IntroductionCharacterisation of banana and plantaingermplasm has until now, been largely based onthe use of phenotypic characters and morerecently on molecular markers such as RFLP andRAPD (see INIBAP Annual Report 1996, p. 24-28). Cytogenetic studies have proved difficult inthe genus Musa because of the small size of thegenome (550 Mbp, Dolezel et al. 1994), just 10%of the barley genome for example, and the largenumber of chromosomes (2n=3x=33 in mostbanana cultivars, compared to 2n=2x=14 inbarley). Molecular cytogenetic studies, which linkdata about the molecular composition andorganisation of the genome with thechromosomes, offer greater understanding ofphylogenetic relationships and improved clarity oftaxonomic discrimination, allowing theidentification of aneuploids and assisting selection.In recent years, there have been rapid advancesin the direct observation and analysis of bananachromosomes using molecular cytogeneticmethods. This focus paper provides someinformation on the applications of such techniquesin relation to banana and plantain research.

In Situ hybridisationThe in situ hybridisation (ISH) technique,developed more than 30 years ago (Gall andPardue 1969, John et al. 1969) allows genes orDNA sequences to be directly localised onchromosomes in cytological preparations. Thedevelopment of user-friendly fluorescenttechniques (Langer-Safer et al. 1982, Pinkel et al.1986) has greatly increased the application of thistechnique during the last 15 years. Fluorescent insitu hybridisation (FISH) allows hybridisation sitesto be visualised directly and moreover, severalprobes can be simultaneously detected with

different fluorochrome, allowing the physical orderon the chromosomes to be determined.

For the FISH technique, DNA sequences to belocalised are first labelled to produce the probe.The probe is coated on the target chromosomewhich is spread in a hybridisation buffer. Aftertreatment to denature the DNA into single strands,the probe and target are allowed to re-anneal. Theprobe will bind specifically to the complementarysite on the chromosome. After washing anddetection with a fluorescent reporter, a discretefluorescent signal is visible at the site of probehybridisation, which can be visualised using afluorescent microscope (Figure 1).

One of the important modifications of the ISHtechnique is genomic in situ hybridization (GISH)(Schwarzacher et al. 1992). GISH is a genomicpainting technique which allows parental genomesin interspecific hybrids to be distinguished(Figure 2). Total genomic DNA from one parent islabelled as a probe and unlabelled total DNA ofthe other parent is used as a block. Alternatively,total DNA from both parents is labelled and theseare both used as probes, each one revealed witha different fluorochrome. This technique is basedon the rapid evolution during speciation ofrepeated sequences, which represent the majorpart of plant DNA. If the species are distantenough, the repeat sequences allow thechromosomes from the two parental species to bedifferentiated.

Applications

Untangling the A, B, S and T genomes by genomicin situ hybridizationThe classification of Musa cultivars into genomicgroups has so far been based on chromosomenumbers and morphological traits (Cheesmann1947, Simmonds and Shepherd 1955) as well as

Figure 1. DoubleFISH showing therDNA sites onsomatic metaphasechromosomes ofNarenga. The 18-25SrDNA site arevisualized in green(FITC) and the 5SrDNA sites arevisualized in red(Texas Red). Thechromosomes arecounterstained withDAPI (blue).(Courtesy of CIRAD)

Fluorescent in situhybridization of plantchromosomes: illuminatingthe Musa genomePat Heslop-Harrison, Julian Osuji, Roger Hull and Glyn HarperJohn Innes Centre, Colney Lane, Norwich NR4 7UH, U.K.

and Angelique D’Hont and Françoise Carreel CIRAD Montpellier and Neufchâteau – France

Focus paper 1

more recently, on molecular markers. GISHhowever provides a powerful complementary toolto molecular markers, enabling the portion of thegenome contributed by each parental species ininterspecific hybrids and their derivatives to bevisualised (Figure 3). This technique has allowedthe chromosomes from the four wild Musaspecies, M. acuminata, M. balbisiana, M.schizocarpa and the Australimusa species,involved in the origins of cultivated bananas to bedifferentiated (Osuji et al. 1997, D’Hont et al. inpress).

The exact genome structure of severalinterspecific cultivars has been examined usingGISH. The results were in most cases consistentwith the chromosome constitution estimatedthrough phenotypic descriptors, with one notableexception. The clone ‘Pelipita’, was found tocontain 8A and 25 B chromosomes, instead of the11A and 22 B predicted (Figure 4).

Using molecular markers, it was recentlyconfirmed that the species M. schizocarpa (Sgenome) and species of the Australimusa section(T genome) have contributed to the origin of somecultivars (Carreel 1994). However, it was notpossible to determine what proportion of thesespecies are present in the genome. Using GISH itwas possible to demonstrate for example, that theS genome contributed a full set of Schromosomes to the cultivar Wompa. Similarly,GISH showed that one basic set of Tchromosomes are present in the cultivars‘Karoina’and ‘Yawa 2’and established theirgenome constitution as AAT and ABBT,respectively (Figure 5).

Identifying individual Musa chromosomes andvisualising DNA sequencesIndividual chromosomes are difficult to identifyconventionally because they are so similar.However individual chromosomes can be definedby the hybridisation of specific cloned or syntheticrepetitive DNA sequences (Osuji et al. 1998,Dolezelová et al. 1998). For example the 18S-25SrDNA is present at a single site in each genomeand can be used to define that chromosome. Thishas a further significant use as this single site ineach genome enables easy assessment of basicploidy levels in hybrid or tissue culture material.The hybridisation pattern obtained can alsoprovide indicators of recent and evolutionaryrearrangements in the genomes (Figure 6).

The development of similar markers (repeatedsequences, BAC, etc.) for the various linkagegroups will enable the different chromosomes tobe assigned to respective linkage groups and willthus efficiently complement genetic mappingefforts. This would also open the way for theinvestigation of structural rearrangements whichare reported to be frequent in bananas (Faure etal. 1993). These rearrangements result inimportant irregularities in meiosis and irregularchromosome transmission and may have beeninvolved in the development of sterility, aprerequisite for edible fruit.

Understanding BSVFISH can be used to analyse the numbers and lociof other chromosomal sequences and it has beenused to analyse the integration of banana streak

Figure 2. Principle of genomic in situhybridisation.(Courtesy of CIRAD)

Figure 3.Metaphase oftriploid plantain MbiEgome (AAB): a. The 33chromosomesstained blue withthe DNA stain DAPI;b. In situhybridisation ofgenomic A DNA(red) and Bgenomic DNA(green); c. Interpretationshows the redlabelled regions on 22chromosomes; the other 11chromosomes arelabelled only withgreen. (Courtesy of John InnesCenter)

Figure 4. GISH onsomatic metaphasechromosomes of‘Pelipita’using total DNAfrom a BB clone revealedin red with Texas Red andtotal DNA from an AAclone revealed in greenwith FITC. (Courtesy ofCIRAD)

Figure 5. GISH onsomatic metaphasechromosomes of‘Yawa 2’ using totalDNA from a AAclone revealed ingreen with FITC,total DNA from a BBclone revealed in redwith Texas Red andDAPI counterstaining(blue). (Courtesy ofCIRAD)

Figure 6. In situ hybridisation to chromosomes of an AAMusa hybrid: a. The 22 chromosomes stained blue withDNA stain DAPI; b. Five sites of hybridisation to 5S rDNAprobe (green); c. Single site hybridisation to 18S-25SrDNA probe on each of the two genomes. (Courtesy ofJohn Innes Center)

virus (BSV) DNA into the Musa genome. Numerouslines of evidence including PCR and genomicSouthern analysis pointed to the possibleintegration of BSV sequences (LaFleur et al. 1996,Ndowora et al. 1997, Harper and Hull 1998). Toexamine whether these BSV sequences in highmolecular weight DNA were actually in the Musanuclear chromosomes, double target in situhybridisation was conducted on chromosomes fromthe plantain cultivar Obino L’Ewai, using a probespecific to BSV and a probe specific to a Musasequence. Both probes gave hybridisation signalson chromosomes of Obino L’Ewai. A majorhybridisation site to BSV was detected on bothchromatids of one chromosome in each metaphaseand at least one weaker hybridisation site wasregularly seen. This clearly demonstrates that viralsequences are integrated in the nuclear genome.The Musa probe showed hybridisation to multiplesites throughout the genome, including near themajor BSV site, but was not uniformly dispersed.

Representatives of AA, AAA and BB genomeMusa were analysed by FISH and all showedclear hybridisation of BSV sequences. Thestrength of the signals indicates that multiplecopies of the target sequence were integrated atmost of the observed sites (Figure 7.) This isfurther compelling evidence that BSV sequencesare integrated into the Musa genome and that thisintegration must have been an ancient event.

Visualisation of fine scale DNA structureThe organisation of gene and DNA structures canbe visualised by a relatively new method, that of in

situ hybridisation of probes to DNA fibres extended to their full molecular length (Fransz et al. 1996, Brandes et al. 1997, see also Schwarzacher et al. in press). Theoreticalconsiderations of the length of the extended DNA molecule andcalibration from hybridisation with probes of known length andinterspersion pattern (Fransz et al.1996, Sjöberg et al. 1997) can relate thelengths of observed fibres to the numbers ofbases (Figures 8 and 9).

This technique was used to investigate thestructure of the integrated BSV sequence. Agenomic clone (Ndowora et al. in press) and PCR-based methods (Harper et al. in press) had shownthat the integrated sequence adjacent to a Musainterspersed sequence was complex, containing aninverted region and some very highly rearrangedstretches. Stretched DNA fibres were prepared onslides from Obino L’Ewai nuclei. Double-targethybridisation with the genomic Musa sequence andBSV showed long rows of hybridisation sites (‘dots’)along stretched DNA fibres. The Musa sequencewas present at sites associated with the BSVhybridisation sites and also independently asvariable lengths of rows of dots (Figure 10). It wasapparent that there were two different lengths ofMusa-BSV chains of dots present in approximatelyequal numbers. Some were 50 µm long,representing structures containing multiple copiesof BSV sequences (150 kb long) and others were17 µm long (about 50 kb structures). Each group offibres, long and short, showed common patterns ofred and green signal sites and gaps, with repeatingunits of BSV sequence adjacent to Musa sequence.The longer structure is considered to correspond tothe major hybridising site seen on metaphasechromosomes while the shorter structure,corresponded to the minor hybridisation site.

Figure 7. Musagenotypes Cavendish(AAA) and ObinoL’Ewai (AAB) showinghybridising(integrated) BSVsequences.In situ hybridisation tometaphase spreadsof Obino L’Ewai: a. The 33chromosomesstained blue with theDNA stain DAPI.b. Hybridisation sitesof BSV (red) showingone major site ineach metaphase(arrowhead) and atleast one minor site(arrow).In situ hybridisation tometaphase spreadsof Dward Cavendish: c. The 33chromosomesstained blue with theDNA stain DAPI.d. Hybridisation sitesof BSV (red) showingat least eight majorsite in eachmetaphase(arrowhead).(Courtesy of JohnInnes Center)Bar = 5 µm

Figure 9. Rye interphasenucleus:a. DAPI stainingshows thestrechted DNA asblue fibres runningdownwards.b. A highlyrepetitive ribosomalDNA probe labelsmultiple sites onsome but not all ofthe fibres. Here, thefibres are toobundled fordetailed analysis ofthe gene structure,but the relationshipbetween nucleus,the fibres and theprobe is evident. (Courtesy of JohnInnes Center)

Figure 8. Cartoon of aninterphase nucleus fixedto a slide: a. The bluechromatin labelled ateight sites by a red in situhybridisation probe; b.After lysis of the nucleusand tilting of the slide theDNA fibres are stretchedto their full molecularlength. They canhybridise with the sameprobe and now clearly showthe relationship between probeand fibre. (Courtesy of JohnInnes Center)

ConclusionMolecular cytogenetic methods are adding apowerful set of tools to those already available tostudy genome organisation, evolution andrecombination. GISH has immense potential foridentification of chromosome origin and can beused to characterise cultivars and hybridsproduced by Musa breeding programmes.Repetitive and single copy DNA probes areyielding insights into the relationship betweengenetic and physical maps of Musa and genomeevolution. Finally, fibre in situ hybridisation can beused to examine the organisation of genes andDNA sequences. Together, these techniquesprovide data for Musa breeders, which can beused to tackle the challenges caused by bananastreak virus, tissue culture and somaclonalvariation, the use of wild germplasm in breedingand the irregular transmission of chromosomesduring meiosis. In situ hybridisation thereforeholds great potential to help scientists developoptimum breeding strategies in order to createhigh quality and disease resistant bananas.

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Figure 10. Fibre stretches ofMusa Obino L’EwaiAAB showinghybridising BSVand associatedMusa sequences.In situ hybridisationto extended DNAfibres from ObinoL’Ewai nuclei.Green and red dotsrepresent probehybridisation sitesto BSV sequencesand associatedMusa sequencesrespectively. Twodifferent patterns ofchains of dots weredetected:a. Threeindependent andaligned long fibresabove a consensusdiagram ofhybridisationpattern showingred sites andchains of greensignals. Both theMusa and BSVsequences arepresent in multiplecopies in thestructure of 150 kb,in at least twodifferent relativeorientations, andare separated bygaps with nohybridisation (nohomology toprobes).b. Three alignedshort fibres aboveconsensusdiagram, showing apattern that can beinterpreted as threesub-repeats. Underthe hybridisation,detection andimagingprocedures used,individual signalsare larger thanexpected from theprobe length, maybe slightlydisplaced from theaxis, and somesupposed targetsites may not havea detectable signal.(Courtesy of JohnInnes Center)Bar = 5 µm,corresponding the15 kb DNA fibrelength.