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Cell Biology |
korová
í Fajkus7
2 Institute of Biophysics, Czech Academy of Sciences, Královopolská 135, 61265 Brno, Czech Republic;
3 Department of Functional Genomics and Proteomics, Masaryk University, Kotláská 2, 61137 Brno, Czech Republic;
4 School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK;
5 Department of Taxonomy, Institute of Plant Genetics and Crop Plant Research, Corrensstraße 3, D-06466 Gatersleben, Germany;
6 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
Received for publication September 6, 2005. Accepted for publication March 7, 2006.
ABSTRACT
Although telomere sequences are considered to be highly conserved, there are switch-points in plant telomere evolution that are congruent with species' phylogenies. When Asparagales diverged, the Arabidopsis-type telomeric minisatellite repeat (TTTAGGG)n was first replaced by a human-type (TTAGGG)n repeat, and both were lost in Allium cepa (Alliaceae). We aimed to discover (1) when this loss occurred during divergence of Alliaceae and, (2) if (TTAGGG)n repeats were replaced by other known telomeric minisatellites. Slot-blot hybridization, fluorescent in situ hybridization, BAL31 digestion, asymmetric PCR, and cloning were used to identify and localize candidate telomeric sequences in species of Nothoscordum, Miersia, Ipheion, Tulbaghia, Gethyum, Gilliesia, Leucocoryne, Tristagma, and representatives of the three major Allium clades. Alliaceae genera other than Allium have human (TTAGGG)-type telomeric repeats that form telomeres. In Allium, only Tetrahymena-type (TTGGGG) repeats were ubiquitous in the genome, but they were not localized to telomeres. Likewise, the consensus telomeric repeats in Arabidopsis, human, Bombyx (TTAGG), Chlamydomonas (TTTTAGGG), and Oxytricha (TTTTGGGG) are absent in Allium telomeres. Alliaceae with human-type telomeres share telomere structures with related Asparagales species. We demonstrate that in the Allium ancestor human-type telomeric repeats were lost from telomeres and were not replaced by any investigated alternative minisatellite repeats. However, human and other types of minisatellite telomeric repeats are interspersed in some Allium genomes and their genomic signatures coincide with Allium clades.
Key Words: alternative telomeres • Asparagales • asymmetric PCR • FISH • minisatellites • onion • slot-blot hybridization • telomere evolution
The ends of eukaryotic chromosomes are capped by a special structure called the telomere. The DNA component of the telomere is typically formed by long arrays of tandemly repeated short minisatellite sequences that are usually thought to be conserved in large groups of organisms, e.g., TTAGGG in vertebrates (first described in humans [Cheng et al., 1989
; Meyne et al., 1989
]), TTTAGGG in plants (Arabidopsis, Richards and Ausubel [1988
]) and TTAGG in insects (Bombyx, Okazaki et al.[1993
]). Until recently (up to 1999), there were only a few exceptions to this view that telomeric repeats can define major eukaryote lineages; the exceptions were in insects of the order Diptera (Biessmann et al., 1990
; Nielsen and Edstrom, 1993
; Biessmann et al., 1996
) and in plants, e.g., Allium cepa (Alliaceae) (Fuchs et al, 1995
). However, this view changed after Sahara et al. (1999)
showed that the telomeric repeat typical of insects was missing in several other insect orders and in a spider. Subsequently, additional insects and other arthropods (Frydrychova and Marec, 2002
; Vitkova et al., 2005
) and two plant groups (the eudicot family Solanaceae [S\#7923;korová et al., 2003a
, b
]) and the monocot order Asparagales [Adams et al., 2001
]) were added to the growing list of organisms lacking the "expected" telomeric motifs.
In land plants, it is likely that the ancestral character condition for telomeric sequences is TTTAGGG since many flowering plants, gymnosperms, ferns, mosses, and liverworts have these sequences (Cox et al., 1993
; Fuchs et al., 1995
; Suzuki, 2004
). Therefore, reported variations in sequences at the telomeres are probably derived conditions (Fig. 1). In the divergence of the monocot order Asparagales, there are two evolutionary switch-points in the minisatellite sequence motif at the telomere. These changes define three clades of Asparagales (S\#7923;korová et al., 2003b
): those plants with (1) the Arabidopsis-type telomeres (e.g., Orchidaceae), (2) the human-type telomeres (e.g. Asparagus), and (3) an unknown type of telomere (as in A. cepa, Alliaceae).
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]), En/Spm-transposable elements, Ty-copia retroelements and/or rDNA (rDNA) (Pearce et al., 1996
; Pich et al., 1996a
, b; Pich and Schubert, 1998
). However, it remains unknown which, if any, of these sequences are at the true telomere or if all chromosomes have the same telomeric sequence. Recently, we observed proteins in A. cepa that specifically bound to the Arabidopsis-type and human-type minisatellite repeats, pointing to conservation of telomere binding proteins and the possible retention of minisatellite repeats at the telomere (Fajkus et al., 2005
). Because the telomere motif has changed at least once in Asparagales evolution from that typical of Arabidopsis to that typical of human, it is possible that a second shift occurred within Alliaceae to give another variant of the telomere motif, or perhaps there has been a complete replacement of the telomerase-based maintenance system. The latter possibility potentially provides a plant model system to study the evolution of an alternative telomere lengthening (ALT) mechanism currently known from only yeast and human cells (Louis, 2002
). To characterize the distribution and occurrence of potential telomere-like minisatellites in Alliaceae, we screened the genomes of Allium species and non-Allium Alliaceae using slot-blot and in situ hybridizations, and highly sensitive primer-extension reactions. Furthermore, we examined whether any of these repeats form chromosome ends using BAL31 nuclease digestion. We show here that the second evolutionary switch-point in telomere divergence in Asparagales occurred with the divergence of Allium from all other species within Alliaceae. We also show that no other investigated minisatellite telomere repeat has replaced these telomeres, but some of the sequence motifs provide characteristic genomic signatures for the three major Allium clades.
MATERIALS AND METHODS
Plant material
Alliaceae sensu stricto (s.s.) (Angiosperm Phylogeny Group, 2003
) consist of the genera Allium (onions), Ipheion, Tulbaghia, Garaventia, Latace, Solaria, Trichlora, Nothoscordum, Miersia, Gilliesia, Gethyum, Leucocoryne, and Tristagma. The plant material used in this study originated from living collections: (1) Allium species collection at IPK Gatersleben, Germany (voucher specimens deposited in herbarium at IPK, accession numbers [TAX], given in Fig. 2); (2) Royal Botanic Gardens, Kew, UK (RBGK)—Gethyum atropurpureum (voucher Chase 639 at K), Miersia chilensis (Chase 18891 at K), Nothoscordum striatum (1993-1508, no voucher), Gilliesia graminea (Chase 450 at K), Leucocoryne coquimbensis (Chase 1710 at K), Tristagma bivalve (1979-783, no voucher) and Allium cepa cv. Ailsa Craig (no voucher); (3) Queen Mary University of London, UK (voucher KY Lim at QMUL)—Ipheion uniflorum cv. Rolf Fiedler (voucher 04-01 Lim at QMUL), Allium cernuum (used for FISH analysis, voucher 04-08 Lim at QMUL), Allium schuberti (voucher 04-09 Lim at QMUL), Allium nigrum (used in BAL31 nuclease and in situ analysis, voucher 04-06 Lim at QMUL, all supplied by Rose Cottage Garden, London) and Iris tectorum (Rangoon Botanical Garden, BIS). (4) Botanical Garden of Osnabruck University, Osnabruck, Germany (OSBN, all kindly provided by Dr. Nikolai Friesen)—A. oreoprasum (01-17-0128-10), A. akaka (02-25-0020-10), A. ochotense (02-05-0093-70), A. moly (01-17-0032-10), A. zebdanense (03-44-0007-80), A. neapolitanum (03-44-0020-70), A. cowanii (04-44-0016-80), A. triquetrum (03-44-0004-80), A. hookeri (01-17-0049-10), A. siculum (01-38-0001-80); (5) Botanic Garden of Masaryk University Brno, Czech Republic (BGMU)—A. ericetorum, kindly provided by Mgr. Magdalena Chytrá.
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). Control concatemers were prepared using a PCR protocol originally described by Ijdo et al. (1991)
and modified as in S\#7923;korová et al. (2003c)
. Genomic DNAs from A. cepa (RBGK) and I. tectorum (QMUL) used as a template in primer-extension reaction (asymmetric PCR) experiments were isolated using a DNeasy Mini kit (QIAGEN, Crawley, UK).
Slot-blot and Southern hybridization
We used slot-blot hybridization with whole-genome DNA from multiple species of Alliaceae to screen for a range of minisatellite telomeric repeat sequences in Alliaceae as described in S\#7923;korová et al. (2003c)
. Slot-blot hybridization on genomic DNA makes it possible to specifically detect a single nucleotide change in the minisatellite repeat unit and cross-hybridization can be reliably avoided (Neplechova et al., 2005
). Briefly, genomic DNA (1 µg) and control telomeric concatemers (100 pg and 500 pg) were loaded onto Biodyne Plus membrane (Pall Gelman Laboratory, Pensacola, Florida, USA) using Bio-Dot apparatus (BioRad Laboratories, Hercules, California, USA) according to manufacturer's instructions. Membranes were hybridized with a 32P-end-labelled oligonucleotide probe specific for the G-rich strand of each telomeric type; Arabidopsis-type (ATSB), human-type (HUSB), Bombyx-type (BOSB), Chlamydomonas-type (CHSB), Tetrahymena-type (TTSB), and Oxytricha-type (OXSB) (Appendix S1, see Supplemental Data with online version of this article).
Hybridization was carried out at 55°C overnight; membranes were washed at 55°C in 1x SCC (0.15% [w/v] sodium chloride, 0.015% [w/v] sodium citrate), 0.1% [w/v) sodium dodecyl sulphate [SDS) [for ATSB probe, 0.5x SSC, 0.1% SDS, cf. Neplechova et al., 2005
]). The use of control concatemers enabled us to normalize the strength of signals between membranes hybridized with different telomeric probes: ATSB, HUSB, BOSB, CHSB, TTSB, and OXSB. The 1.7-kb fragment of 18S rDNA of Solanum lycopersicon (tomato, accession number X51576) (Kiss et al., 1989
) was used for the control re-hybridization of membranes at 65°C. The DNA probe was radioactively labelled using the DecaPrime kit (MBI Fermentas GMBH, St. Leon-Rot, Germany). This hybridization was washed under high stringency conditions (0.2x SSC, 0.1% SDS at 65°C).
In further investigations of selected species, genomic DNA was digested using restriction endonucleases RsaI, MboI, MseI, AluI, HaeIII, and TaqI (New England Biolabs, Beverly, Massachusetts, USA), separated on a 0.9% agarose gel, alkali blotted, and hybridized with different probes. Results were visualized using X-ray film and Phosphoimager STORM 860 (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).
Fluorescence in situ hybridization (FISH)
FISH was carried out as described in Leitch et al. (2001)
with modifications as in Adams et al. (2001)
. The concatemer probes (see section "Slot-blot and Southern hybridization") were labelled by nick translation with either digoxigenin-11-dUTP or biotin-16-dUTP. Alternatively, the human telomeric PNA probe (DAKO A/S, Glostrup, Denmark) was used instead of the HUSB concatemers. In some experiments, 45S rDNA was detected with the pTa71 probe, which includes the 18–5.8–26S rDNA subunits and the intergenic spacer isolated from Triticum aestivum (Gerlach and Bedbrook, 1979
). Chromosome spreads on slides were denatured in 70% formamide in 2x SSC at 70°C for 2 min. The hybridization mix contained 2 µg/ml labelled ATSB, TTSB, or HUSB concatemers in 50% (v/v) formamide, 10% (w/v) dextran sulphate, and 0.1% (w/v) SDS in 2x SSC. After overnight hybridization at 37°C, slides were washed in 20% (v/v) formamide in 0.1x SSC at 42°C at an estimated hybridization stringency of 80–85%. Sites of probe hybridization were detected using 20 µg/ml fluorescein-conjugated, anti-digoxigenin IgG (Roche Diagnostics, Mannheim, Germany, giving green fluorescence) and 5 µg/ml Cy3-conjugated avidin (Amersham Biosciences, giving red fluorescence). Chromosomes were counterstained with 2 µg/ml DAPI (4',6-diamidino-2-phenylindole, giving blue fluorescence) in 4x SSC, mounted in Vectashield (Vector Laboratories, Burlingame, California, USA) medium, examined using a Leica (Heerbrugg, Switzerland) DMRA2 epifluorescence microscope carrying A4, Y3 and L5 filter blocks, photographed with a Orca (Hamamatsu, Japan) ER camera and analyzed using Improvision (Coventry, UK) Openlab software. Images were processed for color balance, contrast, and uniformity of brightness.
The PRINS (primed in situ labelling) reaction was carried out according to Koch et al. (1995)
except that primers Telo2 (5'-[CCCTAA]7-3'); TTTC (5'-[CCCAAC]3-3'), or TTSB (5'-[TGGGGT]4TG-3') were used. Labelled nucleotides were dUTP-conjugated to tetramethylrhodamine-5-2'-deoxy-uridine-5'-triphosphate (Roche Diagnostics). After denaturation (4 min, 94°C), the PRINS reaction was performed for 1 h at 60°C. Alternatively, labelling was performed using dideoxy-PRINS, a modification of PRINS that uses dideoxynucleotide (ddGTP) in the reaction mixture in place of dGTP. This should inhibit elongation of Telo2 at all sites except telomeric DNA, which does not in theory require this nucleotide for the extension reaction to work.
Testing for telomeric localization of minisatellite repeats using BAL31 digestion
Because the detection threshold of FISH techniques may be higher and resolution lower than those of many molecular methods, BAL31 digestion was applied to test for the terminal position of candidate telomeric sequences. Agarose plugs with high-molecular-mass DNA samples were prepared by grinding plant leaves (I. uniflorum, A. nigrum) in liquid nitrogen. The resulting powder was poured into TEM buffer (1 mM Tris-HCl pH 8.0; 100 mM EDTA pH 8.0; 0.4 M mannitol) equilibrated to 46–49°C, and thoroughly homogenized. After addition of an equal volume of 2% low-melting-point agarose (prepared in TEM buffer), the mixture was homogenized again and pipetted into a mold. After solidifying, plugs were incubated in TES buffer (0.5 M EDTA pH 8.0; 10 mM Tris-HCl pH 8.0; 1.0% lauroylsarcosine) for 30 min at 37°C and for 30 min at 50°C in fresh TES buffer. The plugs were then incubated in TES with proteinase K (final concentration 500 µg/ml) at 50°C for 24 h, and the incubation repeated once with fresh TES and proteinase K (Roche Diagnostics). Deproteinized plugs were washed twice in TE (10 mM Tris-HCl pH 8.0; 1 mM EDTA pH 8.0) for 30 min, then twice in TE with 1 mM PMSF (phenylmethylsulphonyl fluoride) for 30 min, and finally in TE.
Samples in agarose plugs were then equilibrated for 30 min in BAL31 nuclease buffer (NEB) and digested with 3 units of BAL31 nuclease (NEB) for 10, 20, 40, 60, or 90 min in a Thermomixer (Eppendorf AG, Hamburg, Germany) at 30°C. Reactions were stopped by buffer exchange with 50 mM EGTA pH 8.0, and BAL31 nuclease was irreversibly inactivated by incubation at 58°C for 15 min. The plugs were incubated in 0.1x TE buffer in the appropriate restriction enzyme buffer. Restriction enzyme digestion was performed as described in Fajkus et al. (1995)
. After digestion, a solution containing low-molecular-mass fractions of digested DNA was precipitated by ethanol and dissolved in TE for analysis by conventional agarose gel electrophoresis and Southern hybridization. High-molecular-mass fractions, which were retained in the agarose plugs, were analyzed by pulse-field gel electrophoresis (PFGE) using the Gene Navigator system (Amersham Biosciences) under the following conditions: 1% FastLane agarose gel in 0.5x TBE buffer, 165 V, pulses 2 s for 4 h followed by 14 h of pulse time ramping from 2–25 s at 13°C. Both conventional and PFGE gels were alkali blotted and hybridized with end-labelled telomeric oligonucleotide probes HUSB or TTSB.
Analysis of genomic arrangement of minisatellite repeats by primer-extension reactions (asymmetric PCR)
Primer-extension experiments have been successfully employed previously to demonstrate the absence of Arabidopsis-type telomeric sequences in A. cepa (Pich et al., 1996b
) and Cestrum species (S\#7923;korová et al., 2003a
). Due to the polarity of telomeric strands, DNA polymerase should elongate the C-rich-strand telomeric sequence primers from the telomere into the inner regions of the chromosomes. This should generate a smear of products after gel electrophoresis when there are long tracts of the minisatellite at the telomere. In addition, bands are generated if the primers bind to internally located sequences with an inverted orientation (S\#7923;korová et al., 2003a
, b). The primers used for primer-extension reactions were HUTC, 5'-(AACCCT)3AAC-3' and TTTC, 5'-(CCCAAC)3-3'. Extension reactions were carried out using the Expand High Fidelity system (Roche Diagnostics) under conditions recommended by the manufacturer for elongating DNA fragments up to 8 kb (S\#7923;korová et al., 2003a
, c). The reaction products were separated in 1% agarose gels, blotted, and hybridized using end-labelled oligonucleotides of the complementary sequence (G-rich strand) as a probe. Fragment sizes were assessed using a 1kb GeneRuler (MBI Fermentas GMBH, Germany). The purified reaction mixture from an extension reaction using the TTTC primer and A. cepa genomic DNA as a template was cloned into the EcoRV site of the pZErO-2 vector (Invitrogen, Carlsbad, California, USA). Clones were sequenced and deposited into the GenBank database (accession number AY940489).
RESULTS
Slot-blot hybridization to screen Alliaceae
Fifty-two Allium species and eight species from other genera of Alliaceae were investigated for the presence of six types of minisatellite repeat sequences typical of the telomeres of different groups of organisms, i.e., Arabidopsis (plant), human (vertebrates), Bombyx (insect), Chlamydomonas (green alga), Oxytricha (ciliate), and Tetrahymena (ciliate) (cf. Appendix S1, see Supplemental Data with online version of this article). Allium species from different subgenera and sections were chosen to cover a representative phylogenetic spectrum of the genus (Friesen et al., 2005
). The slot-blot data, in connection with the phylogenetic scheme of Allium are presented in Fig. 2. Clear differences in hybridization patterns of Alliaceae genomic DNA can be seen when the different telomere repeats were used as probes:
).
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configuration.
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BAL31 nuclease digestion to determine genomic location
Since the above results give low (>1 Mbp) resolution of sequence localization, we employed BAL31 nuclease digestion to investigate the presence of HUSB sequences at chromosome ends in more detail (Fig. 4). This exonuclease progressively shortens DNA fragments at both termini. Therefore, hybridization signal to terminally positioned sequences (terminal restriction fragments [TRFs]) disappears and moves to lower molecular mass during digestion. However, internally positioned sequences are resistant to BAL31 treatment. In I. uniflorum, BAL31-digested DNA that is size fractionated by PFGE and analyzed by Southern hybridization with HUSB probes reveals putative TRFs. These TRFs form a ladder of discrete bands ranging from 12 to 45 kb (Fig. 4, left panel, lane 0). With increasing duration of BAL31 nuclease digestion, there was a gradual decrease in intensity of HUSB signal together with a shortening of TRFs to about 10 kb. This indicates that HUSB is telomeric in nature in I. uniflorum. Since the lower size limit of these TRFs is near the size limit for elution of fragments from agarose plugs during restriction enzyme digestion, DNA from the liquid phase from each digestion reaction was precipitated and analyzed in parallel by conventional gel electrophoresis and Southern hybridization (Fig. 4, middle panel). The analysis showed a single dominant HUSB hybridization band at about 0.5 kb and several weak signals, all of them resistant to BAL31 digestion. These bands are interpreted as internal telomeric-like sequences.
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To characterize sequences associated with interstitial TTAGGG repeats in Allium (cf. Fig. 2), we conducted Southern hybridization experiments to restriction enzyme-digested genomic DNA of A. nigrum, A. mongolicum, and A. schuberti (from clades 2 and 3 of Allium) (Appendix S3 in Supplemental Data with online version of this article). With many restriction enzymes, we found that the HUSB signals were located in short bands of 0.2–4 kb indicating that the TTAGGG motif is part of a conserved repeated sequence. Cloned sequences associated with TTAGGG minisatellites from A. nigrum (accession numbers AY970931–AY970935) did not have any similarities to nucleotide or protein sequences deposited in GenBank. These clones were AT-rich but did not have longer arrays of tandemly arranged minisatellites, which would point at their telomeric origin. In four clones we found only three repeats of TTAGGG and two other minisatellite repeats in close proximity. High-copy sequences derived from clones (AY970927, AY970929) were used as probes for FISH analysis, but they did not give a specific signal indicating a dispersed chromosomal distribution (results not shown).
PCR primer-extension to search for telomeric repeats in Allium
Primer-extension experiments were employed to increase sensitivity of detection of low amounts of minisatellite sequences potentially obscured in Allium species with large 1C genome sizes. HUSB signal was absent in slot-blot and FISH analyses of A. cepa, but slot-blot data indicated that some Tetrahymena-type sequences are present in the genome (Fig. 2). However, we could not localize them by FISH (results not shown). Using primer-extension experiments, we confirmed that HUTC and TTTC sequences are unlikely to be telomeric (Fig. 5). No products were generated when using the human-specific primer HUTC against A. cepa genomic DNA. This is in contrast to the control reaction with Iris tectorum genomic DNA (Fig. 5) possessing the human-type of telomere (Fig. 1). The primer-extension reaction with the Tetrahymena-type specific primer (Fig. 5, TTTC) against A. cepa genomic DNA resulted in a few bands that gave only weak signal when hybridized with TTSB. The same experiment against I. tectorum genomic DNA produced a smear of products and some bands indicating internal telomeric-like sequences in the genome. The TTTC primer-extension products from A. cepa were cloned, but the clones contained only low-copy sequences and their position in the genome could not be determined by FISH. None of these clones had similarity to GenBank nucleotide or protein sequences except for clone C50/14 (GenBank AY940489), which contains an open reading frame with high similarity to proteins of Arabidopsis thaliana (GenBank NP_850186.1) and Oryza sativa (NP_914311). The tblastx search also revealed high similarities to translated sequences from tomato (GenBank BT013552.1, E = 4e-54) and Medicago truncatula (AC18130.9, E = 5e-51).
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Evolution of telomeric consensus sequences in plants
Some species and groups of species that utilize telomerase-based systems to elongate their telomeres have variants of the expected consensus sequence motif at their telomeres. The green alga Chlorella has TTTAGGG sequences at the telomeres (Higashiyama et al., 1995
), but its distant relative Chlamydomonas possesses an altered motif of TTTTAGGG (Petracek et al., 1990
) (see Fig. 1). Two minisatellite variants, TTGGGG and TTTTGGGG, are known at the telomeres of the ciliates Tetrahymena and Oxytricha, respectively (Blackburn and Gall, 1978
; Pluta et al., 1982
). Among multicellular organisms, different variants of telomeric consensus sequences were described in plants in the order Asparagales (Weiss and Scherthan, 2002
; S\#7923;korová et al., 2003c
). The first evolutionary switch-point in Asparagales telomere sequence divergence gave rise to the human-type of telomere repeat (TTAGGG)n. This probably arose from mutation(s) in telomerase (S\#7923;korová et al., 2006
).
We searched for minisatellite telomeric sequences in the Alliaceae genera Nothoscordum, Miersia, Tulbaghia, Ipheion, Gilliesia, Gethyum, Leucocoryne, and Tristagma and found that they all have the human-type telomeric sequence typical of many Asparagales. They also had labelling for the Arabidopsis- and Tetrahymena-type repeats although the human-type is predominant. A mixed arrangement of motifs at the telomere is common in Asparagales with predominantly human-type repeats (S\#7923;korová et al., 2003c
; Rotkova et al., 2004
). Tulbaghia is sister to Gilliesioideae, and this pair is sister to Allium (Fay et al., 2006
). Thus, all of the more distantly related genera of Alliaceae not investigated here are also likely to have the TTAGGG minisatellite telomere. These data reveal a second evolutionary switch-point in telomere sequence divergence in the common ancestor of all extant species of Allium (see section "Loss of minsatellite repeats at Allium telomeres"). However, this switch did not involve a change to any of the investigated minisatellite motifs, including the human-type repeat found in related Asparagales, the Arabidopsis-type repeat in most plants, or the Tetrahymena-type repeat (TTGGGG), which is the most commonly found motif in Allium genomic DNA. It probably arose by further mutation(s) in telomerase causing the complete loss of minisatellite-type of telomeres.
Genomic signatures in Allium
Our data are congruent with the phylogenetic scheme and taxonomy of Allium (Friesen et al., 2005
). Allium can be split into three clades by their genomic signature (Fig. 6). The first clade (Fig. 2, clade 1) is characterized by a variable number of chromosomes (x = 7, [8, 9, 11]), the loss of the minisatellite TTAGGG terminal repeats found in related Asparagales families and other genera of Alliaceae, and only small amounts of the Tetrahymena-type repeat, which are almost certainly interstitial in nature. The second clade (Fig. 2) has a base chromosome number of x = 8 and a higher proportion of human-type of sequences in the genome. However, these sequences are not telomeric. The third clade (Fig. 2) shares the base chromosome number (x = 8) and lacks minisatellite telomeric repeats, but the 1C genome size is on average reduced by about 30%, suggesting genomic downsizing in the ancestor to the group (Appendix S2, see Supplemental Data with online version of this article).
Hybridization patterns corresponded with the subdivisions of subgenera into sections, e.g., similar hybridization patterns occur in species of subgen. Cepa/sect. Cepa—A. proliferum, A. galanthum, A. altaicum, and A. cepa; subgen. Cepa/sect. Schoenoprasum—A. altyncolicum, A. schoenoprasum; subgen. Anguinum/sect. Anguinum—A. victorialis, A. ochotense; subgen. Melanocrommyum/sect. Compactoprason—A. giganteum, A. macleanii; subgen. Melanocrommyum/sect. Melanocrommyum—A. nigrum, A. orientale (Fig. 2). Likewise, species previously investigated by S\#7923;korová et al. (2003c)
had a similar pattern to those investigated in this study; A. subhirsutum var. spathaceum is similar to A. moly (both subgen. Ameralium/sect. Mollium) and A. dregeanum (subgen. Allium/sect. Allium) is similar to A. flavum (subgen. Allium/sect. Codonoprasum).
Allium roylei is difficult to place in phylogenetic schemes, and its position remains unclear, perhaps due to hybrid origin (Friesen, N., University of Osnabrück, Germany, personal communication). Morphological characters indicate relationships with subgen. Polyprason/sect. Oreiprason, but some molecular and chromosomal data support a position in subgen. Cepa/ sect. Cepa, to which it also has a similar hybridization pattern with the telomeric probes used here. In addition, A. roylei, together with other members of the subgen. Cepa/ sect. Cepa group, also share a highly repetitive 375-bp tandem repeat (ACSAT), that has been suggested to be a putative telomeric sequence of A. cepa (Pich et al., 1996b
; Pich and Schubert, 1998
).
Loss of minisatellite repeats at Allium telomeres
Telomeres of all Allium species investigated, with representation across the Allium phylogeny, do not have the consensus telomeric minisatellite repeats found in human, Arabidopsis, Tetrahymena, Chlamydomonas, Bombyx, and Oxytricha. Their absence is supported by results of end-cloning experiments in A. cepa in which several repetitive sequences were isolated but not minisatellites (Pich and Schubert, 1998
). Asparagales species with human-type telomeres possess active telomerase detectable using telomerase repeat amplification protocol (TRAP). However, no TRAP products were obtained in A. cepa (S\#7923;korová et al., 2003c
). Furthermore, primer-extension reactions using primers against Arabidopsis- (Pich et al., 1996a
), human- and Tetrahymena-type telomeric repeats (Fig. 5) either failed to generate a product or gave a product with only a few repeats.
Despite our attempt to analyze a large range of potential minisatellite terminal sequences, it remains possible, although unlikely, that minisatellite variants not investigated in these experiments might still have a telomeric function in Allium. Yeasts can have several different repeats, including e.g., the 25-bp repeats found in Kluyveromyces, which are different from shorter, imprecisely repeated units in Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe (Shampay and Blackburn, 1988
; McEachern and Blackburn, 1994
). Despite this variability, all these telomeric sequences are maintained by telomerase.
Because maintenance and functions of telomeres are largely dependent on their protein components, the evolution of telomeric DNA is likely to have been accompanied by co-adaptation of telomere-binding proteins. As evidence of such co-adaptation in Asparagales with the human-type telomeres, we have observed proteins that bind to either or both of the human- and Arabidopsis-type repeats in Iris and Muscari (Rotkova et al., 2004
). Similar proteins were detected in A. cepa (Fajkus et al., 2005
), despite the absence of all investigated minisatellites at telomeres reported here. The data indicate conservation of telomere-binding proteins in association with divergence and probable loss of minisatellite repeats in evolution.
Allium may not represent an evolutionary dead-end as might be envisaged by the widespread occurrence of telomerase-based systems in eukaryotes. Instead, telomerase-based systems can be considered as evolutionary transitions leading to ALT systems that can evolve when mutation disables the activity of telomerase or associated proteins (Louis, 2002
).
FOOTNOTES
1 The authors thank Mr. T. Hall (Royal Botanic Gardens, Kew), Dr. N. Friesen (the Osnabruck collection), and Mgr. M. Chytra (Allium ericetorum) for plant material; Prof. I. Schubert for encouragement and fruitful discussions; Ms. D. Fridrichova for technical work; and the Grant Agency of the Czech Republic (projects 204/04/P104 to E.S., 521/05/0055 to J.F.), the Grant Agency of the Academy of Science, Czech Republic (IAA600040505), institutional funding (MSM0021622415, AVOZ50040507) and the Leverhulme Trust for support of this work. 
7 Corresponding authors (fajkus{at}sci.muni.cz
, A.R.Leitch{at}qmul.ac.uk
)
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