Institute of Experimental Botany
Laboratory of Molecular Cytogenetics and Cytometry
This chapter provides a short overview of the current status of Musa cytogenetics. Although the history of chromosome studies in Musa goes back to the beginning of the twentieth century, progress has been slow, mainly due to difficulties in analysing the small chromosomes of Musa. Flow cytometry can replace laborious chromosome counting, and it is now used routinely to estimate ploidy levels. The method has also been used to determine the size of the nuclear genome, and can be used to predict genomic constitution. More detailed analysis of the genomic constitution is made possible using genomic in situ hybridisation. The analysis of the long-range organisation of Musa chromosomes has progressed thanks to the application of fluorescence in situ hybridisation. A variety of repetitive DNA sequences have been localised to Musa chromosomes. Nevertheless, their number remains too low to provide a sufficiently complete picture of chromosome organisation. New cytogenetic markers are urgently needed to analyse the behaviour of chromosomes during evolution and in breeding programmes, and to integrate the physical and genetic maps.
The attempts to characterise nuclear genome of Musa at the chromosomal level go back to the first quarter of the twentieth century . Correct determination of chromosome numbers enabled Cheesman  to divide the genus into four sections (Eumusa, Rhodochlamys, Callimusa and Australimusa). This classification, which has withstood the test of time, is based on the basic chromosome number and morphological characters. The sections Eumusa and Rhodochlamys have the basic chromosome number x = 11, the section Australimusa has x = 10, and the section Callimusa has x = 9 or 10. Edible bananas comprise parthenocarpic seed sterile diploid (2n = 2x = 22) and autopolyploid (3x, 4x) clones of M. acuminata as well as allopolyploid clones (3x, 4x) derived from crosses between M. acuminata and M. balbisiana, both belonging to the section Eumusa. Parthenocarpic edible types have also evolved within the section Australimusa and are known as Fe'i or Fehi bananas. Their origin is not clear. Despite a long history, the development of Musa cytogenetics was rather slow. The determination of chromosome number, the analysis of chromosome structure, and pairing behaviour in meiosis were hampered by difficulties in analysing the small chromosomes of Musa.
2. PLOIDY DETERMINATION
As the accurate determination of chromosome number and/or ploidy by chromosome counting is laborious, a variety of phenotypic traits including stomata size, stomata density and pollen size were used as alternative approaches to estimate ploidy [3,4]. They were not found to be reliable, mainly due to the strong influence of the genotype [5,6]. Doleel et al. [7,8] demonstrated that rapid and reliable ploidy screening in Musa could be performed using DNA flow cytometry. The method was quickly adapted in many laboratories and its application has led to many unexpected discoveries. For instance, several well known clones previously believed to be tetraploid were found to be triploid [9,10]. As mitotic activity in vitro is usually low, flow cytometry is an attractive alternative to chromosome counting to monitor karyological instability of cell and tissue cultures, and to characterise regenerated plants.
In addition to germplasm characterisation, ploidy screening using flow cytometry finds important applications in breeding. Some of the improvement programmes are based on crossing plants with different ploidy levels, giving rise to progenies representing combinations of male and female gametes with various chromosome numbers [11,12]. DNA flow cytometry represents an ideal tool for high throughput selection of progenies of desired ploidy. In conjunction with artificial polyploidisation, flow cytometry may be used for the production of tetraploids that are employed in 4x × 2x crosses to obtain seed sterile triploids . Van Duren et al.  developed an integrated procedure involving in vitro polyploidisation and flow cytometric ploidy screening for mass production of non-chimeric autopolyploids. The process of chimera dissociation during clonal propagation in vitro has been recently analysed in detail by Roux et al. .
3. GENOME SIZE
The size of the nuclear genome of Musa was not known until recently. The first reliable estimate was made using flow cytometry . The nuclear genome of Musa was found to be small (552-607 Mbp), with the A genome being larger than the B genome. The results suggested that genome size might be used to discriminate both genomes. Using a larger set of diploids, Lysák et al.  demonstrated that the B genome is smaller by 12% on average. No intraspecific variation of genome size was found among the accessions of M. balbisiana, with an average size of 537 Mbp. On the other hand, small but statistically significant variation (591-615 Mbp) was found among the subspecies and clones of M. acuminata . The differences may reflect distinct areas of origin of individual accessions of M. acuminata and may be due to variation in the copy number of repetitive DNA sequences .
4. MIXOPLOIDY AND ANEUPLOIDY
While mixoploidy may be induced artificially after a treatment of multicellular structures with antimicrotubule drugs [14,15] or after gamma irradiation , it is not clear how frequently this phenomenon occurs spontaneously in vivo. Shepherd  and Shepherd and da Silva  reported the occurrence of aneuploid cells in root meristems of a range of triploid clones. The extent of mixoploidy varied among the clones and the frequency of normal triploid cells ranged from 8% to 95%. The results obtained in other laboratories [21,22] do not support the concept of widespread occurrence of mixoploidy in vivo. The disagreement may be, in part, due to artefacts during slide preparation and/or erroneous counting of satellites as 'minichromosomes' . Doleel et al.  showed that in Musa, satellites are often separated from the rest of the chromosome body by an extended and weakly stained secondary constriction. The constriction may easily break during slide preparation. Weakly stained secondary constrictions can be detected using fluorescence in situ hybridisation (FISH) with a probe for 45S rDNA [22,25].
5. GENOMIC CONSTITUTION
Genomic constitution in Musa has traditionally been determined from morphological parameters . According to Lysák et al. , nuclear DNA content may be used to predict genomic constitution. However, the interpretation of flow cytometric data may be compromised by differences among individual A and B genomes, and/or the involvement of other Musa genomes. A powerful alternative approach involves genomic in situ hybridisation (GISH), where labelled genomic DNA is used as a probe . Osuji et al.  were the first to demonstrate the potential of GISH for Musa. Although substantial cross-hybridisation between A and B genome DNA was observed, the authors were able to discriminate between chromosomes of A-genome and B-genome origin in cultivated clones and artificial hybrids. Subsequently, D'Hont et al.  showed that the method might also be used to discriminate chromosomes representing the S (M. schizocarpa) and the T (M. textilis) genomes. As only broad centromeric regions were labelled [25,28], GISH did not allow the identification of inter-genomic chromosome translocations.
6. CHROMOSOME STRUCTURE
Little is known about the structure of Musa chromosomes; most of the information comes from the studies on chromosome pairing during meiosis [29,30]. A comprehensive summary of meiotic studies in Musa is given by Shepherd . Local wild seeded species and subspecies were found to show regular chromosome pairing with 11 bivalents, indicating the absence of detectable structural heterozygosity. On the other hand, diploid parthenocarpic clones showed aberrant chromosome pairing with univalents, trivalents, and multivalents, indicating heterozygosity for one or more translocations or inversions . This information is important for mapping studies, as chromosome structural changes may influence recombination and affect the interpretation of results.
7. FLUORESCENCE IN SITU HYBRIDISATION
Fluorescence in situ hybridisation (FISH) has been very useful for physical mapping of DNA sequences and to study large-scale organisation of plant genomes. Doleelová et al.  and Osuji et al.  used FISH to physically map rRNA genes in M. acuminata and M. balbisiana. In both species, 18S-5.8S-26S rDNA was localised to nucleolus organising regions (NORs) of one pair of satellited chromosomes. Both research groups detected variation in the number of 5S rDNA sites. The finding of odd numbers of 5S rDNA sites in vegetatively propagated diploid edible varieties  is in line with the data on structural chromosome heterozygosity  and indicates their hybrid origin. Osuji et al.  used FISH with a (CCCTAAA)6 probe to demonstrate that chromosome ends in Musa consist of Arabidopsis-type telomeric repeats. Plant genomes contain a large proportion of various classes of mobile genetic elements. In Musa, a Copia-like element was detected by Baurens et al. . Balint-Kurti et al.  isolated an element, named monkey, that shows homology to gypsy-like retroelements. While the chromosomal distribution of the Copia-like element is not known, the monkey retroelement localises predominantly to nucleolus organising regions. Using FISH, Harper et al.  demonstrated that sequences of banana streak virus (BSV) are integrated in the nuclear genome. Recently, new repetitive DNA sequences were isolated and characterised by Valárik et al. . Most of them clustered in centromeric regions. Copy number analysis suggested that they contribute to the difference in genome size between M. acuminata and M. balbisiana.
8. CHROMOSOME-SPECIFIC MOLECULAR MARKER
The 18S-5.8S-26S rDNA provides the first, and so far the only, chromosome-specific molecular marker in Musa, while the 5S rDNA can only be used to detect a subgroup of chromosomes. Further work should concentrate on obtaining cytogenetic markers for remaining chromosomes or chromosome arms. Identification of individual chromosomes using physically mapped DNA sequences will permit analysis of their behaviour and segregation during evolution and in breeding programmes. Physically mapped single- and low-copy DNA sequences will also provide anchor sites needed to integrate physical and genetic maps. As the isolation of repetitive DNA sequences did not result in chromosome-specific landmarks , the most promising route seems to be isolation of chromosome-specific clones from BAC (bacterial artificial chromosome) libraries.
I am grateful to my colleague, Dr. Hana Šimková, for critical reading of the manuscript. This work was undertaken as a part of the Global Programme for Musa Improvement (PROMUSA) and was supported by the Research Contract No. 8145/RB from the International Atomic Energy Agency and by the Research Grant No. A6038204 from the Grant Agency of the Academy of Sciences of the Czech Republic.
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