In the past decade the development of genetic and physicalmaps has greatly benefited from improvements in molecularbiological tools. Thanks to the development of innovative
methods for mapping numerous markers and processing complexdata sets, the analysis of relatively long DNA molecules has be-come relatively routine. As a consequence, genetic maps of repre-sentative species of most plant and animal classes have now beenestablished. Physical maps have also become available for largeDNA contigs, although the occurrence of large blocks of tandemrepeats and abundant dispersed repetitive sequence elements con-tinues to impede the establishment of large-scale sequence infor-mation for most higher eukaryotic chromosomes. In general, therelative positions (and not the order) of loci on such maps, greatlydiffers from their corresponding sites on the genetic map. Studieson recombination nodules in spread-synaptonemal complex prep-arations1, and on chiasmata in diplotene cells, have revealed thatthe discrepancy between genetic and physical maps is a result of thenon-random distribution of crossover events along the chromosomes.
Cytogenetic or chromosome maps serve as intermediates betweenphysical and genetic maps in displaying the approximate positionsof genes and molecular markers, relative to structural markers,such as centromeres, telomeres, heterochromatin bands and sec-ondary constrictions. For a long time, such maps were based onthe results of classical cytogenetic strategies using deletions,translocations and trisomics. Cytogenetic maps are unique inbringing together information from linkage maps and the distri-bution of crossover-events and physical (DNA) maps, but requirethe presence of a well-defined chromosome portrait and a largecollection of deletions, translocations and trisomics or other chro-mosome variants. Maize, tomato, wheat, rye and rice are all modelspecies for such cytogenetic studies. However, other plant speciesfor which genetic maps have been developed, depict complexkaryotypes, and most of the individual chromosomes generallylack unique banding patterns, and so cannot be identified. In addi-tion, spontaneous structural and numerical chromosome mutantsare scarce, and are laborious and time consuming to generate. Analternative analysis strategy is fluorescence in situ hybridization(FISH), which allows distinct positioning of unique sequencesand repetitive DNA on chromosomes.
Fluorescence in situ hybridization – general featuresThe detection of chromosomal DNA in cytological preparationswas initially based on the application of isotopic labelled RNAs
and DNAs. A major breakthrough came with the introduction offluorescent labels, (in)directly linked to DNA probes and visual-ized under the fluorescence microscope(see Ref. 2 for an exampleof the use of this technique with plant DNA). The technology ofFISH appeared superior to previous in situ technologies in pro-viding better spatial resolution along with the potential of simul-taneously using different fluorescence systems for multi-probeanalysis. The number of targets to be analysed simultaneously,depends on the number of fluorochromes with different excitationand emission wavelengths3.
Basic FISH makes use of green and red fluorochromes forprobe detection and a third, blue fluorescing 49,6-diamidino-2-phenylindole (DAPI), for counterstaining the chromosomalDNA. In advanced multicolour systems, such as multicolour FISH(mFISH)4 and spectral karyotyping (SKY)5, up to five differentfluorescent images can be sequentially captured by a high sensi-tivity charge-coupled device (CCD) camera and the images digitallyprocessed and pseudo-coloured to visualize .50 probes at thesame time. The success of assaying DNA sequences by FISH hasalso been accelerated by improved detection sensitivity and spatial resolution. Detection sensitivity denotes the smallest DNA sequence on a target that can be detected unambiguously(J. Wiegant, PhD thesis, Leiden University, 1994). In practice,this corresponds to .10–20% of the observed targets that displayhybridization signals on identical positions of the two sister chro-matids of a chromosome, or on the corresponding positions onhomologous chromosomes. Under optimal hybridization anddetection conditions, and with the most sensitive CCD camera, FISHsensitivity remains dependent principally on the accessibility ofthe targets, and, therefore, on the extent of DNA condensation.Regular fluorescence photomicrography of human metaphasechromosomes6 allowed the detection of targets smaller than 30 kb,whereas capturing images with cooled CCD cameras7 enhanceddetection of targets as small as 1–3 kb. In plants, similar sensitiv-ities have been reported for Petunia hybrida8,9, tobacco10 andrice11. Because of the inconsistency of mapping small DNAprobes, the use of large insert DNA clones, especially BACs andYACs, has become a powerful alternative to FISH mapping12,13.
The second parameter, spatial resolution, is defined as thesmallest physical distance between adjacent target sequences thatcan be resolved using the fluorescence microscope. Under theoptical limit of a 1.4 numerical aperture for a conventional micro-scope objective, a maximum distance of only 0.2 mm can be
High resolution FISH in plants –techniques and applicationsJ. Hans de Jong, Paul Fransz and Pim Zabel
Fluorescence in situ hybridization (FISH) is an effective and accurate cytogenetic tool formapping single copy and repetitive DNA sequences on chromosomes. Attempts to increasethe detection sensitivity of very small chromosomal targets, and to improve the spatial resol-ution of signals derived from flanking sequences, have led to the development of a variety ofnovel techniques: it is now possible to perform in situ hybridizations on interphase nuclei,meiotic pachytene chromosomes and isolated chromatin (DNA fibres). The recent applicationof these techniques has indicated that a spatial resolution of 1 kb between adjacent targetsand a sensitivity of targets smaller than 1 kb is now feasible. Here, we describe the benefits ofthese novel chromosome analysis techniques and discuss their relevance for the study ofplant genomes.
resolved. Thus, the mapping resol-ution of target sequences in a FISHexperiment is determined by theirnative positions on the chromosomalDNA. This distance fluctuatesstrongly as chromatin condensationlevels change, and therefore variesbetween nuclei from various celltypes and at different stages of mitoticor meiotic divisions. In addition,spatial resolution depends on theway in which chromosome materialhas been pre-treated and spread orstretched onto the microscope slide.
Metaphase and interphase – FISHThe first material to be used forFISH experiments were mitotic meta-phasechromosomes. However, res-olution remained limited, generally inthe order of 2–5 Mbp (Refs 14,15).Interphase nuclei provide better res-olution, with values of as little as50–100 kb, but lack recognizablechromosome structures and differbetween cell types in terms ofendopolyploidization, chromosomedomain dispositions and chromatincondensation patterns. This inabilityto identify individual chromosomesin the karyotype has posed a seriousrestriction to FISH studies, wherehigh resolution and positioning ofthe signals with respect to cen-tromere, telomeres and other chro-mosome markers is essential.Chromosome banding before or fol-lowing in situ hybridization canovercome this drawback, but thecombination of both has as yet beenrealized for only a few mammalianand plant species16.
Pachytene – FISHMeiotic prophase chromosomes of plant species, in particular atpachytene, exhibit specific features, which make them particularlyattractive for FISH studies. First, they are far longer than theirmitotic metaphase counterparts, and so have better spatial resolution.Comparing the chromosome lengths at pachytene with those atmitotic metaphase of representative species with either a large or asmall genome size, a greater than sevenfold length difference wasfound for rye; whereas the difference for maize was 10-fold; tomato,15-fold; Arabidopsis, .20-fold; and rice, 40-fold. Apparently,genome size is the primary factor for this pachytene:metaphase ratio.However, a closer look at chromosome morphology reveals that thedistribution and organization of heterochromatin also contribute tothis variation, although not directly. This becomes clear if we com-pare the pachytene morphology of rice with that of Arabidopsis. Inrice, with a genome size four times that of Arabidopsis, pachytenechromosomes lack the dense pericentromeric heterochromatinblocks found in Arabidopsis(Fig. 1), but instead have numeroussmaller chromomeres scattered along their euchromatin arms.
Resolution of FISH in heterochromatin is distinct from euchro-matin areas. In tomato, for example, 77% of the chromatin is
heterochromatic17, occupying ~116 mm in an average pachytenecomplement. The remaining 23% is euchromatic and measures368 mm. With a microscopic resolution limit of 0.1 mm, FISH canresolve 1.2 Mb in heterochromatin and 120 kb in euchromatin,which is far better than the estimated 4–5 Mb for mitoticmetaphase chromosomes (Table 1). In Arabidopsis, which onaverage has even less condensed chromatin, spatial resolution isin the order of 60 and 140 kb for euchromatin and hetero-chromatin, respectively (X-B. Zhong, PhD thesis, WageningenAgricultural University, 1998).
Further improvements in spatial resolution might be realizedwith meiotic prophase nuclei at the short-lasting diffuse diplotenestage, when chromosomes shed their synaptonemal complex (SC)and form a network of fine threads. Diffuse diplotene nuclei,which might have the potential of mammalian pro-nuclei18, are ofspecial interest for studying repeats in heterochromatin, whichbecomes, at least for tomato, most decondensed at this stage (J.H. de Jong and P.F. Fransz, unpublished). Another source ofplant cells that are of significance for high resolution FISH are poly-tene interphase chromosomes that are known to occur in suspensor
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Fig. 1. Pachytene chromosomes in higher plants. (a) Pachytene chromosomes of tomato(Lycopersicon esculentum) hybridized with the telomeric probe pAtT4 from Arabidopsis thaliana(red fluorescence) and the species-specific subtelomeric repeat TGR1 (green fluorescence). TheDAPI (49,6-diamidino-2-phenylindole) counterstained chromosomes have brightly fluorescent het-erochromatin (Het) around the centromeres (Cen) and a few distal and interstitial sites, and weaklyfluorescing euchromatin (Eu) in most of the chromosome arms. The nucleolar organizer region(NOR) is the distal part of the satellite chromosome containing the 45S rDNA genes. (b) Pachytenebivalent 4 of Arabidopsis, probed with the 45S rDNA probe at the NOR (white signal), the 5S rDNAat the centromere heterochromatin (green signal) and the 480 kb CIC7C3 YAC (red signal). Thebivalent was digitally dissected from a whole pachytene complement using computer imaging soft-ware. (c) Complete pachytene complement of Arabidopsis. The bright fluorescing regions are theheterochromatin regions around the centromeres and the NOR. Note that most of the chromosomesare euchromatin. (d) Pachytene nucleus of Allium cepa. The very long pachytene chromosomes ofthis large-genome species demonstrate the very high proportion of heterochromatin. Scale bars 5 5 mm.
cells of Phaseolus19. In addition, synaptonemal complex-spreadpreparations, which contain long stretches of chromatin loopsextending from the SCs, might be preferable in FISH studieswhere the highest possible resolution is required20.
The second reason for choosing pachytene chromosomes astargets in FISH is the conspicuous differentiation of euchromatin,chromomeres and larger heterochromatin blocks. In most plantspecies, heterochromatin is confined to large chromosome seg-ments around the centromeres, telomeres and nucleolar organi-zers, and their pattern of diagnostic heterochromatin areas can behelpful in identifying individual chromosomes in the comple-ment. In addition, typical pairing configurations, such as loops indeletion heterozygotes, and multivalents with pairing partnerexchanges in translocations and trisomics, are helpful in providinginformation about specific chromosomal regions. Because het-erochromatin fulfils specific chromosomal house-keeping func-tions and is rich in repetitive DNA sequences in which meioticrecombination is being suppressed, the positioning of FISH sig-nals for coding sequences in such areas might be helpful in betterunderstanding epigenetic phenomena, such as position-effect variegation, gene silencing and DNA methylation.
The suitability of pachytene cells for FISH studies is based ontheir capacity to spread, such that chromosomes can be identifiedin the nucleus. Chromosome spreading is optimal at latepachytene when chromosomes detach from their typical earlyprophase clump of chromatin. In addition to technical consid-erations, such as the use of cell wall digestion enzymes and aceticacid maceration, which facilitate chromosome spreading, thequality of late pachytene complements is also known to differbetween species and even between genotypes of the same species.Tomato is a well-known example of a plant species with superiorpachytene morphology21 (Figs 1 and 2). However, Petunia hybrida,another representative of the Solanaceae, has notoriously recalcitrantpachytene nuclei. Maize has the best studied pachytene chromo-somes of all the monocots (generally far better chromosomespreading, and more detailed morphology at late pachytene, isseen in the KWS line than most other lines). A comparable differ-ence in pachytene morphology between genotypes has recentlybeen described for Arabidopsis thaliana22. Well-spread pachytenecomplements were observed in the Arabidopsis WS, and C24lines (Fig. 1), in contrast to the Landsberg erectaand Columbiaecotypes, which show very poor pachytene morphology.
In spite of these advantages, pachytene FISH also suffers froma few disadvantages. For example, raising a plant to flowering
stage requires more time and effort than simply germinating seedsor growing young plants for mitotically active tissue. Moreover,the selection of anthers with pollen mother cells at the right stagemakes the production of pachytene-spreads relatively time con-suming and laborious, and the number of microsporocytes in ananther can be low in insect- and self-pollinating species. In addi-tion, the analysis of pachytene chromosomes in polyploid species isparticularly difficult because of numerous complex pairing configu-rations, asynapsis and chromosome stickiness. Finally, the tracingand identification of individual chromosomes in pachytene nucleican also be a major problem in Lilium longiflorum, Allium cepa(Fig. 1), rye and other species with long chromosomes.
Extended DNA fibre analysisA major improvement in FISH detection and sensitivity has beenachieved with techniques known as FISH to extended DNA fibresand FISH to stretched interphase chromatin. These techniquesboth make use of linearized chromosomal DNA, on which FISHsignals of different probes directly reflect the physical position ofthe probe DNA along the DNA molecule. Well-known examplesare DNA halo preparations23 and single chromatin fibres24, whichare obtained from interphase nuclei on microscope slides. DNAfibres are released after disrupting the nuclear matrix with highsalt, sodium hydroxide and ethanol, or an EDTA-SDS buffer, andthen spread. Hybridization and detection follows standard FISHprotocols. With a stretching close to that of native DNA, spatialresolution of neighbouring DNA targets is strongly enhanced. In acalibration experiment with three contiguous cosmids of chromo-some 4 of Arabidopsis, the lengths of the fluorescent signals in themicroscope were gauged using the respective molecular size ofthe targets. Quantitative analysis revealed an extension degree of3.27 kb per mm (Ref. 25), which is only 28% higher than the 2.97 kb per mm value for native duplex B-DNA. Slightly lowervalues of 2.53–3.22 kb per mm were obtained in a second, compa-rable fibre-FISH study of Arabidopsis26. Because the mapping resol-ution for flanking signals is only limited by the optical resolutionof the fluorescence microscope, targets only ~1 kb apart can nowbe resolved23, a value that is far better than the 1.2 Mb to 120 kbfor pachytene chromosomes (Table 1).
The advent of fibre FISH technology also brought about abreakthrough in detection sensitivity. Because the accessibility ofthe probe to the target DNA is no longer hindered by densely con-densed, intact chromosome structures, a strong detection gain forFISH signals is to be expected. Using probes hybridized to targetsin the 45S rDNA genes of tomato, detection of DNA target se-quences as small as 700 bp can unequivocally be demonstrated25,and for human DNA even 200 bp can be resolved on DNA fibres27.
A more recent technology, referred to as molecular combing,provides a challenging alternative to the standard molecular map-ping procedure, by applying FISH directly to isolated, usuallysmall, DNA targets. This technique involves the immobilizationof purified DNA molecules, generally YACs, BACs and cosmids,which are stretched across the receding meniscus of a waterdroplet on lysine-coated or positively charged microscopeslides28,29. Smaller fragments, typically plasmids or restrictionfragments, are hybridized to the target molecules and their pos-itions mapped by two-colour FISH (Fig. 3).
Because fluorescent tracks directly reflect the lengths of targetsequences on the DNA fibre, reliable quantitative analysis is pro-vided. However, there are a few important sources of variation,giving rise to fluorescent tracks of different length and structure:• Breakage of the fibres might occur during preparation of the
DNA fibres, thereby necessitating the quantitative analysis oftracks in the same preparation.
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Table 1. Mapping resolution and detection sensitivity of metaphase chromosomes, pachytene
aData from Refs 17,21,25 and X-B. Zhong, unpublished.
• Fibres can differ in their stretching degree, and, if insufficientlystretched, fibres are seen as a solid line rather than the beadednature of a completely stretched fibre30.
• DNA fibres can be locally damaged, covered with proteins, orfail to hybridize with the probe DNA.
• Polymorphism of tandem array lengths between homologues.• Dynamic changes in the repeat length during cell differenti-
ation and the ageing process.• Closely linked cosmid and l-clones hybridized to fibres from
large genome species (for example, rye and lily) might generatediscontinuous FISH tracksin the case of numerousblocks of repeat sequencesinterspersed in or betweenunique sequences.
To avoid misinterpretation ofthe FISH results, it is crucial tocompare various fluorescentsignals of the same target.Two- or three-colour FISH toadjacent or overlapping targetscan be helpful in providinginternal landmarks for recog-nizing full hybridization sig-nals (Figs 2 and 3). Generally,a target of 10 kb, which gener-ates a 3 mm fluorescent string,is the minimum size that can bedistinguished unambiguously.Shorter stretches can bedemonstrated only with good
flanking markers, making them easily distinguishable from smallbackground signals. As a rule of thumb, extended DNA fibre FISH is most powerful for hybridization signals from~1–300 mm, which roughly corresponds to a target range of1–1000 kb (X-B. Zhong, PhD thesis, Wageningen AgriculturalUniversity, 1998).
ApplicationsThe major advantage of using pachytene chromosomes ratherthan mitotic chromosomes or interphase nuclei for FISH studies is
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Fig. 3. Fluorescence in situ hybridization (FISH) to phage l-DNA molecules. Preparations were preparedaccording to the molecular combing technique28 (slightly modified). (a) Overview of microscopic preparation.(b) Restriction map with positions of the probes. (c) A l-molecule stained with YoYo. (d) A l-DNA moleculehybridized with the BglII restriction fragments, as shown in Fig. 1b. Scale bar 5 10 mm.
Fig. 2.Fluorescence in situ hybridization to tomato chromosomes and extended DNA fibres. Figures 2a and 2b are at the same magnification.(a) DAPI (49,6-diamidino-2-phenylindole) stained mitotic metaphase chromosome 6, measuring 2.6 mm, with a distal green signal of the sub-telomeric TGR1 repeat. The arrow indicates the position of the centromere. (b) Pachytene bivalent of tomato chromosome 6, measuring 39.3 mm, with telomere and TGR1 repeats (see Fig. 1a). Heterochromatin and euchromatin are well differentiated. The centromere is indicatedby an arrow. (c) Examples of FISH for different probes hybridized to extended DNA fibres. (c i) The 16 kb telomere repeat (red) and 408 kb subtelomere repeat TGR1 (green) of the distal end of the short arm of chromosome 6 of tomato34. (c ii) The 5S rDNA repeat (657 kb) at the pericentromeric heterochromatin of the short arm of tomato chromosome 1. (c iii) Three partly overlapping cosmids in a contig of Arabidopsis.
the enhanced resolution without loss of recognizable chromosomestructure. The commonly used DAPI counterstain generates a reproducibly bright fluorescing pattern of heterochromatin andAT-rich segments, which aids the identification of individualchromosomes. The differentiation of cytological markers alongthe pachytene chromosomes provides unique information on thedistribution of coding DNA sequences, as well as repetitive DNAelements, with respect to heterochromatin blocks, chromomeres,nucleolar organizer regions and other specialized chromosomeregions. Where such DNA sequences are concurrently hybridizedto extended DNA fibres, the molecular organization of long arraysof various sequence elements are established. Arabidopsis, withits limited number of heterochromatin segments, and relativelylow content of repeat families, is probably one of the most suitablespecies for such an analysis. Thus, pachytene FISH with the tandem sequence pAL1 demonstrated that this repeat occurs in the pericentromeric heterochromatin areas of all chromosomes(P.F. Fransz et al., unpublished). Two-colour FISH of Ty1-copia31
and the 164B repeat32 with the pAL1 tandem sequence to extendedDNA fibres, resolved the molecular organization of these repeatsin the pericentromeric DNA. Comparable FISH experiments onterminal heterochromatin areas of Allium cepachromosomes33
were carried out to demonstrate the physical positions of the Ty1-copia retro-element and the En/Spm-transposable element withintandem repeats in these chromosome segments.
In an extensive study of the molecular organization of telo-meric heterochromatin in tomato34, FISH analysis of pachytenechromosomes was effective in resolving the distribution of theTTTAGGG telomere repeat and the tomato-specific TGR1 subtelomeric repeats on all chromosome arms. The combinedapplication of FISH to pachytene complements and extendedDNA fibres revealed that each chromosome end possesses aunique molecular organization in terms of molecular size andsequence composition.
The use of FISH to map DNA sequences containing genesassigned to centromere regions in genetic mapping studies are ofparticular interest. Plant chromosomes have long pericentromericregions, which are rich in heterochromatin and generally display asuppression of meiotic recombination. This lack of recombinationaround the centromere impedes precise genetic mapping in thisregion. A clear example was given in a mapping study on the pos-ition of Mi and Aps-1: two important marker genes in tomato thatgenetically mapped to the centromere region of chromosome 6(Ref. 35). A FISH study of DNA sequences covering the genesprobed to pachytene chromosomes confirmed their assignment tochromosome 6. Pachytene analysis also revealed that the fluor-escent signals of the BAC spanning the Mi-region were observed atthe junction of euchromatin and pericentromeric heterochromatinin the short arm, whereas the YAC containing the Aps-1gene waslocated in the pericentromeric heterochromatin of the long arm, ata distance of .40 Mb from the Mi-region13.
Because extended DNA fibres directly display linear positionsof DNA sequences in a construct or contig, in situhybridization isparticularly informative in evaluating the precise localization andordering of clones, resolving overlaps and distances, and provid-ing a detailed image of the integrity and co-linearity of probes onchromosomal-target DNAs. In a FISH study about the position of complex T-DNA constructs in transgenic potato plants, analysisof complex T-DNA hybridized to extended DNA fibres36 revealedmultiple T-DNAs that are closely integrated. Using differentlabelled probes of T-DNA and vector sequences, the compositionof complex loci has been determined and the copy number and arrangement of T-DNAs assessed more accurately than bySouthern blot analysis alone.
ConclusionIt is clear from these examples that high resolution FISH mappingbroadens our view of how single copy and repetitive sequencesare organized along the chromosome arms. In a large-scale studyinvolving all major classes of repeats, patterns that are representativeand unique for each chromosome can be produced. In a compre-hensive study of the sugar beet (Beta vulgaris) genome, the pos-itions of major classes of repeats and gene-rich regions were assessedwith regard to the heterochromatic pericentromere regions andgene-rich distal euchromatin regions of the chromosomes37. Thus,in summary, FISH adds a powerful new tool to the existing batteryof cytological techniques, and will play an important role in futureefforts to determine genome structure.
AcknowledgementsOur thanks to Xiao-Bo Zhong for his important contribution to thisstudy. Mrs Peng Zhang provided Fig. 3a–c and her experimentalwork on the molecular combing and FISH to extended DNA fibresis greatly appreciated. PF was financially supported by a grant fromthe Commission of the European Communities (No. PL960443).
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J. Hans de Jong* is at the Laboratory of Genetics, WageningenAgricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands; Paul Fransz and Pim Zabelare at the Laboratoryof Molecular Biology, Wageningen Agricultural University,Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
Since the early days of genetics, plant biologists have wonderedabout the fate of broken chromosomes. Barbara McClintock,using cytological tools, showed that the outcome of X-ray-
induced chromosome breaks is translocations, deficiencies, ringchromosomes and end-fusions1. She reached the conclusion that‘broken ends of chromosomes will fuse, 2-by-2, and any brokenend with any other broken end’. In modern-day terminology, shehad characterized what we call DNA double-strand break (DSB)repair. Amazingly, she had the insight to realize that any end couldfuse with any end, a phenomenon known today as non-homologousend-joining or as illegitimate recombination. In plants, natural
DSBs-inducing factors include transposons, irradiation, endonu-cleases (Fig. 1) or mechanical pulling as with dicentric chromo-somes. Meeting the challenge of DSB repair is not a simple taskbecause both strands are damaged and the missing informationcannot easily be reconstituted from a complementary strand asthey can be in an excision or mismatch repair. DNA ends mightnot be compatible for ligation and exonuclease activity probablyenlarges the break into a gap. Simple rejoining of broken endsmight therefore result in loss of genetic information.
So what to do at an end? Utilizing a homologous template for DSBrepair, such as a sister chromatid, or a homologous chromosome
How plants make ends meet: DNAdouble-strand break repairVera Gorbunova and Avraham A. Levy
DNA double-strand breaks (DSBs) lead to serious genomic deficiencies if left unrepaired.Recent studies have provided new insight into the mechanisms, the mutants and the genesinvolved in DSB repair in plants. These studies indicate that high fidelity DSB repair via hom-ologous recombination is less frequent than non-homologous end-joining. Interestingly,non-homologous end-joining in plants is more error-prone than in other species, being asso-ciated with various rearrangements that often include deletions and insertions (filler DNA).We discuss the mechanism of error-prone DSB repair, which is probably an important drivingforce in plant genome evolution.
