cytogenetic tools for arabidopsis thaliana

Cytogenetic tools for Arabidopsis thaliana

Maarten Koornneef1, Paul Fransz2 & Hans de Jong1*1Wageningen University, Laboratory of Genetics, Arboretumlaan 4, 6703 BD Wageningen,the Netherlands; E-mail: [email protected]; 2University of Amsterdam, SwammerdamInstitute 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 waspaid to its cytogenetics for a long time. The spectacular interest in chromosome research for this species thatnow is the model plant species by excellence came with the introduction of molecular cytogenetical researchincluding FISH technology, genome sequence data and immunodetection of chromatin proteins. In thispaper, we present an overview of the most important cytogenetic tools that were developed for Arabidopsisin 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, telotrisomicsand translocations for assigning linkage groups to chromosomes. Its small genome and chromosome sizeand relatively simple organization of heterochromatin have been the key to a successful characterizationof the molecular organization of repetitive and single copy sequences on the chromosomes, both in metaphaseand pachytene complements, but also in interphase nuclei and extended DNA fibres. Finally, Arabidopsis isthe first plant species in which a heterochromatin knob could be analysed in full detail and in which chromo-some painting with BAC clones covering whole chromosome arms could be established. All these achieve-ments are probably only the very first steps in a promising new era in plant cytogenetics andchromatin research yet to come.

Introduction

Arabidopsis thaliana (L.) Heynhold (2n¼ 2x¼ 10)is presently the most common plant species forgenetic and molecular studies. Major advantagesfor genetic studies include the small plant size andshort generation time, and its propagation by self-fertilization, while the combination of easy trans-formation and the availability of the almostcomplete genomic DNA sequence make the model

unsurpassed for genome research (Meinke et al.1998). Until a few years ago however, Arabidopsisreceived very little attention for cytogeneticresearch, in spite of its simple karyotype of only ¢vechromosomes, as already described by Laibach(1907) (referred to as Stenophragma thalianum(L.); see Figure 1). Relevant information in this ¢rstscienti¢c paper on Arabidopsis, not dealing withtaxonomy, is the observation of some pairing ofhomologues during mitosis, con¢rmed later by

Chromosome Research 11: 183�194, 2003. 183# 2003 Kluwer Academic Publishers. Printed in the Netherlands

Steinitz-Sears (1963) and the presence of 10chromocentres in interphase nuclei, studied inmuch more detail almost a century later by Franszet al. (1998a, 2002).

Subsequent work on Arabidopsis cytogenetics(reviewed in Re¤ dei 1970) provided little newinformation until Steinitz-Sears (1963) published adetailed description of mitotic and meioticchromosomes, which included the analysis ofpolyploids and trisomics. Chromosome morphol-ogy at that time included only centromere position(median or submedian), chromosome length andthe presence of a nucleolar organizer on the longestchromosome (Steinitz-Sears 1963) and on a secondchromosome (Sears & Lee-Chen 1970), nowadaysassigned to the chromosomes 2 and 4, respectively.However, in the ¢rst Giemsa-stained C-bandingkaryotype (Ambros & Schweizer 1976), only onesatellite chromosome was observed, which had amedian centromere, which is surprising knowingthat both chromosome 2 and 4 have submediancentromeres that carry large terminal rDNAclusters at the end of their short arms. Thesecon£icting results indicate the di⁄culty of studyingthe small Arabidopsis chromosomes ranging from1.5�2.8 mm. Only with the advent of molecularcytogenetics and the use of the DNA-speci¢c DAPI

£uorochrome could a reliable karyotype forsomatic metaphase chromosomes be establi-shed (Heslop-Harrison & Maluszynska 1994,Maluszynska & Heslop-Harrison 1993). The use ofrDNA as probes established unambiguously thepresence of two NOR-carrying chromosomes. A£ow cytometry-determined karyotype, usingtrisomics for chromosome reference, was inagreement with the previously determinedkaryotype (Samoylova et al. 1996).

Meiosis

Meiosis was ¢rst studied by Laibach (1907) andlater far more extensively by Steinitz-Sears (1963).Chromosomes at metaphase I, and even more atlate prophase I displaying chiasmata and othermorphological details are popular for pairinganalysis and are helpful in the identi¢cation oftrisomics and other aneuploids (Ross et al. 1996,Sears & Lee-Chen 1970). Sears & Lee-Chen (1970)mentioned the superiority of the long pachytenechromosomes for karyotype analysis. However, atthat time, this technique was not su⁄cientlydeveloped to reveal well-di¡erentiated fully ana-lysable complements. Further improvements

Figure 1. First drawing of Arabidopsis thaliana chromosomes at late metaphase II (Laibach 1907).

184 M. Koornneef et al.

revealed more details in premeiotic phases ofmeiosis (Kla¤ �ssterska¤ & Ramel 1980) but couldnot demonstrate chromosomal details for karyo-typing. However, the detailed description ofmale meiosis by Ross et al. (1996), using animproved cell-spreading procedure and DAPIstaining, showed that pachytene chromosomesare ideal subjects for cytogenetics in Arabidopsisbecause of their size and the amount of detailthey show with respect to the distribution of eu-and heterochromatin. Albini (1994) alreadyshowed that the electron microscopic analysis ofsynaptonemal complexes (SCs) from meiotic pro-phase I cells allowed the construction of apachytene SC karyotype in which the two NORchromosomes are the smallest chromosomes, aswas con¢rmed by the complete DNA sequence(Arabidopsis Genome Initiative 2000). Comparedto light microscopy of pachytene complements, theanalysis of SC spreads is technically very demandingand time consuming.

Steinitz-Sears (1963) gave a general descriptionof meiosis in Arabidopsis. Numerical data weregiven by Armstrong & Jones (2001) who estimatedchiasma frequency in diakinesis and metaphase Icomplements at 9.7 for male meiosis and 8.5 forfemale meiosis. Sanchez-Moran et al. (2001) whostudied meiosis of pollen mother cells in combi-nation with £uorescence in-situ hybridization(FISH) using the 45S and 5S rDNA repeats asprobes found on average 9.24 chiasmata per cell inthe Wassilewskija (Ws) accession. Because theirprocedure allowed the identi¢cation of individualbivalents, they could provide chiasma estimates forindividual chromosomes. These numbers mayrepresent a slight underestimation in view of thetotal genetic map length of around 600 cM, which isequivalent to approximately 12 chiasmata. Whenperforming a similar analysis for 8 di¡erent Ara-bidopsis accessions, Sanchez-Moran et al. (2002)showed that there is signi¢cant variation betweenaccessions for chiasma frequencies and that thisvariability is chromosome-speci¢c. Di¡erencesbetween female and male meiosis seem to a¡ectrecombination in speci¢c regions as was deducedfrom comparing recombination in reciprocalcrosses of heterozygotes with homozygote recessivetester lines (Vizir & Korol 1990) or by comparingmale meiosis with the genetic map based on bothfemale and male meiosis (Copenhaver et al. 1998).

Numerical chromosome variants and their use

Haploids are considered useful in genetics, espe-cially for the selection of recessive mutants at thetissue culture level. Haploid Arabidopsis plantsderived from anther culture were reported byGressho¡ & Doy (1972) and later by Amos &Scholl (1978), but neither procedure could repro-ducibly generate mature haploid plants for vege-tative propagation (reviewed in Morris & Altmann1994). The e¡ectiveness of mutant selection basedon M2 generations and the use of reverse geneticshas abolished the need for using tissue culture toselect for conditional lethal mutants.

Tetraploid (2n¼ 4x¼ 20) and hexaploid (2n¼ 6x¼ 30) Arabidopsis plants were found upon col-chicine treatment (Bouharmont & Mace¤ 1972,Re¤ dei 1964) and also occurred frequently in tissueculture regenerants (Morris & Altmann 1994,Negrutiu et al. 1978). The latter resulted in manyunwanted tetraploids upon transformation whentissue-culture-based protocols were used for this. Inaddition to the tissue culture environment, the highlevels of endopolyploidy reported by Galbraithet al. (1991) may increase the frequency of suchpolyploids. Spontaneous tetraploids have beenfound in some accessions (Heslop-Harrison &Maluszynska 1994, Koornneef unpublishedobservations). Diploids and tetraploids show fewmorphological di¡erences (Bouharmont & Mace¤1972). The leaves of tetraploids are more sphericaland have thicker stems. An easier way to distin-guish tetraploids from the diploids is the relativelylarge seeds and pollen grain size in the former(Altmann et al. 1994, Bronckers 1963). Fertility issomewhat reduced in tetraploids and seed set intetraploid� diploid crosses relatively good, leadingto vigorous growing and partial fertile triploids.The selfed progeny of these triploids showed avariation of chromosome numbers including tri-somics. Hexaploid� tetraploid crosses in contrastto hexaploid� diploid crosses were rather suc-cessful (Re¤ dei 1964). The ease of such ploidycrosses allows the transfer of genetic alleles fromhigher polyploids into diploids.

Trisomics and telotrisomics

Trisomic plants carrying an extra chromosomecompletely homologous to one of the ¢ve chro-

Cytogenetic tools for Arabidopsis thaliana 185

mosome pairs (2n¼ 2xþ 1) can be found at highfrequency in the progeny of triploid Arabidopsis(Steinitz-Sears 1963, R˛bbelen & Kribben 1966,Koornneef & van der Veen 1983). Telotrisomicsthat possess an extra chromosome arm with afunctional centromere occur at low frequency inthe progeny of primary trisomics. A trisomic planthas a speci¢c phenotype characteristic for thechromosome or chromosome arm involved, whichcan be used for identi¢cation of this aneuploidwithout further chromosome analysis. Transmis-sion of pollen with the extra chromosome is lessthan 15% or even absent but, with only an extrachromosome arm, transmission can be as high as32% as found for telotrisomic Tr3A (Koornneef &van der Veen 1983). The rare and sterile tetra-somics (2n¼ 2xþ 2) have a phenotype similar tobut more severe than that of the correspondingprimary trisomics. The ease of phenotypic iden-ti¢cation depends very much on the uniformity ofthe genetic background and therefore trisomicanalysis to locate linkage groups is most con-venient with morphological markers in the geneticbackground of the trisomics (Lee-Chen & Steinitz-Sears 1967, Koornneef & van der Veen 1983).Markers segregating in the progeny of trisomicsare associated with the extra chromosome by theirspeci¢c trisomic segregation ratios. In the selfedprogeny of a duplex (AAa) plant, all trisomicprogeny is wild type (A..) and the diploid progenysegregates 8 (A.) to 1 (aa). When the polymorphicmarker is not located on the extra chromosome, anormal disomic 3 : 1 ratio, both among the tri-somic and diploid progeny, is observed.

Chromosome nomenclature in Arabidopsis isbased on the six mainly short linkage groupsdescribed by Re¤ dei (1965) which were assigned tothe chromosomes by trisomic analysis (Lee-Chen& Steinitz-Sears 1967, Sears & Lee-Chen 1970).The incorrect assignment of linkage group 4 haslater been corrected in a trisomic and linkageanalysis by Koornneef & van der Veen (1983), whoalso assigned the linkage groups described byMcKelvie (1965) to the ¢ve chromosomes.Therefore, chromosome nomenclature does notfollow the order of decreasing length, as was donefor many other organisms, but is based onassignment of linkage groups. Consequently,chromosome 1 is the longest in the complementand chromosome 2 the shortest. This linkage-

group-based nomenclature is now widely acceptedin the Arabidopsis research community and thesequence programme (Arabidopsis GenomeInitiative 2000) and clearly di¡ers from thenomenclature based on chromosome length ofAmbros & Schweizer (1976) as discussed bySchweizer et al. (1987) and Heslop-Harrison &Maluszynska (1994).

The location of centromeres on the linkage maps

Telotrisomics have been used to locate centromeresin Arabidopsis and are available for both arms ofchromosome 1 and 5, and one for the lower arm ofchromosome 3 (Sears & Lee-Chen 1970, Koornneef& van der Veen 1983, Koornneef 1983, Samoylovaet al. 1996). The absence of telotrisomics ofchromosomes 2 and 4 is most likely explained bythe fact that primary trisomics for these chromo-somes are less di¡erent from wild type than theother primary trisomics. Therefore, a telotrisomicof the short arm might not be distinguishable fromwild type, whereas a trisomic for the long armprobably will be similar in phenotype to the pri-mary trisomic.

Telotrisomics can be used for centromerelocation in di¡erent ways. When one of two linkedmarkers segregates trisomic and the other disomic,only the former is associated with the extrachromosome arm and, consequently, the cen-tromere lies between the two markers. The seg-regation ratios for telotrisomics are slightlydi¡erent from those of primary trisomics, becausethe two complete chromosomes predominantlysegregate to opposite poles at anaphase I ofmeiosis. In the case of a marker located on theadditional chromosome arm, sel¢ng of a genotypeAtAa results in a 1 : 0 segregation among the(telo)trisomic progeny and 3 : 1 among the diploidprogeny. In the case of a crossover event betweenthe marker and the centromere, these ratios changeand hence allow mapping of centromeres based onrecombination. Models predicting these ratios andtheir experimental veri¢cation were described inKoornneef (1983) and allowed a rather accuratelocation of the centromere in chromosome 1 and 5.Since molecular markers were not available in theearly eighties, mapping was performed only withmorphological markers. Molecular markers, which


are now abundantly available in Arabidopsis, canbe used in a similar way. Alonso-Blanco et al.(1998) used dose dependency, a method comparingband intensity of AFLP markers in the chromo-some 3 telotrisomic with the band intensity in thediploid and the primary trisomic for centromerelocation. Accordingly, the centromere of chro-mosome 3 could be mapped between two markersthat were completely linked in a recombinantinbred line population of 162 lines. This procedureis especially powerful because AFLP markersobtained with certain primer combinations tendto cluster predominantly around centromeres(Alonso-Blanco et al. 1998).

Other methods to map centromeres were thetetrad analysis using the quartet(qrt1) mutationthat allowed analysis of all four pollen grainsderived from a single tetrad (Copenhaver et al.1998, 2003 (this issue)), and FISH with clonescontaining centromere sequences on pachytenechromosomes that are also mapped on the linkageand physical maps (Fransz et al. 1998a, 2000,Tabata et al. 2000, Haupt et al. 2001).

Structural chromosome variants

Structural chromosome variants such as translo-cations, inversions and deletions could hardly be

studied in mitotic cell complements but werecytogenetically identi¢ed at pachytene and meta-phase I where speci¢c multivalent chromosomeassociations allowed cytogenetic identi¢cation oftranslocation heterozygotes and other structuralvariants (Sree Ramulu & Sybenga 1979, 1985,Vergunst et al. 2000). Selection for translocationscould also be done e⁄ciently on the basis of semi-sterility of the translocation heterozygotes (SreeRamulu & Sybenga 1979). Genetic methods basedon changes in linkage relationships betweenchromosomes, which include speci¢c DNAsequences and FISH technology are other ways forassessing chromosomal rearrangements.

The Arabidopsis sequence map is based on theColumbia (Col) accession and most of the linkagemapping was performed on Columbia�Landsbergerecta (Ler) cross, assuming collinearity of geneorder between the accessions. Recent cytogeneticand genetic analyses, however, demonstratedvarious polymorphisms for structural rearrange-ments, including small tandem duplications andinsertions, as were con¢rmed by comparing DNAsequence data of Ler and Col (Arabidopsis Gen-ome Initiative 2000). It is likely that this high rate ofpolymorphism was partly due to transposonactivity in speci¢c chromosomal regions as wasdemonstrated for the complex RPP5 resistancecluster region (No€eel et al. 1999).

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.


2

DAPI karyotyping of pachytene chromosomesrevealed a polymorphism for a heterochromaticknob, hk4S, in the short arm of chromosome 4 thatwas shown in the Col and WS but not in the C24and Ler accessions (Fransz et al. 1998a). FISH alsorevealed polymorphism for the 5S rDNA cluster,which is (1) near the centromere of chromosomearm 3S in Col, Cape Verde Islands (Cvi) andKashmir (Kas1), (2) in the lower arm of chro-mosome 3 in Ler and (3) absent in WS (Fransz et al.1998a). Linkage analysis of the F2 derived from thecross Hannover/Mˇnden�Ws showed a trans-location between chromosomes 3 and 4 (Kowalskiet al. 1994). Since genetic and cytogenetic analysisthus far involved only a limited number ofaccessions, many more examples of chromosomalpolymorphisms are yet to be expected.

Irradiation is a classical way to induce chro-mosomal aberrations and was shown to occur withrelatively high frequency after treatment with bothX-rays and fast neutrons (Sree Ramulu & Sybenga1979, 1985). About half of the translocations inthese studies showed a reduced frequency or evenabsence of translocation homozygotes in the pro-geny of heterozygotes, presumably due to damageat the breakpoints (Sree Ramulu & Sybenga 1985).This interpretation is supported by the ¢nding ofextended deletions at break ends during doublestrand break repair (Kirik et al. 2000). Speci¢cchromosome markers linked to the translocationbreakpoint made it possible to identify the twochromosomes involved in the translocation(Koornneef et al. 1982). Unfortunately thesetranslocations are no longer available for directlylocating genes to speci¢c regions of the chromo-somes.

Other structural variants include deletions thatcan be generated by irradiating haploid wild-typepollen and using this pollen to irradiate recessivemutants (Timpte et al. 1994, Vizir et al. 1994, Vizir& Mulligan 1999). Such recessive phenotypesoccurred at approximately 1%, when irradiatedpollen is used to pollinate diploids, but increased tovalues between 3 and 10%, when a tetraploidmarker line was pollinated instead (Vizir &Mulligan 1999). The major part of the deletionsthat behave as dominant lethals was rescued intriploids. Vizir & Mulligan (1999) provided indirectevidence that many of these deletions are inter-stitial, spanning on average 100 kbp in size, and

transmit through the male line to subsequentgenerations only at very low frequencies. Thisexplains why, in general, only smaller deletions aredetected for irradiation-induced mutants that wereisolated in M2 generations and show regularMendelian inheritance (Shirley et al. 1992,Bruggemann et al. 1996). These deletions were usedfor clonal analysis (Furner et al. 1995) and forcloning of the GA1 and RGA1 genes via genomicsubtraction (Sun et al. 1992, Silverstone et al.1998). Deletions are also helpful in map-basedcloning because they are easily detected. Comparedto chemical mutagenesis, the fact that two tan-demly repeated homologous genes can be knockedout by a deletion o¡ers an important advantage fordetermination of gene functions (Li et al. 2001).

An important and often unwanted source ofstructural variants comes from transformationexperiments. Upon transformation with Agro-bacterium tumefaciens, Negruk et al. (1996)reported minor deletions less than 17 bp in severalindependent cer2mutants, while Castle et al. (1993)estimated that 20% of T-DNA mutants from agenetic analysis of embryo defective mutants wereinvolved in translocations. Detailed analysis ofsome transformants indicated complex rearrange-ments, which may occur especially when multipleT-DNA sequences are integrated. Probably manyof these rearrangements such as the 26 cM para-centric inversion bordered by two T-DNAs, mayhave occurred during the T-DNA integ-ration process rather than resulting from intra-chromosomal recombination between homologousT-DNA insertions (Laufs et al. 1999). Nacry et al.(1998) reported for a single line a translocationbetween chromosome 2 and 3, a paracentricinversion on chromosome 2 and deletions onchromosome 2 and 3 near the T-DNA integrationsites. Tax & Vernon (2001) described a region onchromosome 5 larger than 40 kbp and containing aT-DNA that had inserted into a locus on chro-mosome 1. Such complex rearrangements causeproblems in the identi¢cation of the genes, dis-ruption of which leads to the phenotype understudy. Furthermore, paracentric inversionsstrongly suppress recombination; as an example,co-segregation of the mutant phenotype to both T-DNAs bordering the 26 cM inversion was observed(Laufs et al. 1999). A special case of translocationsand inversions using transformation is their


induction by the Cre/lox recombination system,where the Cre protein induces recombinationbetween lox sites. When a genome contains lox siteson di¡erent chromosomes, translocations betweenthese sites can occur, whereas their presence on thesame chromosome may lead to deletions andinversions (Vergunst et al. 2000).

FISH technology

A major breakthrough inArabidopsis chromosomeresearch was the introduction of FISH technologyfor direct detection of DNA sequences on chro-mosomal targets. Initially only hybridizations onmitotic metaphase complement were carried outusing repeat DNAs as probes (e.g. Maluszynska &Heslop-Harrison 1991, Schwarzacher & Heslop-Harrison 1991, Murata & Motoyoshi 1995, Heslop-Harrison et al. 1999). These repetitive sequences,which are straightforward and diagnostic markersin karyotype analyses, and represent about 10% ofthe genomic DNA, include especially the ribosomalrepeats (18S, 26S and 5S rDNAs), centromererepeats (180 bp pAl1 and the 106B repeats), peri-centromeric repeats (such as the Athila-like

repeats) and the telomere repeat at all chromosomeends. Although FISH studies revealed considerableinformation on the molecular organization ofArabidopsis chromosomes, the spatial resolutionbetween adjacent targets of metaphase comple-ments of only 2MB was insu⁄cient for detailedchromosome mapping studies (Murata &Motoyoshi 1995).

Considerable improvement of resolution wasachieved with FISH on spread pachytene chro-mosomes. These 20�25 times longer meiotic pro-phase complements measuring 50�80 mm forindividual chromosome pairs (Table 1), now allowresolution values of 50 kbp in euchromatin (Franszet al. 1998a). The chromosomes also displayconspicuous di¡erentiation of heterochromatin andcentromere regions (Figure 3) and are thereforemore accurate and informative for karyotyping andphysical mapping and genome studies (Fransz et al.1998a, de Jong et al. 1999). Speci¢c disadvantagesof pachytene analysis in Arabidopsis are: (1) thenumber of microsporocytes in an anther is relativelow; (2) development of microsporocytes is not verysynchronous, which means that cells in the pre-paration are at di¡erent meiotic stages; (3) ability tospread at late pachytene when chromosomesresolve from the clustered state in the synizetic knot

Table 1. Overview of genomic, genetic and chromosomal data of Arabidopsis thaliana

chr. 1 chr. 2 chr. 3 chr. 4 chr. 5 Total Average

Genetic map length (cM) 135 97 101 125 139 597 119.4Physical length (kb)a 29205 17463 23560 22140 26170 118538 23707.6Physical length (% genome) 24.6 14.7 19.9 18.7 22.1 100.0 20Physical/genetic length ratio 216.3 180.0 233.3 177.1 188.3 198.6 199.0Number of genesb 7041 4390 5623 3817 5847 26718 5344Chromosome length mitotic metaphase (mm)c 1.5�2.8Chromosome length pachytene (mm)d 80.8 52.1 69.3 52.7 76.3 331.2 66.2Relative length 24.4 15.7 20.9 15.9 23.0 100.0Centromere index (% s/sþ l) 49 23 39 24 45(Peri)centromeric heterochromatin (mm) 4.1 4.1 4.3 3.4 4.3 20.2 4.0Knob length (mm) 0.8 0.8Heterochromatin total (mm) 4.1 4.1 4.3 4.2 4.3 21.0 8.5NOR heterochromatin length (mm) 1.6 1.7 3.3Centromere region (mm)e 2.2 1.5 1.3 1.5 1.9 8.3Euchromatin 76.7 46.6 65.0 46.8 72.0 307.1 61.4

ahttp://www.arabidopsis.org/servlets/mapper;bhttp://biolinx.bios.niu.edu/t80maj1/arab chromos.htm;cHeslop-Harrison & Maluszynska 1994;dFransz et al. 1998;eHaupt et al. 2001; chr-chromosome.


is strongly genotype dependent (cf. maize KWSline): WS and C24, the accessions lacking 5S loci onchromosome 3, spread better than Col and Ler etc.(Fransz et al. 1998a). Examples of both types areshown in Figure 4.

Increasing microscopic resolution of chromatinto molecular dimensions has been obtained withFISH on extended DNA and on isolated DNAmolecules immobilized on coated slides. Using threeoverlapping cosmid clones hybridized to chromatin¢bres from interphase leaf nuclei, Fransz et al.(1996) gauged the DNA size�FISH signal ratio at3.27 kb/mm, a stretching degree close to that of thenative Watson-Crick conformation. Shortly later,Jackson et al. (1999) carried out direct physicalmapping of BAC inserts by FISH of circular BACmolecules immobilized onmicroscope slides.With astretching ratio of 2.44 kb/mm and a detectionsensitivity of 2 kb, even small linear distances candirectly be transformed into kb values. Fibre-FISHthat can spanDNA targets of more than 1.7Mbp ina single preparation (Jackson et al. 1998) providesunsurpassed spatial resolution of values as close as 1kb and a detection sensitivity better than 700 bp.However, the absence of any structural landmarkthat can position a FISH signal on the chromosomerequires probes from adjacent or overlappingregions for proper interpretation (Fransz et al.1998b). Fibre-FISH has proven especially suc-cessful for establishing the molecular size of dis-persed andpartly overlapping repeats (Brandes etal.1997b, Fransz et al. 2000) and has been shown to ¢llin the gaps in physical maps (Jackson et al. 1998).

FISH karyotyping and repeat polymorphisms

Individual chromosomes at pachytene are about25 times longer compared to mitotic metaphase(Table 1) and are unambiguously identi¢ed usingthe 45S rDNA and 5S rDNA repeats as additionalchromosomal markers (Figure 2). More recentFISH analysis with YACs and BACs painted onthe short arm demonstrated that the heterochro-matic knob hk4S resulted from a paracentricinversion in the proximal part of the chromosomearm 4S, bringing pericentromeric heterochromatinto an interstitial position (Fransz et al. 2000). Thisunique heterochromatic island was the subject ofan extensive comparative DNA sequence�FISHstudy and revealed detailed structural, molecularand functional properties of this specializedchromosome region (Fransz et al. 2000, CSHL/WUGSC/PEB Arabidopsis Sequencing Con-sortium 2000).

Major tandem repeats are 45S rDNA on thenucleolar organisers of chromosomes 2 and 4 and5S rDNA on chromosomes 4 and 5 and poly-morphic sites on chromosome 3 (Maluszynska &Heslop Harrison 1991, Murata et al. 1997, Franszet al. 1998a). All other tandem repeats could bemapped in the centromere and pericentromereregions (Brandes et al. 1997a). Heslop-Harrisonet al. (1999) analysed AtCon (pAL1 repeat) inArabidopsis, a 178-bp tandem repeat with sequencesimilarity to yeast CDEI and human CENP-BDNA-protein binding motifs, located at thecentromeres of all chromosomes. Parts of the

Figure 3. Photomontage of chromosomes at meiotic metaphase (a) and pachytene (b). A comparison is also made between FISH signalsof cosmid E4-6 (red) and cosmid E4-11 (green) on a pachytene bivalent (b) and on an extended DNA fibre (c) at roughly the samemagnification (bar¼ 10 mm).


less-conserved regions were detected on speci¢cchromosomes, indicating that there are chromo-some-speci¢c variants of AtCon. Of particularinterest is the molecular organization of theproximal chromosome regions including cen-tromere structure and pericentromeric hetero-chromatin region. Table 1 gives an overview ofgenomic, genetic and chromosomal data for allchromosomes in Arabidopsis. Centromeres aredemonstrated with the 180-bp centromere repeat

and 106B probes that are organized in interspersednon-separated blocks (Thompson et al. 1996).Centromere length ranges from 1.3 to 2.2 mm, andis signi¢cantly proportional to chromosome size, acorrelation previously found for other plant speciesby Bennett et al. (1981). Pericentromere hetero-chromatin regions, which are painted with the17A20 BAC containing retrotransposons andmeasure 4.1�4.4 mm, are roughly the same for allchromosomes.

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) and45S rDNA (red); (D) Col , hybridized with 5S rDNA (green) and GA1 (red). NOR, nucleolus organizing region; chr.4, centromere regionof chromosome 4 (bar¼ 10 mm).


Chromosome painting strategies

True chromosome painting or chromosome in-situsuppression (CISS) is based on FISHwith ampli¢edarbitrary DNA sequences from isolated chromo-somes and blocked with unlabelled genomic repeat(Cot) fraction, and has been applied successfully tomammals, birds, reptiles and insects. In spite ofmany e¡orts, the CISS technique could not beadapted directly to plant species (Schubert et al.2001). An attractive alternative way to studyindividual chromosomes is genomic in-situ hybri-dization (GISH, or genome painting) painting alienchromosomes in monosomic additions, but theseinterspeci¢c aneuploids are available only for a fewcrop�wild species combinations. A more versatilechromosome painting strategy is FISH with large-insert single-sequence clones like BACs and YACson pachytene chromosomes. Fransz et al. (2000)demonstrated the power of this approach bypainting the entire chromosome arm 4S with ¢vecontiguous YAC clones, spanning in total 2.8 Mbpand covering almost the entire arm. Later, BACclones were used instead, painting speci¢c chro-mosomes (Lysak et al. 2001, Fransz et al. 2002).BAC FISH painting works best for small-genomespecies and for tomato, maize and other specieswith larger genomes requires blocking with un-labelled Cot10�Cot100 repetitive DNA fractionsto suppress cross hybridization of repeats to non-speci¢c chromosome regions (unpublished data).BAC FISH to homoeologous chromosomes ofrelated species, comparable to ZOO-FISH inmammalian cytogenetics has now also beendemonstrated with Arabidopsis BACs hybridizedto 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 thestudy of chromosome organization in interphasenuclei. Fransz et al. (2002) studied parenchymainterphase nuclei by FISH using rDNA clones, andBACs painting the entire chromosome 4, andimmunostaining of methylated DNA and acety-lated histones. Interphase nuclei display a variablenumber of clearly distinguishable chromocentrescomposed of one or more centromeric hetero-chromatin regions, regions that contain heavilymethylated repeat sequences from which di¡erent

0.2�2Mbp chromatin loops emanate. This studyrevealed frequent associations of homologouschromocentres, alignment of homologous regionsand variable-sized loops.

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