table 1) and the total DNA content in the nuclei of quiescent cells (Table 1). Each parental chromosome had, on average, 75 fg of DNA.
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Mapping 45S and 5S rDNA loci
The 18S–5.8S–25S (NOR) and 5S rDNA multigene families were located simultaneously on chromosomes of the three selected cultivars using pTa71 and pTa794, respectively. Using these loci (NOR and 5S RNA) as chromosome markers to identify homologous chromosomes (Fig. 1A, B", C"), these experiments also allowed the investigation of aspects of genome remodelling. As expected from the positions of the loci previously mapped in sugarcane (D’Hont et al., 1998; Ha et al., 1999; Acevedo et al., 2002), the 5S and the 45S rDNA were located on different sets of chromosomes. The 5S were always interstitial while the 45S rDNA lay at terminal (S. officinarum) and interstitial positions (S. spontaneum). For both gene families, sites of various intensities were detected, suggesting an unbalanced distribution of the full complement of ribosomal copies (Fig. 1A). The stronger signals were observed with pTa71 in all cultivars studied, i.e. the NORs were associated with a nucleolar constriction. Size heterogeneity between the chromosomes carrying an rDNA site was observed, some chromosomes being markedly longer than others (compare Fig. 1B with B" and C with C").
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Chromosome number, as well as the number of NORs and 5S genes clusters were recorded (Table 1). Between 102 and 106 chromosomes were counted in the ‘My5514' preparations. FISH detected ten signals with pTa71 (large rDNA precursor) and another ten with the pTa794 (5S ribosomal gene) on 20 different chromosomes. Differences in the intensity of the signals were minimal in ‘My5514' (Fig. 1E). Nine of the ten NOR (18S–5.8S–25S) sites lay in a terminal position, while the other was interstitial (Fig. 1C").
About 110 chromosomes were counted in the ‘B42231' preparations. Ten NORs (18S–5.8S–25S rDNA sites) were observed, four associated with the nucleolar constriction of four satellitized chromosomes. Eight signals were located at distal positions on eight chromosomes, two of them showing very faint signals (arrowheads in Fig. 1A). The other two NOR signals were seen at interstitial positions on two other chromosomes. Ten 5S rDNA sites were detected, all showing different intensities, on ten chromosomes different to those carrying the NORs (Fig. 1A).
Between 113 and 117 chromosomes were counted in ‘C236-51’ preparations. Twelve signals were detected using pTa71 and another 12 using pTa794 in 24 chromosomes of ‘C236-51’. The NORs (18S–5.8S–25S rDNA genes) were localized in an interstitial position in only two chromosomes. Differences in the intensity of the signals were observed between all these loci. The strongest signals were seen in the nucleolar constriction of four satellitized chromosomes. The 5S rDNA genes were localized in an interstitial position in another 12 chromosomes. Differences in the intensity of these loci were also observed (Fig. 1B").
Identification of parental chromosomes in sugarcane cultivars
Comparative genomic in situ hybridization using total DNA from S. officinarum as one probe and S. spontaneum as another, allowed the contribution of parental chromosomes in preparations of the three sugarcane clones to be analysed. Due to the high homology between the genomes of the two species, all chromosomes were labelled with the same probes. However, sequence differences between the genomes of the two species enhanced fluorescence in two groups of chromosomes (Fig. 1B', C'). Reprobing was performed on the same slides simultaneously using the 18S–5.8S–25S and 5S rRNA genes as probes (Fig. 1B", C"). Comparison between the sequential hybridizations confirmed that the interstitial signal observed with pTa71 in the three clones belonged to S. spontaneum chromosomes and revealed the identity of the parental chromosomes with 5S rDNA loci (Fig.1B, C").
An example of the results obtained with ‘C236-51’ is shown in Fig. 1B–B". In situ hybridization was performed using total biotin-labelled DNA of S. officinarum as one probe and total digoxigenin-labelled DNA of S. spontaneum as another. In this metaphase, 116 chromosomes were scored (Fig. 1B). Ninety-seven were entirely labelled red-orange, showing their origin in S. officinarum. Some of the chromosomes clearly showed a red-orange terminal segment. Seventeen were labelled yellow demonstrating their origin in S. spontaneum. Two clearly showed a red-orange interstitial segment. Only two chromosomes derived from the two species were observed in this metaphase (Fig. 1B'). A second hybridization was performed on the same slide with the 18S–5.8S–25S rRNA (green signals) and 5S rRNA genes (red) (Fig. 1B"). Comparison of Fig. 1B' and B" shows that two of the 18S–5.8S–25S sites are located in an interstitial position on two S. spontaneum chromosomes, the other ten localized terminally on the chromosomes of S. officinarum (in some cases associated with a nucleolar constriction). Also, two of the 12 5S sites observed in this clone were localized on two S. spontaneum chromosomes. Comparison between sequential hybridizations showed that some of the red-orange segments observed terminally on the S. officinarum chromosomes, and all of the interstitials ones observed on two S. spontaneum chromosomes, correspond to major rDNA sites.
Successive hybridization was performed on chromosome preparations of ‘Cristalina’ (control), ‘My5514' and ‘B42231'. Comparison between the sequential hybridizations of S. officinarum (‘Cristalina’) (data not shown) revealed that, as for the hybrids (Fig. 1B', D), some of the red-orange segments observed terminally on S. officinarum chromosomes corresponded to major rDNA sites. These highly conserved genes hybridized to DNA of both species. Their detection in red-orange suggests that the red coloration was more intense than the yellow-green. The remaining red-orange segments observed must correspond to other highly conserved sequences of both species or S. officinarum-specific sequences with exclusive homology to S. officinarum DNA.
Among the chromosomes of ‘My5514' and ‘B42231', about 17% and 16%, respectively, were identified as having been contributed by S. spontaneum, and about 5% and 4% as chromosomes derived from S. officinarum and S. spontaneum. Figure 1C–C" shows an example of the results obtained with ‘My5514’. Of the 105 chromosomes in this metaphase (Fig. 1C), 15 originated from S. spontaneum (arrows in Fig. 1C) while another five were derived from S. officinarum and S. spontaneum chromosomes (arrowheads in Fig. 1C). Comparison of Fig. 1C' and C", after reprobing the same slide with the rDNA probes, confirmed that one S. spontaneum chromosome bore one of the ten 18S–5.8S–25S loci, while two of the ten 5S rDNA loci were localized interstitially on two other S. spontaneum chromosomes.
Saccharum chromosomes at the mitotic mid-metaphase stage were evenly condensed to form short rod shapes (Fig. 1B). However, the pro-metaphase stage, during which chromosomes are relatively long, facilitates the characterization of chromosomes derived from translocation or recombination between S. officinarum and S. spontaneum. Different types of chromosomes were identified in these hybrids. The most frequent were chromosomes of S. spontaneum with a terminal S. officinarum segment (Fig. 1D), chromosomes of S. officinarum with a terminal S. spontaneum segment (Fig. 1D), and a few chromosomes with a yellow signal (S. spontaneum) surrounded by two red segments (S. officinarum), probably the result of a double exchange event (Fig. 1C').
Organization of ribosomal gene clusters in nucleoli
An attempt was made to analyse the potential activity of the different NORs. The use of 4% paraformaldehyde fixation did not interfere with the FISH. On the contrary, it allowed the simultaneous observation of nucleoli and hybridization sites (Fig. 1E, I). The 18S–5.8S–25S and 5S ribosomal genes were located simultaneously on the interphase nuclei of proliferating roots. While the 5S rDNA genes showed no nucleolar association (Fig. 1E), the majority of the 18S–5.8S–25S hybridization sites were associated with the nucleolus (Fig. 1F, I). Exceptionally, all NORs (pTa71 sites) were found associated with an individual nucleolus (Fig. 1H). This indicates that no ribosomal gene clusters were silenced in these materials. However, nucleolar fusion was frequently observed. Though three to five nucleoli were normally found in all cultivars (Fig. 1I), in a small percentage of cells, all NORs were associated with a single large nucleolus (Fig. 1F).
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Discussion |
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The fusion of single cells from two different species, irrespective of their origin as gametes (interspecific hybridization) or somatic cells (artificial cell fusion), produces a new cellular system with two chromosome sets. Such cells can suffer certain genomic changes that ensure an harmonious behaviour and activity of both genomes. In fact, somatic cell genetics is based in the selective loss of certain chromosomes. Such loss occurs gradually during the process of cell divisions following interspecific cell fusion (Harris, 1974). Hybridization is frequent, particularly in plants. The origin of a new specie via hybridization (diploidy or allopolyploidy) requires rapid genomic changes to co-ordinate genomes to act in harmony (Rieseberg et al., 1995; Ozkan et al., 2001). In diploid species, such as wild sunflowers, hybrid speciation is accompanied by chromosome reorganization apparently induced by recombination (Rieseberg et al., 2003). In wheat, the rapid elimination of DNA is a general phenomenon in newly synthesized allopolyploids. Changes after hybridization seem to take place in the F1 or in the first generation which follows (Shaked et al., 2001). When vegetative propagation occurs, as in sugarcane, this remodelling can continue for long periods of time during the somatic cellular divisions of non-sexual processes.
Currently, cultivated sugarcane clones are essentially derived from an interspecific hybridization performed at the beginning of the 20th century between S. officinarum and S. spontaneum. Because the species are highly polyploid (an autopolyploid origin is suspected), and because the chromosomes within and between species are similar, little information has been available on the precise behaviour of wild chromosomes during introgression processes and their exact contribution to today’s cultivars. In addition, the relatively symmetrical condition of the karyotype, the mostly median chromosomes, their large number (between 100 and 130), and small size (1–3 µm at mid-metaphase) hinders their identification. The lack of precision in determining the chromosome number is due to the difficulty in accurately assessing such small and numerous chromosomes, although somatic instability cannot be excluded.
Among the chromosomes of the three hybrids, about 16% were identified as having been contributed by S. spontaneum, and less than 5% as recombinant and/or translocated chromosomes between S. officinarum and S. spontaneum. These chromosomes could arise from recombination events in the few meiotic opportunities that occur in the interspecific hybrids and their early generation descendants. Structural interchanges might also be explained by spontaneous translocations accumulated during vegetative propagation. However, the origin of interspecific chromosome interchanges cannot be deduced from karyological observations. Chromosome pairing behaviour has not been definitively clarified in sugarcane. Nonetheless, the occurrence of meiotic recombination between S. officinarum and S. spontaneum chromosomes cannot be disregarded, nor can the common assumption that no recombination occurs between the chromosomes of the two species be discarded (Berding and Roach, 1987).
Aneuploidy occurs when parts or whole chromosomes are absent from a genome, or present in excess. Sugarcane is characterized by aneuploidy. These results show both parental genomes are aneuploid. Thus, in ‘My5514', one S. spontaneum chromosome bears one of the ten 18S–5.8S–25S loci, while there are two S. spontaneum chromosomes with two of the ten 5S rDNA loci. At least in this cultivar, there is a genome unbalance.
The preferential loss of S. spontaneum from the hybrids studied might be explained as a consequence of the 2n+n transmission. S. officinarum when pollinated by S. spontaneum normally transmits its full somatic number (Bremer, 1961). However, n+n transmission also occurs (Roach, 1969; Ming et al., 2002). If there is n+n transmission in any of the hybrids analysed in this study, the selective loss of S. spontaneum chromosomes may result from a failure in the recognition of paternal centromeres by the maternal kinetochore proteins in mitosis.
It is assumed that, in these autopolyploid species, there is one locus for each rDNA family in the basic chromosome number (x=10 and x=8 in S. officinarum and S. spontaneum, respectively). Size heterogeneity between the chromosomes carrying the rDNA genes was particularly noticeable in all the hybrids, with some chromosomes being clearly longer than others. The differences in morphology seen between the homologous chromosomes of S. officinarum carrying an rDNA site seem to confirm that remodelling occurred (at least in these homologous chromosomes) by chromosomal interchange events. The increase in the mean DNA content per chromosome in the ‘B42231’ and ‘C236-51’varieties supports that genome remodelling in these hybrids involves the introgression of additional pieces of DNA between chromosomes of the same genome.
A striking feature of this comparative study is the strict correlation between the number of NORs and the number of clusters of 5S ribosomal genes (independent of parental origin). This correlation was untouched by the general genome instability of these hybrids. The integration of the rRNA transcripts into the ribosomes may explain why they have been respected. However, their maintenance requires a special fail-safe mechanism since the ribosomal 5S genes are quite autonomous in their behaviour during the somatic cell cycle (Cuadrado et al., 1998). The images obtained in metaphase, when the NORs are still dormant and condensed, show that there is some heterogeneity in signal size, probably due to the different number of genes in each NOR. Only the major ones were associated with a nucleolar constriction. However, the simultaneous visualization of nucleoli and NORs reveals the participation of multiple NORs in the formation of a single nucleolus. The FISH images directly demonstrate the functional redundancy of the rDNA genes in the sugarcane genome, and that all rDNA repeats are potentially active, i.e. associated with a nucleolus.
A striking observation was that nucleolar dominance (amphiplasty) (Lacadena et al., 1988) is not a prevalent epigenetic mechanism in sugarcane hybrids. This differs with other observations on Brassica (Chen and Pikaard, 1997) and other interspecific hybrids, and with that observed after experimental NOR hypomethylation (De la Torre et al., 1991). There is a lack of amphiplasty despite the obvious redundancy and large size of the NORs displayed in sugarcane hybrids.
During the last decade, genetic maps have been produced for S. spontaneum, S. officinarum and modern sugarcane (see revision of Grivet and Arradu, 2001). None of the published genetic maps of modern sugarcane cultivars is saturated. In the more refined map, about 10% of the cosegregation groups involve only S. spontanneum specific molecular markers, and about other 10% have a double S. spontaneum and S. officinarum origin (Hoarau et al., 2001). These results show that about 16% of the chromosomes are inherited from S. spontaneum and less than 5% are recombinant or translocated chromosomes, containing sequences of both S. spontaneum and S. officinarum. The quantitative difference could be a consequence of the divergent origin of the material used in both studies. However, it cannot be discarded that genomic in situ hybridization may have a resolution power that is insufficient to identify all recombinant chromosomes, specially when the introgression affects small pieces of chromosomes.
These data provide detailed information on the genome structure of modern sugarcane, perhaps the most complex structure found in crop plants, and give quantitative insight into the importance of meiotic recombination and/or translocation events in the remodelling of their genomes.
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Acknowledgements |
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This work has been supported by the Spanish DGES (projects PB98-0647 and BMC 2001-2195) by the CSIC/CITMA agreement (project 99CU0010) and the University of Alcalá (E041/2001). Ricardo Acevedo was financed by a Mutis fellowship from the Spanish Agency of International Cooperation (AECI). We wish to thank the Instituto de Investigaciones de la Caña de Azucar (INICA) for providing the sugarcane cultivars used in this work and Mr Adrian Burton for revision of the English.
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