Chromosome Research 11: 183194, 2003. # 2003 Kluwer Academic Publishers. Printed in the Netherlands
183
Cytogenetic tools for Arabidopsis thaliana
Maarten Koornneef1, Paul Fransz2 & Hans de Jong1* 1 Wageningen University, Laboratory of Genetics, Arboretumlaan 4, 6703 BD Wageningen, the Netherlands; E-mail:
[email protected]; 2 University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, 1098 SM Amsterdam, the Netherlands *Correspondence
Key words: Arabidopsis, cytogenetics, DNA sequences, FISH, heterochromatin, meiosis
Abstract Although the first description of chromosomes of Arabidopsis dates as far back as 1907, little attention was paid to its cytogenetics for a long time. The spectacular interest in chromosome research for this species that now is the model plant species by excellence came with the introduction of molecular cytogenetical research including FISH technology, genome sequence data and immunodetection of chromatin proteins. In this paper, we present an overview of the most important cytogenetic tools that were developed for Arabidopsis in recent decades. It shows the power of meiosis for studying synaptic mutants and FISH technology, and the development of numerical and structural chromosome mutant series like trisomics, telotrisomics and translocations for assigning linkage groups to chromosomes. Its small genome and chromosome size and relatively simple organization of heterochromatin have been the key to a successful characterization of the molecular organization of repetitive and single copy sequences on the chromosomes, both in metaphase and pachytene complements, but also in interphase nuclei and extended DNA fibres. Finally, Arabidopsis is the first plant species in which a heterochromatin knob could be analysed in full detail and in which chromosome painting with BAC clones covering whole chromosome arms could be established. All these achievements are probably only the very first steps in a promising new era in plant cytogenetics and chromatin research yet to come.
Introduction Arabidopsis thaliana (L.) Heynhold (2n ¼ 2x ¼ 10) is presently the most common plant species for genetic and molecular studies. Major advantages for genetic studies include the small plant size and short generation time, and its propagation by selffertilization, while the combination of easy transformation and the availability of the almost complete genomic DNA sequence make the model
unsurpassed for genome research (Meinke et al. 1998). Until a few years ago however, Arabidopsis received very little attention for cytogenetic research, in spite of its simple karyotype of only ¢ve chromosomes, as already described by Laibach (1907) (referred to as Stenophragma thalianum (L.); see Figure 1). Relevant information in this ¢rst scienti¢c paper on Arabidopsis, not dealing with taxonomy, is the observation of some pairing of homologues during mitosis, con¢rmed later by
M. Koornneef et al.
184
Figure 1. First drawing of Arabidopsis thaliana chromosomes at late metaphase II (Laibach 1907).
Steinitz-Sears (1963) and the presence of 10 chromocentres in interphase nuclei, studied in much more detail almost a century later by Fransz et al. (1998a, 2002). Subsequent work on Arabidopsis cytogenetics (reviewed in Re¤dei 1970) provided little new information until Steinitz-Sears (1963) published a detailed description of mitotic and meiotic chromosomes, which included the analysis of polyploids and trisomics. Chromosome morphology at that time included only centromere position (median or submedian), chromosome length and the presence of a nucleolar organizer on the longest chromosome (Steinitz-Sears 1963) and on a second chromosome (Sears & Lee-Chen 1970), nowadays assigned to the chromosomes 2 and 4, respectively. However, in the ¢rst Giemsa-stained C-banding karyotype (Ambros & Schweizer 1976), only one satellite chromosome was observed, which had a median centromere, which is surprising knowing that both chromosome 2 and 4 have submedian centromeres that carry large terminal rDNA clusters at the end of their short arms. These con£icting results indicate the di⁄culty of studying the small Arabidopsis chromosomes ranging from 1.52.8 mm. Only with the advent of molecular cytogenetics and the use of the DNA-speci¢c DAPI
£uorochrome could a reliable karyotype for somatic metaphase chromosomes be established (Heslop-Harrison & Maluszynska 1994, Maluszynska & Heslop-Harrison 1993). The use of rDNA as probes established unambiguously the presence of two NOR-carrying chromosomes. A £ow cytometry-determined karyotype, using trisomics for chromosome reference, was in agreement with the previously determined karyotype (Samoylova et al. 1996).
Meiosis Meiosis was ¢rst studied by Laibach (1907) and later far more extensively by Steinitz-Sears (1963). Chromosomes at metaphase I, and even more at late prophase I displaying chiasmata and other morphological details are popular for pairing analysis and are helpful in the identi¢cation of trisomics and other aneuploids (Ross et al. 1996, Sears & Lee-Chen 1970). Sears & Lee-Chen (1970) mentioned the superiority of the long pachytene chromosomes for karyotype analysis. However, at that time, this technique was not su⁄ciently developed to reveal well-di¡erentiated fully analysable complements. Further improvements
Cytogenetic tools for Arabidopsis thaliana revealed more details in premeiotic phases of meiosis (Kla¤sterska¤ & Ramel 1980) but could not demonstrate chromosomal details for karyotyping. However, the detailed description of male meiosis by Ross et al. (1996), using an improved cell-spreading procedure and DAPI staining, showed that pachytene chromosomes are ideal subjects for cytogenetics in Arabidopsis because of their size and the amount of detail they show with respect to the distribution of euand heterochromatin. Albini (1994) already showed that the electron microscopic analysis of synaptonemal complexes (SCs) from meiotic prophase I cells allowed the construction of a pachytene SC karyotype in which the two NOR chromosomes are the smallest chromosomes, as was con¢rmed by the complete DNA sequence (Arabidopsis Genome Initiative 2000). Compared to light microscopy of pachytene complements, the analysis of SC spreads is technically very demanding and time consuming. Steinitz-Sears (1963) gave a general description of meiosis in Arabidopsis. Numerical data were given by Armstrong & Jones (2001) who estimated chiasma frequency in diakinesis and metaphase I complements at 9.7 for male meiosis and 8.5 for female meiosis. Sanchez-Moran et al. (2001) who studied meiosis of pollen mother cells in combination with £uorescence in-situ hybridization (FISH) using the 45S and 5S rDNA repeats as probes found on average 9.24 chiasmata per cell in the Wassilewskija (Ws) accession. Because their procedure allowed the identi¢cation of individual bivalents, they could provide chiasma estimates for individual chromosomes. These numbers may represent a slight underestimation in view of the total genetic map length of around 600 cM, which is equivalent to approximately 12 chiasmata. When performing a similar analysis for 8 di¡erent Arabidopsis accessions, Sanchez-Moran et al. (2002) showed that there is signi¢cant variation between accessions for chiasma frequencies and that this variability is chromosome-speci¢c. Di¡erences between female and male meiosis seem to a¡ect recombination in speci¢c regions as was deduced from comparing recombination in reciprocal crosses of heterozygotes with homozygote recessive tester lines (Vizir & Korol 1990) or by comparing male meiosis with the genetic map based on both female and male meiosis (Copenhaver et al. 1998).
185 Numerical chromosome variants and their use Haploids are considered useful in genetics, especially for the selection of recessive mutants at the tissue culture level. Haploid Arabidopsis plants derived from anther culture were reported by Gressho¡ & Doy (1972) and later by Amos & Scholl (1978), but neither procedure could reproducibly generate mature haploid plants for vegetative propagation (reviewed in Morris & Altmann 1994). The e¡ectiveness of mutant selection based on M2 generations and the use of reverse genetics has abolished the need for using tissue culture to select for conditional lethal mutants. Tetraploid (2n ¼ 4x ¼ 20) and hexaploid (2n ¼ 6x ¼ 30) Arabidopsis plants were found upon colchicine treatment (Bouharmont & Mace¤ 1972, Re¤dei 1964) and also occurred frequently in tissue culture regenerants (Morris & Altmann 1994, Negrutiu et al. 1978). The latter resulted in many unwanted tetraploids upon transformation when tissue-culture-based protocols were used for this. In addition to the tissue culture environment, the high levels of endopolyploidy reported by Galbraith et al. (1991) may increase the frequency of such polyploids. Spontaneous tetraploids have been found in some accessions (Heslop-Harrison & Maluszynska 1994, Koornneef unpublished observations). Diploids and tetraploids show few morphological di¡erences (Bouharmont & Mace¤ 1972). The leaves of tetraploids are more spherical and have thicker stems. An easier way to distinguish tetraploids from the diploids is the relatively large seeds and pollen grain size in the former (Altmann et al. 1994, Bronckers 1963). Fertility is somewhat reduced in tetraploids and seed set in tetraploid diploid crosses relatively good, leading to vigorous growing and partial fertile triploids. The selfed progeny of these triploids showed a variation of chromosome numbers including trisomics. Hexaploid tetraploid crosses in contrast to hexaploid diploid crosses were rather successful (Re¤dei 1964). The ease of such ploidy crosses allows the transfer of genetic alleles from higher polyploids into diploids. Trisomics and telotrisomics Trisomic plants carrying an extra chromosome completely homologous to one of the ¢ve chro-
186 mosome pairs (2n ¼ 2x þ 1) can be found at high frequency in the progeny of triploid Arabidopsis (Steinitz-Sears 1963, R˛bbelen & Kribben 1966, Koornneef & van der Veen 1983). Telotrisomics that possess an extra chromosome arm with a functional centromere occur at low frequency in the progeny of primary trisomics. A trisomic plant has a speci¢c phenotype characteristic for the chromosome or chromosome arm involved, which can be used for identi¢cation of this aneuploid without further chromosome analysis. Transmission of pollen with the extra chromosome is less than 15% or even absent but, with only an extra chromosome arm, transmission can be as high as 32% as found for telotrisomic Tr3A (Koornneef & van der Veen 1983). The rare and sterile tetrasomics (2n ¼ 2x þ 2) have a phenotype similar to but more severe than that of the corresponding primary trisomics. The ease of phenotypic identi¢cation depends very much on the uniformity of the genetic background and therefore trisomic analysis to locate linkage groups is most convenient with morphological markers in the genetic background of the trisomics (Lee-Chen & SteinitzSears 1967, Koornneef & van der Veen 1983). Markers segregating in the progeny of trisomics are associated with the extra chromosome by their speci¢c trisomic segregation ratios. In the selfed progeny of a duplex (AAa) plant, all trisomic progeny is wild type (A..) and the diploid progeny segregates 8 (A.) to 1 (aa). When the polymorphic marker is not located on the extra chromosome, a normal disomic 3 : 1 ratio, both among the trisomic and diploid progeny, is observed. Chromosome nomenclature in Arabidopsis is based on the six mainly short linkage groups described by Re¤dei (1965) which were assigned to the chromosomes by trisomic analysis (Lee-Chen & Steinitz-Sears 1967, Sears & Lee-Chen 1970). The incorrect assignment of linkage group 4 has later been corrected in a trisomic and linkage analysis by Koornneef & van der Veen (1983), who also assigned the linkage groups described by McKelvie (1965) to the ¢ve chromosomes. Therefore, chromosome nomenclature does not follow the order of decreasing length, as was done for many other organisms, but is based on assignment of linkage groups. Consequently, chromosome 1 is the longest in the complement and chromosome 2 the shortest. This linkage-
M. Koornneef et al. group-based nomenclature is now widely accepted in the Arabidopsis research community and the sequence programme (Arabidopsis Genome Initiative 2000) and clearly di¡ers from the nomenclature based on chromosome length of Ambros & Schweizer (1976) as discussed by Schweizer et al. (1987) and Heslop-Harrison & Maluszynska (1994).
The location of centromeres on the linkage maps Telotrisomics have been used to locate centromeres in Arabidopsis and are available for both arms of chromosome 1 and 5, and one for the lower arm of chromosome 3 (Sears & Lee-Chen 1970, Koornneef & van der Veen 1983, Koornneef 1983, Samoylova et al. 1996). The absence of telotrisomics of chromosomes 2 and 4 is most likely explained by the fact that primary trisomics for these chromosomes are less di¡erent from wild type than the other primary trisomics. Therefore, a telotrisomic of the short arm might not be distinguishable from wild type, whereas a trisomic for the long arm probably will be similar in phenotype to the primary trisomic. Telotrisomics can be used for centromere location in di¡erent ways. When one of two linked markers segregates trisomic and the other disomic, only the former is associated with the extra chromosome arm and, consequently, the centromere lies between the two markers. The segregation ratios for telotrisomics are slightly di¡erent from those of primary trisomics, because the two complete chromosomes predominantly segregate to opposite poles at anaphase I of meiosis. In the case of a marker located on the additional chromosome arm, sel¢ng of a genotype AtAa results in a 1 : 0 segregation among the (telo)trisomic progeny and 3 : 1 among the diploid progeny. In the case of a crossover event between the marker and the centromere, these ratios change and hence allow mapping of centromeres based on recombination. Models predicting these ratios and their experimental veri¢cation were described in Koornneef (1983) and allowed a rather accurate location of the centromere in chromosome 1 and 5. Since molecular markers were not available in the early eighties, mapping was performed only with morphological markers. Molecular markers, which
Cytogenetic tools for Arabidopsis thaliana are now abundantly available in Arabidopsis, can be used in a similar way. Alonso-Blanco et al. (1998) used dose dependency, a method comparing band intensity of AFLP markers in the chromosome 3 telotrisomic with the band intensity in the diploid and the primary trisomic for centromere location. Accordingly, the centromere of chromosome 3 could be mapped between two markers that were completely linked in a recombinant inbred line population of 162 lines. This procedure is especially powerful because AFLP markers obtained with certain primer combinations tend to cluster predominantly around centromeres (Alonso-Blanco et al. 1998). Other methods to map centromeres were the tetrad analysis using the quartet(qrt1) mutation that allowed analysis of all four pollen grains derived from a single tetrad (Copenhaver et al. 1998, 2003 (this issue)), and FISH with clones containing centromere sequences on pachytene chromosomes that are also mapped on the linkage and physical maps (Fransz et al. 1998a, 2000, Tabata et al. 2000, Haupt et al. 2001).
Structural chromosome variants Structural chromosome variants such as translocations, inversions and deletions could hardly be
187 studied in mitotic cell complements but were cytogenetically identi¢ed at pachytene and metaphase I where speci¢c multivalent chromosome associations allowed cytogenetic identi¢cation of translocation heterozygotes and other structural variants (Sree Ramulu & Sybenga 1979, 1985, Vergunst et al. 2000). Selection for translocations could also be done e⁄ciently on the basis of semisterility of the translocation heterozygotes (Sree Ramulu & Sybenga 1979). Genetic methods based on changes in linkage relationships between chromosomes, which include speci¢c DNA sequences and FISH technology are other ways for assessing chromosomal rearrangements. The Arabidopsis sequence map is based on the Columbia (Col) accession and most of the linkage mapping was performed on Columbia Landsberg erecta (Ler) cross, assuming collinearity of gene order between the accessions. Recent cytogenetic and genetic analyses, however, demonstrated various polymorphisms for structural rearrangements, including small tandem duplications and insertions, as were con¢rmed by comparing DNA sequence data of Ler and Col (Arabidopsis Genome Initiative 2000). It is likely that this high rate of polymorphism was partly due to transposon activity in speci¢c chromosomal regions as was demonstrated for the complex RPP5 resistance cluster region (No€el et al. 1999).
2 Figure 2. Ideogram of the five chromosomes of Arabidopsis thaliana, accession Ler, showing chromosome length (in mm) at pachytene, centromeres, pericentromeric heterochromatin, nucleolar organiser regions, euchromatin and (polymorphic) heterochromatin knobs.
188 DAPI karyotyping of pachytene chromosomes revealed a polymorphism for a heterochromatic knob, hk4S, in the short arm of chromosome 4 that was shown in the Col and WS but not in the C24 and Ler accessions (Fransz et al. 1998a). FISH also revealed polymorphism for the 5S rDNA cluster, which is (1) near the centromere of chromosome arm 3S in Col, Cape Verde Islands (Cvi) and Kashmir (Kas1), (2) in the lower arm of chromosome 3 in Ler and (3) absent in WS (Fransz et al. 1998a). Linkage analysis of the F2 derived from the cross Hannover/Mˇnden Ws showed a translocation between chromosomes 3 and 4 (Kowalski et al. 1994). Since genetic and cytogenetic analysis thus far involved only a limited number of accessions, many more examples of chromosomal polymorphisms are yet to be expected. Irradiation is a classical way to induce chromosomal aberrations and was shown to occur with relatively high frequency after treatment with both X-rays and fast neutrons (Sree Ramulu & Sybenga 1979, 1985). About half of the translocations in these studies showed a reduced frequency or even absence of translocation homozygotes in the progeny of heterozygotes, presumably due to damage at the breakpoints (Sree Ramulu & Sybenga 1985). This interpretation is supported by the ¢nding of extended deletions at break ends during double strand break repair (Kirik et al. 2000). Speci¢c chromosome markers linked to the translocation breakpoint made it possible to identify the two chromosomes involved in the translocation (Koornneef et al. 1982). Unfortunately these translocations are no longer available for directly locating genes to speci¢c regions of the chromosomes. Other structural variants include deletions that can be generated by irradiating haploid wild-type pollen and using this pollen to irradiate recessive mutants (Timpte et al. 1994, Vizir et al. 1994, Vizir & Mulligan 1999). Such recessive phenotypes occurred at approximately 1%, when irradiated pollen is used to pollinate diploids, but increased to values between 3 and 10%, when a tetraploid marker line was pollinated instead (Vizir & Mulligan 1999). The major part of the deletions that behave as dominant lethals was rescued in triploids. Vizir & Mulligan (1999) provided indirect evidence that many of these deletions are interstitial, spanning on average 100 kbp in size, and
M. Koornneef et al. transmit through the male line to subsequent generations only at very low frequencies. This explains why, in general, only smaller deletions are detected for irradiation-induced mutants that were isolated in M2 generations and show regular Mendelian inheritance (Shirley et al. 1992, Bruggemann et al. 1996). These deletions were used for clonal analysis (Furner et al. 1995) and for cloning of the GA1 and RGA1 genes via genomic subtraction (Sun et al. 1992, Silverstone et al. 1998). Deletions are also helpful in map-based cloning because they are easily detected. Compared to chemical mutagenesis, the fact that two tandemly repeated homologous genes can be knocked out by a deletion o¡ers an important advantage for determination of gene functions (Li et al. 2001). An important and often unwanted source of structural variants comes from transformation experiments. Upon transformation with Agrobacterium tumefaciens, Negruk et al. (1996) reported minor deletions less than 17 bp in several independent cer2 mutants, while Castle et al. (1993) estimated that 20% of T-DNA mutants from a genetic analysis of embryo defective mutants were involved in translocations. Detailed analysis of some transformants indicated complex rearrangements, which may occur especially when multiple T-DNA sequences are integrated. Probably many of these rearrangements such as the 26 cM paracentric inversion bordered by two T-DNAs, may have occurred during the T-DNA integration process rather than resulting from intrachromosomal recombination between homologous T-DNA insertions (Laufs et al. 1999). Nacry et al. (1998) reported for a single line a translocation between chromosome 2 and 3, a paracentric inversion on chromosome 2 and deletions on chromosome 2 and 3 near the T-DNA integration sites. Tax & Vernon (2001) described a region on chromosome 5 larger than 40 kbp and containing a T-DNA that had inserted into a locus on chromosome 1. Such complex rearrangements cause problems in the identi¢cation of the genes, disruption of which leads to the phenotype under study. Furthermore, paracentric inversions strongly suppress recombination; as an example, co-segregation of the mutant phenotype to both TDNAs bordering the 26 cM inversion was observed (Laufs et al. 1999). A special case of translocations and inversions using transformation is their
Cytogenetic tools for Arabidopsis thaliana induction by the Cre/lox recombination system, where the Cre protein induces recombination between lox sites. When a genome contains lox sites on di¡erent chromosomes, translocations between these sites can occur, whereas their presence on the same chromosome may lead to deletions and inversions (Vergunst et al. 2000).
FISH technology A major breakthrough in Arabidopsis chromosome research was the introduction of FISH technology for direct detection of DNA sequences on chromosomal targets. Initially only hybridizations on mitotic metaphase complement were carried out using repeat DNAs as probes (e.g. Maluszynska & Heslop-Harrison 1991, Schwarzacher & HeslopHarrison 1991, Murata & Motoyoshi 1995, HeslopHarrison et al. 1999). These repetitive sequences, which are straightforward and diagnostic markers in karyotype analyses, and represent about 10% of the genomic DNA, include especially the ribosomal repeats (18S, 26S and 5S rDNAs), centromere repeats (180 bp pAl1 and the 106B repeats), pericentromeric repeats (such as the Athila-like
189 repeats) and the telomere repeat at all chromosome ends. Although FISH studies revealed considerable information on the molecular organization of Arabidopsis chromosomes, the spatial resolution between adjacent targets of metaphase complements of only 2MB was insu⁄cient for detailed chromosome mapping studies (Murata & Motoyoshi 1995). Considerable improvement of resolution was achieved with FISH on spread pachytene chromosomes. These 2025 times longer meiotic prophase complements measuring 5080 mm for individual chromosome pairs (Table 1), now allow resolution values of 50 kbp in euchromatin (Fransz et al. 1998a). The chromosomes also display conspicuous di¡erentiation of heterochromatin and centromere regions (Figure 3) and are therefore more accurate and informative for karyotyping and physical mapping and genome studies (Fransz et al. 1998a, de Jong et al. 1999). Speci¢c disadvantages of pachytene analysis in Arabidopsis are: (1) the number of microsporocytes in an anther is relative low; (2) development of microsporocytes is not very synchronous, which means that cells in the preparation are at di¡erent meiotic stages; (3) ability to spread at late pachytene when chromosomes resolve from the clustered state in the synizetic knot
Table 1. Overview of genomic, genetic and chromosomal data of Arabidopsis thaliana
Genetic map length (cM) Physical length (kb)a Physical length (% genome) Physical/genetic length ratio Number of genesb Chromosome length mitotic metaphase (mm)c Chromosome length pachytene (mm)d Relative length Centromere index (% s/s þ l) (Peri)centromeric heterochromatin (mm) Knob length (mm) Heterochromatin total (mm) NOR heterochromatin length (mm) Centromere region (mm)e Euchromatin a
chr. 1
chr. 2
chr. 3
chr. 4
chr. 5
Total
Average
135 29205 24.6 216.3 7041 1.52.8 80.8 24.4 49 4.1
97 17463 14.7 180.0 4390
101 23560 19.9 233.3 5623
125 22140 18.7 177.1 3817
139 26170 22.1 188.3 5847
597 118538 100.0 198.6 26718
119.4 23707.6 20 199.0 5344
52.1 15.7 23 4.1 0.8 4.1 1.6 1.5 46.6
69.3 20.9 39 4.3
52.7 15.9 24 3.4 0.8 4.2 1.7 1.5 46.8
76.3 23.0 45 4.3
331.2 100.0
66.2
20.2
4.0
4.3
21.0 3.3 8.3 307.1
8.5
4.1 2.2 76.7
http://www.arabidopsis.org/servlets/mapper; http://biolinx.bios.niu.edu/t80maj1/arab chromos.htm; c Heslop-Harrison & Maluszynska 1994; d Fransz et al. 1998; e Haupt et al. 2001; chr-chromosome. b
4.3 1.3 65.0
1.9 72.0
61.4
190
M. Koornneef et al.
Figure 3. Photomontage of chromosomes at meiotic metaphase (a) and pachytene (b). A comparison is also made between FISH signals of cosmid E4-6 (red) and cosmid E4-11 (green) on a pachytene bivalent (b) and on an extended DNA fibre (c) at roughly the same magnification (bar ¼ 10 mm).
is strongly genotype dependent (cf. maize KWS line): WS and C24, the accessions lacking 5S loci on chromosome 3, spread better than Col and Ler etc. (Fransz et al. 1998a). Examples of both types are shown in Figure 4. Increasing microscopic resolution of chromatin to molecular dimensions has been obtained with FISH on extended DNA and on isolated DNA molecules immobilized on coated slides. Using three overlapping cosmid clones hybridized to chromatin ¢bres from interphase leaf nuclei, Fransz et al. (1996) gauged the DNA sizeFISH signal ratio at 3.27 kb/mm, a stretching degree close to that of the native Watson-Crick conformation. Shortly later, Jackson et al. (1999) carried out direct physical mapping of BAC inserts by FISH of circular BAC molecules immobilized on microscope slides. With a stretching ratio of 2.44 kb/mm and a detection sensitivity of 2 kb, even small linear distances can directly be transformed into kb values. Fibre-FISH that can span DNA targets of more than 1.7 Mbp in a single preparation (Jackson et al. 1998) provides unsurpassed spatial resolution of values as close as 1 kb and a detection sensitivity better than 700 bp. However, the absence of any structural landmark that can position a FISH signal on the chromosome requires probes from adjacent or overlapping regions for proper interpretation (Fransz et al. 1998b). Fibre-FISH has proven especially successful for establishing the molecular size of dispersed and partly overlapping repeats (Brandes et al. 1997b, Fransz et al. 2000) and has been shown to ¢ll in the gaps in physical maps (Jackson et al. 1998).
FISH karyotyping and repeat polymorphisms Individual chromosomes at pachytene are about 25 times longer compared to mitotic metaphase (Table 1) and are unambiguously identi¢ed using the 45S rDNA and 5S rDNA repeats as additional chromosomal markers (Figure 2). More recent FISH analysis with YACs and BACs painted on the short arm demonstrated that the heterochromatic knob hk4S resulted from a paracentric inversion in the proximal part of the chromosome arm 4S, bringing pericentromeric heterochromatin to an interstitial position (Fransz et al. 2000). This unique heterochromatic island was the subject of an extensive comparative DNA sequenceFISH study and revealed detailed structural, molecular and functional properties of this specialized chromosome region (Fransz et al. 2000, CSHL/ WUGSC/PEB Arabidopsis Sequencing Consortium 2000). Major tandem repeats are 45S rDNA on the nucleolar organisers of chromosomes 2 and 4 and 5S rDNA on chromosomes 4 and 5 and polymorphic sites on chromosome 3 (Maluszynska & Heslop Harrison 1991, Murata et al. 1997, Fransz et al. 1998a). All other tandem repeats could be mapped in the centromere and pericentromere regions (Brandes et al. 1997a). Heslop-Harrison et al. (1999) analysed AtCon (pAL1 repeat) in Arabidopsis, a 178-bp tandem repeat with sequence similarity to yeast CDEI and human CENP-B DNA-protein binding motifs, located at the centromeres of all chromosomes. Parts of the
Cytogenetic tools for Arabidopsis thaliana
191
Figure 4. Differences of chromosome spreading at pachytene in four accessions: (A) WS hybridized with 5S rDNA (green) and CIC7C3 (red); (B) C24 hybridized with several BACs from the distal part of chromosome arm 4S; (C) Ler, hybridized with 5S rDNA (green) and 45S rDNA (red); (D) Col , hybridized with 5S rDNA (green) and GA1 (red). NOR, nucleolus organizing region; chr.4, centromere region of chromosome 4 (bar ¼ 10 mm).
less-conserved regions were detected on speci¢c chromosomes, indicating that there are chromosome-speci¢c variants of AtCon. Of particular interest is the molecular organization of the proximal chromosome regions including centromere structure and pericentromeric heterochromatin region. Table 1 gives an overview of genomic, genetic and chromosomal data for all chromosomes in Arabidopsis. Centromeres are demonstrated with the 180-bp centromere repeat
and 106B probes that are organized in interspersed non-separated blocks (Thompson et al. 1996). Centromere length ranges from 1.3 to 2.2 mm, and is signi¢cantly proportional to chromosome size, a correlation previously found for other plant species by Bennett et al. (1981). Pericentromere heterochromatin regions, which are painted with the 17A20 BAC containing retrotransposons and measure 4.14.4 mm, are roughly the same for all chromosomes.
M. Koornneef et al.
192 Chromosome painting strategies True chromosome painting or chromosome in-situ suppression (CISS) is based on FISH with ampli¢ed arbitrary DNA sequences from isolated chromosomes and blocked with unlabelled genomic repeat (Cot) fraction, and has been applied successfully to mammals, birds, reptiles and insects. In spite of many e¡orts, the CISS technique could not be adapted directly to plant species (Schubert et al. 2001). An attractive alternative way to study individual chromosomes is genomic in-situ hybridization (GISH, or genome painting) painting alien chromosomes in monosomic additions, but these interspeci¢c aneuploids are available only for a few cropwild species combinations. A more versatile chromosome painting strategy is FISH with largeinsert single-sequence clones like BACs and YACs on pachytene chromosomes. Fransz et al. (2000) demonstrated the power of this approach by painting the entire chromosome arm 4S with ¢ve contiguous YAC clones, spanning in total 2.8 Mbp and covering almost the entire arm. Later, BAC clones were used instead, painting speci¢c chromosomes (Lysak et al. 2001, Fransz et al. 2002). BAC FISH painting works best for small-genome species and for tomato, maize and other species with larger genomes requires blocking with unlabelled Cot10Cot100 repetitive DNA fractions to suppress cross hybridization of repeats to nonspeci¢c chromosome regions (unpublished data). BAC FISH to homoeologous chromosomes of related species, comparable to ZOO-FISH in mammalian cytogenetics has now also been demonstrated with Arabidopsis BACs hybridized to chromosomes of several Brassicaceae species (Lysak et al. 2003, Comai et al. 2003 (this issue)) and to the Arabis holboellii (¼Boechera holboellii) chromosome complements (unpublished results). Chromosome painting is also outstanding for the study of chromosome organization in interphase nuclei. Fransz et al. (2002) studied parenchyma interphase nuclei by FISH using rDNA clones, and BACs painting the entire chromosome 4, and immunostaining of methylated DNA and acetylated histones. Interphase nuclei display a variable number of clearly distinguishable chromocentres composed of one or more centromeric heterochromatin regions, regions that contain heavily methylated repeat sequences from which di¡erent
0.22 Mbp chromatin loops emanate. This study revealed frequent associations of homologous chromocentres, alignment of homologous regions and variable-sized loops.
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