COMMENTARY

The molecular cytogenetics of plants

J. S. HESLOP-HARRISON

Karyobiology Group, Cell Biology Department, John Innes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, UK

Introduction

Over the last ten years, major advances have been made inthe molecular understanding of plant genomes -both withrespect to cloning of genes and with better insight into thestructural organization of the DNA. The results achievednow enable the molecular genetics of defined systems to betackled directly, and the directed manipulation of geneexpression during development - whether of flowerdevelopment (see Coen, 1991), wound responses (e.g. seeStanford et al. 1990) or phytohormome action (e.g. seeHuttly and Baulcombe, 1989). The increasing size of thesequence database permits comparison of the DNA fromdifferent species through coding regions, introns andrepetitive sequences. It ia not always easy to recognizefully many of the answers to major biological questionsthat have been published following the plethora oftechnical developments that have enabled the research -in cloning and transformation, PCR (polymerase chainreaction), transposon tagging, and in sequence analysis.

In the rapid acquisition of information about DNAsequences, a gap has appeared between our knowledge ofthe genome at the molecular level and that at thechromosome level. Molecular cytogenetics is now permit-ting the linking of molecular genetics with cytogenetics,using a combination of tools from both disciplines, and, inparticular, direct in situ hybridization of labelled probes tointerphase nuclei and chromosomes prepared for light(Trask, 1991) or electron microscopy (Leitch et al. 1990).The work is providing novel answers about genome andgene organization, gene activities and even meioticrecombination. Here, I aim to discuss some of the recentprogress in this area from the viewpoint of a plantcytogeneticist. Many of my examples are taken from twocontrasting groups of species: the small-grained cerealsare grasses, and include barley, rye and wheat, whileArabidopsis thaliana, usually referred to as Arabidopsis,is a small rapidly growing species in the Brassica family.

The Nucleotype

Cells of different eukaryotic plants are involved in thesame set of basic processes, and probably contain DNAthat encodes about 50000 genes. Higher animals have asimilar number of genes, if those related to nervous andimmunological systems are excluded. Assuming that eachgene encodes a protein of average length 300 amino acids,and that introns, regulatory or other sequences flankingthe DNA encoding these genes are about 500 base pairsJournal of Cell Science 100, 15-21 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

(bp) for each gene, the minimum number of base pairs ofDNA required is some 80 000 000 bp, or 80 Mbp (megabasepairs). Various trees (including horse chestnut, Aesculushippocastanum, and oaks, Quercus) and Arabidopsisspecies have amounts of DNA in their nucleus that arelittle greater than this value (Bennett and Smith, 1976;Bennett et al. 1982); the nematode Caenorhabditis eleganshas a similar genome size. At the opposite extreme of DNAcontents, the diploid lily Fritillaria davisii has some 86 000Mbp. Between the extremes lie diploid species such asmung bean {Vigna sinensis; 530 Mbp), rice (Oryza sativa;960 Mbp), human (Homo sapiens; 3000 Mbp), barley(Hordeum vulgare; 5300 Mbp), rye (Secale cereale; 7000Mbp) and wheat (Triticum aestivum; 17 000 Mbp). The vastrange of DNA contents, and the lack of correlation withthe perceived complexity of an organism, make compari-sons at the DNA level between species difficult. Theremust be significant differences in the organization andpositioning of the genes, and in the non-coding DNA thatis responsible for most of the variation between thespecies. The huge variation in DNA amounts alsonecessitates differences in technical approaches to genomestudies. For example, in Arabidopsis, the complete genomeis cloned in yeast artificial chromosomes (YACs) andavailable as a library in a few dozen 96-well microtitreplates (Ward and Jen, 1990; Grill and Somerville, 1991). Inthe small-grained cereals, the molecular genetics isrestricted to a few dozen cloned genes (e.g. see Futers et al.1990), augmented by a few hundred repetitive DNA clones(see Flavell, 1985) and short cloned DNA sequences thatdetect restriction fragment length polymorphisms(RFLPs; e.g. see Liu et al. 1991). The molecular genetics ofrice and maize, with intermediate genome sizes, is slightlymore extensive (see e.g. Coe, E. (ed.), 1991; Maize GeneticsCooperation Newsletter, 65; unpublished), but still limitedcompared with that in Arabidopsis.

Structural sequences of chromosomes

The number of chromosomes, which in the diploid speciesmentioned above ranges from 10 to 46, has littlecorrelation with the species DNA content. Chromosomestructure, in both plants and animals, is similar, with afew exceptions. The chromatin, visualized in wholenuclear preparations using the light or electron micro-scope, represents the DNA double helix wrapped around

Key words: cytogenetics, plant genome, genome organization.

15

the nucleosome core and then coiled several times;metaphase and interphase chromosomes differ in one levelof coiling (Manuelidis and Chen, 1990). Hence examin-ation of chromosomes at the structural level in one speciescan give more general conclusions. Nevertheless, there aresignificant, and not fully explained, differences betweenplant and animal chromosomes; for example, only thelatter show banding patterns after particular treatments(Greilhuber, 1977), which presumably reflects differencesin DNA organization, protein associations or replication.

At metaphase, cytological preparations of chromosomesshow the telomeres ('ends') and primary constrictions atthe centromeres. The telomere is now among the bestcharacterized DNA sequences at the molecular level andconsists of a short tandemly repeated DNA sequence(where the same 6 or 7 base pair unit is repeated manyhundreds of times; Zakian, 1989). In Arabidopsis, theconsensus sequence is TTTAGGG (Richards and Ausubel,1988), and in situ hybridization of the labelled telomeresequence localizes the sequence to the physical ends ofcereal chromosomes (Schwarzacher and Heslop-Harrison,1991). The telomeric DNA does not have a Watson-Crickstructure, and it is not replicated by a template-dependentDNA polymerase, but rather by a reverse transcriptasewhere the RNA template is an integral part of theribonucleoprotein (a telomerase; e.g. see Blackburn, 1991).Some of the information revealed by in situ localization ofthe telomere sequence would be very difficult to obtain byother methods. For example, the location at everychromosome end, and a few intercalary sites, would bedifficult to conclude from Southern hybridization data, andthe variation in copy number of the telomere repeat unitfrom cell to cell and chromosome to chromosome could notbe measured using techniques that only analyse thesequence from large numbers of pooled nuclei.

The molecular cytogenetics of other structures on thechromosome is being investigated. The centromere, wheremicrotubules bind at metaphase before separation of thetwo chromatids at anaphase, has a key structural role (seeRattner, 1991) where DNA sequence organization relatesto associated proteins and to centromere function. Many ofthe sequences near or at the centromere are highlyrepeated, and hence can be localized at interphase andused to elucidate interphase nuclear architecture (Heslop-Harrison and Bennett, 1990; Maluszynska and Heslop-Harrison, 1991).

Origins of replication have been a major area of researchin yeast (see Murray and Szostak, 1983), and there aremany replication origins that are under tight geneticcontrol in plants (Kidd et al. 1989). The molecularcharacterization of sequences that associate with DNAbinding proteins is far ahead of their characterization atthe cytogenetical level. Where are these sequences alongthe chromosome? Where are they spatially within thenucleus?

A final class of structural features of chromosomes isthat relating to meiotic pairing and recombination. Moensand Pearlman (1990) have shown the association oftelomere and centromere DNA with the synaptonemalcomplex core of meiotic prophase chromosomes, but whatare the sequences associated with the synaptonemalcomplex along the whole chromosome length? Are se-quences related to chromosome structure also associatedwith recombination sites? New evidence suggests that therecombination events may occur prior to synapsis me-diated by the synaptonemal complex, at least in yeast(Haber et al. 1991); if these results prove general, then

different classes of structural sequences may be associatedwith homologue recognition, recombination and thenpairing.

Physical organization of repetitive sequences

At interphase, light and low-power electron-microscopestudies allow the nucleolus (reviewed by Jordan, 1991) andchromatin to be resolved. Pieces of condensed chromatin,usually referred to as heterochromatin, are visible, and (atleast in species with more than some 1000 Mbp of DNA)chromatin axes can be followed. The physical organizationof repetitive DNA is important, since such sequencesmake up most of the DNA in most higher organisms — eventhe Arabidopsis genome is 25 % repetitive DNA (Leutwileret al. 1986), while the wheat genome (17 000 Mbp) consistsof more than 80% middle and highly repetitive DNA(Flavell, 1985).

Studies of gross nuclear morphology in many, althoughnot all, tissues, show a configuration recognized as early as1885 by Rabl, where the nucleus has a pole at which thecentromeres are preferentially located opposite the telo-mere pole. In species with higher DNA contents, there isoften a strong gradient in the proportion of the nucleusfilled by chromatin (see Anamthawat-J6nsson and Heslop-Harrison, 1990; and Fig. 1), with about half the volume ofthe nucleus near the centromere pole filled by chromatin,while the telomere zone is only 15 % filled.

Many of the heterochromatic segments are supercoiled,tandemly repeated DNA sequences. Staining propertiesindicate that they contain repetitive DNA (Schweizer,1981), and in situ hybridization confirms the co-location ofparticular repetitive sequences. For example, light micro-graphs of stained DNA and electron microscope picturesshow large heterochromatic segments near the centro-meres at metaphase. Using in situ hybridization toArabidopsis, Maluszynska and Heslop-Harrison (1991)have shown by in situ hybridization that a repetitivesequence (cloned by Martinez-Zapater et al. 1986) co-localizes with many of the heterochromatic knobs, andthat it appears as similarly condensed units both onmetaphase chromosomes and at interphase. The probeproduces such discrete hybridization signals at interphasethat it can be used to count centromeres, and hencechromosome, numbers in non-dividing tissues.

The type of DNA sequence found in knobs consists oflarge numbers of copies of the same (or closely similar)sequence in tandem arrays, but another class of highlyrepetitive DNA is found dispersed throughout the genome.An example of such a sequence in plants was described byMoore et al. (1991), who found an element of the genomethat comprised some 5 % of the total DNA of barley. Usingin situ hybridization, they were able to show that thesequence was interspersed and located over most chromo-some arms, although excluded from the telomeric, nu-cleolar organizing and paracentromeric regions (Fig. 2).Again, without direct examination of chromosomal lo-cation, the organization of the element would be difficult todetermine, and many questions arise from the discovery.Why is the sequence excluded from some chromosomalregions? Does it tend to flank particular types or classes ofsequence? What differences are there in locations andtypes of sequences in the excluded regions? Indications ofthe answer to another question, *How does a dispersedrepeat become distributed on all chromosomes?', come

16 J. S. Heslop-Harrison

Fig. 1. A electron micrograph ofa section through an interphasenucleus from a rye root-tip. Thenucleus shows a strong gradientwith the centromeres (arrows)lying in a region where a largeproportion of the nuclear volumeis filled by chromatin. Thetelomeres (arrowhead indicatessub-telomeric heterochromatin)lie in a zone with a muchsmaller proportion of chromatin(see Anamthawat-J6nsson andHeslop-Harrison, 1990) x 12 000.

from sequence analysis, since it has similarities toretrotransposon-like elements in animals.

In situ hybridization of both tandemly repeated anddispersed sequences is proving to be not only of use forplant genome analysis but also of practical value in plantbreeding. Lapitan et al. (1986) have demonstrated the useof a species-specific cloned dispersed repeat from rye toidentify rye chromosomes and chromosome segments inhybrids with wheat, which are of potential agriculturalimportance. Total genomic DNA, where many repetitive

sequences act as the probe, can also be used to identify theparental origin of chromosomes or chromosome segmentsin hybrids and breeding lines (Fig. 3; Anamthawat-J6nsson et al. 1990), and the method is now beingdeveloped further for use in screening plant breeding lines(Heslop-Harrison, Miller and colleagues, unpublisheddata). Tandem arrays are of interest in this context. In rye,the size of the sub-terminal knobs (and hence copy numberof the tandem repeat) on many chromosome arms can vary(Gustafson et al. 1983), and may be selected in rye itself

Molecular cytogenetics of plants 17

Fig. 2. In situ localization of a cloned DNA sequence, BIS1, tobarley chromosomes. DAPI staining (left) shows the DNA onthe chromosomes, while a biotin-labelled fragment of BIS1(right) hybridizes in situ over most of the chromosomes exceptat centromeric and telomeric regions. The sequence may be (ormay have been) capable of movement and amplification in thegenome through reverse transcription, as is indicated by itsdispersion over the chromosomes (Moore et al. 1991).

Fig. 3. A partial metaphase spread from a root tip of a wheatvariety, Custom, which includes a rye chromosome armtranslocated onto a wheat arm. The spread has been probed insitu with total genomic DNA from rye, with non-species-specific hybridization blocked with wheat DNA. Allchromosomes fluoresce with the counterstain (light grey), whilethe strongly labelled chromosome arm from rye fluorescesbrightly (white; Heslop-Harrison et al. 1990). Despite therelatively close relationship of the two species, there areenough different dispersed sequences to permit discriminationof the rye chromosome arm, and the point of recombinationand presence of other recombination events can be examined.

and in the hybrid crop, triticale, with potential effects onagronomic performance.

Chromosomal localization of genes

For mapping genes in human, Ferguson-Smith (1991) hasrecently stated that in situ hybridization 'is now themethod of choice for assigning a cloned DNA sequence toits parent chromosomes'. In plants, this statement is nowonly true for one class of genes, those present in multiplecopies in tandem arrays, such as the 18 S, 26 S and 5.8 SrRNA gene subunits (Mukai et al. 1991) and the 5 S rRNAgenes (Mukai et al. 1990). These are present in tandemarrays of thousands of copies at one or more chromosomallocus, but some whole loci may be unexpressed, while onlya subset of the genes is expressed at other loci (Flavell,1986; Flavell and O'Dell, 1990). There is clear cytologicalevidence for the expression of rRNA genes at interphase,through the production of the nucleolus where the sizecorrelates with activity, and at metaphase chromosomesthat have recently expressed the rDNA genes normallyshow a secondary constriction at the nucleolar organizingregion (NOR). Thus the expressed sequences can belocated on chromosomes by cytology and hence mappedphysically at metaphase. The presence of non-expressingrRNA genes in rDNA-containing heterochromatin lyingadjacent to the nucleolus or elsewhere in the interphasenucleus has been shown by in situ hybridization (Appels etal. 1986; Leitch et al. unpublished data). A similarrepression of genes by condensation may be found infemale mammals, where one X chromosome is largelyinactive, and sometimes visible as a condensed Barr body.Because of the large number of tandem repeats, and thelimited variation within the repetitive element, and lackof expression of some loci, the total number of chromo-somes carrying rRNA genes cannot easily be found usingsegregation or RFLP analysis or by examination of thechromosomes. Thus, the presence and locations of rDNAon five pairs of wheat chromosomes (Mukai et al. 1991) andtwo Arabidopsis chromosomes (Maluszynska and Heslop-Harrison, 1991) have only been shown by in situhybridization.

There are two basic maps of a genome: the physical orcytogenetic map, and the genetic map. The physical mapshows the morphology of the chromosomes and thephysical location of genes and other chromosome markersalong the length of the chromosomes. The genetic map isbased on the chromosome linkage groups on which genes,restriction fragment length polymorphisms (RFLPs), andother markers, lie. The genetic or recombination distancesbetween the markers on each chromosome give the mapdistances (see O'Brien, 1990). As well as using analysis oflarge segregating families for genetic mapping, genes andRFLPs can be mapped to chromosomes using the widerange of aneuploid stocks available in plants. Arabidopsishas a complete set of trisomic lines (with one chromosomepresent in an extra copy) that permit direct gene mapping.Wheat has many aneuploids that enable each gene ormarker to be assigned to a particular chromosome orchromosome arm. Below the level of the chromosome arm,connection of genetical map to the physical map isdifficult. Although, of course, the physical and geneticmaps show markers in the same order along thechromosome, the physical location of most markers isunknown except for specific points such as the centromeresand secondary constrictions. When physical and genetical

18 J. S. Heslop-Harrison

locations are known, there is often little correlationbetween the separation distances of markers on the twotypes of map. One of the first studies of the relationships ofthe maps in cereals was by Linde-Laursen (1979), whoexamined barley populations segregating for C-bands. Hefound almost no recombination between two bands thatwere widely separated on one chromosome arm, whileneither physical band was linked with an esterase locus onthe same arm, implying a high level of recombination.Now, physical technologies are beginning to link the mapsmore closely, and to answer questions about genelocalization.

Using the estimated total length of the Arabidopsisgenetic map, in cM (centiMorgans), derived from recombi-nation studies, and dividing the length by the genome sizeindicates that each cM occupies an average of 150 kb. Grilland Somerville (1991) have cloned the genome of Arabi-dopsis in YACs, and are probing the genome with manyRFLP markers (Chang et al. 1988), that have been mappedin segregating populations. One clone of 170 kb containedthree markers that mapped over 2.9 cM, while a second180 kb clone included two RFLP markers that mapped1.6 cM apart, both quite similar to the expectation.Nevertheless, correlation of the genetic and physicaldistances suggests that there are probably regions,perhaps near the centromeres and telomeres, whererecombination is suppressed, and the frequency isincreased in other regions of the genome.

In cereals, with genomes 50 or more times larger thenArabidopsis, a similar physical mapping approach isimpractical. Even measurement of the genetic length ofthe chromosomes is difficult, since there is no simplemethod to map genetically the end of a chromosome;chance discovery of a terminal RFLP can greatly increasegenetic length. Data from deletion mapping and in situhybridization of cloned genes are tending to show thatgenes are not randomly distributed along chromosomearms. Certain genes, and other markers, that are mappedgenetically midway along an arm, or towards thecentromere, seem to be physically located in the terminalchromosome region, and hence all markers distal to thesemust also be located in this region. For example, Tsujimotoand Noda (1990) examined lines of wheat that had shortdeletions, detected by C-banding, on the long arm of thechromosome designated 5A. They found that a deletion of13 % of the physical length of the arm caused loss ofmarkers representing at least 83 % of the genetic length ofthe arm. Similarly, Curtis and Lukaszewski (1991) foundthat the distal half of the physical arm of another wheatchromosome accounted for almost 90% of the recombi-nation. A clone for the secalin genes in rye can be used toprobe a major gene on the nucleolar organizing chromo-some. Gustafson et al. (1990) found that the gene wasphysically located close to the NOR, although genetic datashow that recombination is frequent between the two(Fig. 4). High-resolution mapping of a short chromosomalregion was possible with the in situ hybridizationtechnique. There is very limited recombination in the 90 %of the chromosome length that physically lies between thecentromere and the NOR (Gustafson et al. 1990) in rye, inagreement with Snape et al. (1985), who found thatrecombination was much more frequent in regions ofwheat chromosomes distal to the centromere. Anotherapproach to correlation of genetic and physical maps wasused in another cereal, maize, by Dooner (1986; Dooner etal. 1985), i.e. a transposon-tagging strategy. He estimatedthat one cM in the region of the gene Bronze represented

•XGIi

-Gpi

—XNor-Xpsr161

-Xpsr325-XPpdk XEm

—XLec XGIu

-Xpsr330

-XAdh

Fig. 4. A comparison of two physical, cytogenetic maps andthe genetic map of chromosome 1R of rye; C-band map (left, R.Schlegel and R. Kynast, personal communication), in situhybridization map (centre, from Gustafson et al. 1990) andgenetic or RFLP map (right, from Wang et al. 1991). All mapsshow the nucleolar organizing region (NOR) where rRNAgenes are tandemly arrayed, the centromere and telomeres.Telomeres have not been mapped genetically (right). The twophysical maps (left, centre) separate the centromere and NORwidely, while genetic linkage analysis places them closetogether, indicating little recombination between them.

only 14 kb of DNA, compared with an average value ofmore than 2300 kb in the genome as a whole. Not all areasnear genes have greatly elevated recombination fre-quencies (recombination hot spots); Cheung et al. (1991)have mapped the small multi-gene family of a-amylasegenes in wheat by pulsed-field gel electrophoresis, andfound that 1 cM approximates to 1000 kb within the locus,which is close to the 3000 kb average over the genome as awhole.

It is becoming clear that physical and genetic distancesbetween loci along chromosomes need not be correlated inlarger plant genomes, as in the human genome (e.g. seePetersen et al. 1991). The discovery and significance ofsuch discrepancies, and examination of differences be-tween male and female recombination maps, will beimportant for understanding genetic recombination andphysical sequence organization on chromosomes. Only byknowing the physical positions of genes along chromo-somes can genetic linkage, and gene and sequenceinterdependence, be understood. Genes are not randomlyspaced along the chromosome arms, so division of genomesize by number of cM may give little indication of thephysical separation of genes - although knowledge of thephysical distance is important for, say, cloning strategiesinvolving cloning (walking) between an RFLP marker anda linked gene.

Conclusions

Technical progress to increase both the resolution andsensitivity of gene, marker and sequence mapping is now

Molecular cytogenetics of plants 19

required. Current research using in situ hybridization isleading to better labelling technology (e.g. multiple targetlabelling; Leitch et al. 1991), and the detection of shortersequences. Flow cytometry provides a complementaryapproach to molecular cytogenetics. Although the tech-nology has proved to be of major importance in mam-malian cytogenetics, it has not yet been usefully applied toplants. Gray and Cram (1990) described why the analysisand sorting of plant chromosomes would be useful: flowkaryotyping is accurate and fast, while chromosomesorting enables the construction of chromosome-specificlibraries and hence gene mapping. They also consider thatthe work is of considerable economic interest, sincechromosome transplantation via fusion techniques andmicroinjection would be possible with sorted plant chromo-somes. While some progress towards the sorting of alimited number of plant species has been made, there areformidable difficulties in generating the high metaphaseindex required for maintaining stable cell cultures, and inisolation of chromosomes from cells with walls; inmammalian cytogenetics all these are established tech-niques or are not required. Nevertheless, there is immensepotential in the method for analysing large genomes, suchas those of the cereals.

The production of stable transgenic plants requires thephysical insertion of genes into the chromosomes. Does thesite of incorporation affect gene expression (see Stief et al.1989)? Do features, such as co-suppression of pairs ofinserted genes (Matzke and Matzke, 1990), relate to geneposition? Hager and Miller (1991) reported tight linkage ofcertain developmentally co-regulated Drosophila genes,and speculate whether such grouping is a commonoccurrence. In a plant breeding context, perhaps areas oflow recombination could be used to keep desirable geneclusters together through a crossing programme.

Developments in gene cloning and antibody technologyover the last decade have denned structures and localizedDNA and proteins within the nucleus, enabling aspects ofthe physical organization of the nucleus to be elucidated inthree dimensions (Heslop-Harrison and Bennett, 1990).Knowledge from molecular cytogenetics can help answermany of the fundamental problems of both plant geneticsand molecular biology. Physical knowledge of locations ofgenes and DNA sequences, whether active in control,expression, recombination or evolution, is helping in theunderstanding of genome behaviour and gene action.When will we be able to look directly at a chromosome andread off the sequence of DNA pairs, while assaying theactivity and purpose of each sequence?

I am grateful to Dr Andrew Leitch, Dr Trude Schwarzacher andKesara Anamthawat-J6nsson for their immense help in ourmolecular cytogenetics reseach programme and providing theplates. I also thank Ilia Leitch, Jola Maluszynska, Shi Min,Mingli Wang, Michael Bennett, Michael Gale, David Laurie,Graham Moore and many other colleagues for helpful discussionsabout the uses of molecular cytogenetics and for access to pre-publication data, and Gill Harrison for drawing Fig. 4. The workwas supported by BP and Venture Research International, andAFRC through grants 111/569 and 570 under the PMB initiative.

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