Isolation, characterization and chromosome

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To detect clones carrying repetitive DNA sequences, the library was hybridized with 'Pisang Mas' genomic. DNA labelled with digoxigenin by random priming.
Chromosome Research 10: 89^100, 2002. # 2002 Kluwer Academic Publishers. Printed in the Netherlands

89

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.)

M. Vala¤rik, H. S imkova¤, E. Hr› ibova¤, J. S afa¤r› , M. Dolez›elova¤ & J. Dolez›el* Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovska¤ 6, CZ-77200 Olomouc, Czech Republic; Tel: +420 68 5228521; Fax: +420 68 5228523; E-mail: [email protected] *Correspondence Received 13 August 2001; received in revised form and accepted for publication by Pat Heslop-Harrison 15 October 2001

Key words: chromosome structure, genome size, in-situ hybridization, Musa, repetitive DNA

Abstract Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa.

Introduction Edible bananas (Musa spp. L.) are giant perennial herbs growing mostly in humid parts of the tropics. They are commonly divided into desert bananas, cooking bananas and plantains, and beer

bananas. Dessert bananas are palatable when eaten raw and constitute a major export commodity second only to citrus in terms of the world fruit trade. Other bananas are grown for local consumption and are generally processed by cooking or fermentation. Together with dessert

M. Vala¤rik et al.

90 bananas they provide the staple diet for millions in developing countries. Recently, various pests and the epidemic spread of diseases have threatened seriously the maintenance of this vital production (Gowen 1995, Robinson 1996). Genetic improvement of banana for resistance to pests and diseases or fruit quality is very dif¢cult as most of cultivars are seed-sterile diploid, triploid or tetraploid parthenocarpic hybrids of two diploid species, Musa acuminata Colla. (A genome, 2n ¼ 2x ¼ 22) and Musa balbisiana Colla. (B genome, 2n ¼ 2x ¼ 22) (Simmonds & Shepherd 1955). The evolution of some hybrids involved crossing with M. schizocarpa Simmonds (S genome, 2n ¼ 2x ¼ 22) and Australimusa species (T genome, 2n ¼ 2x ¼ 20) (Carreel 1994, D’Hont et al. 2000). Several strategies that permit breeding at polyploid level have been introduced and have led to the release of disease-resistant cultivars (Vuylsteke et al. 1993, 1997). Nevertheless, it became obvious that effective breeding will require better knowledge of the Musa genome evolution and structure. Various types of biochemical and molecular markers have been used to investigate the evolution and systematic relationship between various Musa cultivars and species. The use of biochemical markers based on isozymes (Jarret & Litz 1986) and £avonoides (Horry & Jay 1988) demonstrated great divergence among Musa species and a high level of differentiation of M. acuminata. Subsequently, molecular markers have been developed and enabled detailed analysis of the genetic relationship between various accessions of Musa (Jarret et al. 1992, Grapin et al. 1998, Howell et al. 1994, Carrel 1994, Kaemmer et al. 1997) and construction of genetic linkage maps (Faure¤ et al. 1993a). Segregation distortions were observed for a number of loci and were explained by the presence of chromosome structural rearrangements (Faure¤ et al. 1993a). Indeed, chromosome inversions and translocations have been detected by analysing meiosis in diploids (Wilson 1946, Faure¤ et al. 1993b). Structural heterozygosity interferes with efforts to recombine and transfer desirable traits from wild and cultivated diploids to new improved hybrids. A better knowledge of chromosome structure and karyotype evolution in Musa is thus needed. Unfortunately, the analysis of Musa chromosomes

has been complicated by their small size (Dolez›el et al. 1998) and scarcity of suitable molecular cytogenetic markers. This has impeded both the development of a reliable karyotype of Musa and the establishment of physical cytogenetic maps. Until now, only a few DNA sequences have been mapped to Musa chromosomes. Using £uorescence in-situ hybridization (FISH), Dolez›elova¤ et al. (1998) and Osuji et al. (1998) demonstrated the presence of 18S^25S rDNA in the secondary constriction of one pair of chromosomes in M. acuminata and M. balbisiana. The number of 5S rDNA sites varied from 4 to 8 per diploid chromosome set. In addition to telomeric repeats (Osuji et al. 1998), LTR retrotransposon monkey has been mapped to Musa chromosomes. Copies of monkey were found concentrated in the nucleolus organizer regions and colocalized with rRNA genes. Other copies of monkey appeared to be dispersed throughout the genome (Balint-Kurti et al. 2000). Although other repetitive DNA sequences have been described in Musa (Baurens et al. 1997a, b, 1998), their genomic distribution has not been studied. In this work we have isolated clones carrying 26S and 5S rDNA sequences, which will enable the analysis of distribution of rDNA sequences in Musa using homologous probes for the ¢rst time. We have also isolated DNA clones carrying various types of repetitive DNA sequences, which were sequenced and compared with other known DNA sequences. Their genomic distribution was established using £uorescence in-situ hybridization in two clones representing M. acuminata and M. balbisiana. The results presented in this study provide ¢rst insights into the long-range organization of chromosomes and karyotype evolution in Musa.

Materials and methods Plant material All Musa clones used in the present study were obtained from the INIBAP Musa Transit Centre (ITC, Katholieke Universiteit, Leuven, Belgium) as in-vitro rooted plantlets. After transfer to soil, plants were maintained in a heated greenhouse. M. acuminata ‘Pisang Mas’ (ITC 0653) and

Repetitive DNA sequences in bananas M. balbisiana ‘Cameroun’ (ITC 0246) were used for construction of genomic libraries, while M. acuminata ‘Calcutta 4’ (ITC 0249) and M. balbisiana ‘Tani’ (ITC 1120) were used for mapping DNA sequences to metaphase chromosomes. Construction of partial genomic libraries Genomic DNA isolated from ‘Pisang Mas’ and from ‘Cameroun’ was digested separately with 6 U per mg of TaqI restriction endonuclease (MBI Fermentas) at 65MC for 4 h. The DNA was precipitated and subsequently dephosphorylated with 0.25 U CIAP (MBI Fermentas) per mg DNA for 30 min at 37MC. CIAP was removed by phenol^chloroform extraction and subsequently DNA was ethanol precipitated. Digested Musa DNA (600 ng) was mixed with 200 ng of cloning vector pBluescript II SK+ (Stratagene) digested with restriction endonuclease Bsp106I (Stratagene) and ligated for 15 h at 12MC with 5 U of T4 DNA Ligase (MBI Fermentas). Afterwards the ligase was inactivated by incubation at 65MC for 10 min. Non-recombined vector was removed from the ligation mixture by digestion with the Bsp106I. Ligation mixture was used to transform to Escherichia coli XL1Blue mrF0 (Stratagene) competent cells using the heat shock method (Ausubel et al. 1998). Recombinant clones were selected on plates with X-gal/IPTG.

91 The 5S rDNA probe was labelled with digoxigenin using PCR with a pair of speci¢c primers (RICRGAC1, RICRGAC2), which amplify 303 bp in rice (Ohmido & Fukui 1995). To detect clones carrying repetitive DNA sequences, the library was hybridized with ‘Pisang Mas’ genomic DNA labelled with digoxigenin by random priming. Clones providing a visible hybridization signal were collected and used for subsequent analyses.

Southern hybridization All hybridizations were performed on Hybond N+ membranes (Amersham) and detection of hybridization signals was done using antidigoxigenin-AP (Roche). Signal visualization was performed by membrane incubation with CDP Star chemiluminiscent substrate (Amersham) followed by exposure to X-ray ¢lm ranging from 1 to several hours at room temperature. For Southern hybridization, 2 mg of ‘Pisang Mas’ genomic DNA digested with various enzymes were fractionated on 1% agarose gel and subsequently blotted onto the membrane by capillary transfer. The inserts of selected clones were PCR-labelled by digoxigenin using a pair of speci¢c primers (T3 and T7) and used as probes for hybridization. Clones giving strong hybridization signals were selected as repetitive sequences.

Library screening Copy number Partial genomic libraries were replicated on Hybond N+ nylon membranes (Amersham) placed on agar plates with appropriate antibiotics and cultivated at 37MC for 10 h. The colonies were lysed and the DNA was ¢xed on the membrane and puri¢ed as described by Ausubel et al. (1998). To detect banana 45S ribosomal DNA sequences, the ‘Pisang Mas’ library was screened with the probe VER17 consisting of 5.8S, parts of the 18S and most of 25S genes of Vicia faba (Yakura & Tanifuji 1983). The VER17 probe was labelled with digoxigenin using Random Primed DNA Labelling Kit (Roche). The ‘Cameroun’ DNA library was hybridized with a probe for 5S rRNA gene of Oryza sativa (Ohmido & Fukui 1995).

To estimate copy number of newly isolated repeats in M. acuminata ‘Calcutta 4’ and in M. balbisiana ‘Tani’, serial dilutions of genomic DNAs were dot-blotted, together with serial dilutions of PCR-ampli¢ed DNA of isolated repeats as a set of standards. Individual membranes were prepared separately for each repeat and hybridized with labelled probes. The images were captured by a CCD camera and analysed by ScionImage software (Scion). The copy number of each repetitive sequence per genome was estimated densitometrically as a number of molecules of the probe hybridized with one genome. Presence of plastid and mitochondrial DNA was ignored.

M. Vala¤rik et al.

92 Sequencing and homology search Clones selected as repetitive sequences were sequenced (D. Trnka Sequenation Service, Prague) and searched for homologies in GenBank with BLASTX 2.2.1 and BLASTN 2.1.3 (Altschul et al. 1997). The homologies described as signi¢cant in this paper have a maximal expectation value of at most 2  1015 . Chromosome preparations Metaphase spreads were prepared as described by Dolez›el et al. (1998). Actively growing root tips were pretreated in 0.05% 8-hydroxyquinoline for 3 h and ¢xed in 3:1 ethanol:acetic acid. Fixed roots were washed in a solution of 75 mmol/L KCl and 7.5 mmol/L EDTA (pH 4). Meristem tips were digested in a mixture of 2% pectinase and 2% cellulase for 90 min at 30MC. Protoplast suspension was then ¢ltered through a 150-mm nylon mesh and pelleted. The pellet was resuspended in 75 mmol/L KCl and 7.5 mmol/L EDTA (pH 4) and incubated for 5 min at room temperature. After pelleting, the protoplasts were washed three times with 70% ethanol, and 5 ml of suspension were dropped onto a slide. Shortly before drying out, 5 ml of 3:1 ¢xative were added to the drop to induce protoplast bursting. Finally, the slide was rinsed in 100% ethanol and air-dried. In-situ hybridization Localization of DNA sequences was performed according to Dolez›elova¤ et al. (1998). Chromosome preparations were denatured in a solution of 70% formamide in 0.6  SSC for 2 min at 72‡C, and 20 ml of hybridization mixture were applied to each slide. The hybridization mixture consisted of 50% formamide, 10% dextran sulphate in 2  SSC, 250 mg/ml sheared calf thymus DNA, and 1 mg/ml labelled probe. All probes were digoxigenin labelled by PCR as described above. The mixture was denatured at 76‡C for 15 min shortly before use. The hybridization was carried out at 37‡C overnight. After removing the cover slips, slides were washed with 50% formamide in 1  SSC at 42‡C for 10 min, and three times in 2  SSC at room temperature. The sites of digoxigenin-labelled probe hybridization were

detected using anti-digoxigenin^FITC (Roche) and the signal was ampli¢ed using antisheep^FITC (Vector Laboratories). Chromosome preparations were counterstained with 0.2 mg/ml 40 ,6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield antifade solution (Vector Laboratories). Fluorescence microscopy The slides were examined with Olympus AX 70 £uorescence microscope and the images of DAPI, and FITC £uorescence were acquired separately with a cooled high-resolution black-and-white CCD camera. The camera was interfaced to a PC running the MicroImage software (Olympus). Image processing consisted exclusively of signal intensity, contrast, and background adjustments that affected the whole image.

Results Screening for repetitive DNA sequences Partial genomic libraries of M. acuminata ‘Pisang Mas’ and M. balbisiana ‘Cameroun’ contained approximately 500 and 1200 clones, respectively. The average insert size was 480 bp. The libraries were screened for DNA clones carrying ribosomal DNA sequences and anonymous repetitive DNA sequences. Several clones from the ‘Pisang Mas’ library showed signals after hybridization with the probe for V. faba 45S rDNA. A clone with the strongest hybridization signal, named Radka1, was chosen for subsequent analyses. Only one clone was detected in the ‘Cameroun’ library giving positive signal after hybridization with the probe for rice 5S rDNA. The clone has been named Radka2. Twenty clones named Radka3 to Radka22 were selected from the ‘Pisang Mas’ library based on visible signals after hybridization with labelled genomic DNA. Inserts of all 22 clones were ampli¢ed by PCR, digoxigenin labelled and used as probes for Southern hybridization with digested genomic DNA. Ten of them showed strong hybridization signals and were classi¢ed as high copy repetitive sequences. Their insert lengths and GenBank

Repetitive DNA sequences in bananas

Figure 1. Examples of strong Southern hybridization signals of newly isolated repetitive DNA sequences exhibiting ladder-like pattern characteristic for tandem organized repetitive sequences. (a) DNA clone Radka14, found clustered in secondary constriction and for which no homology to known sequence has been found. (b) DNA clone Radka8 carrying part of a £anking region of monkey retrotranspozon.

accession numbers are given in Table 2. Ladder-like banding pattern, which is characteristic of tandemly organized sequences, was observed in six clones (Radka1, 2, 7, 8, 9 and 14) (Figure 1). Remaining sequences showed smears with few visible bands (data not shown). Sequence analysis All twelve repetitive sequences were sequenced and compared with accessions in GenBank to estimate homology with known DNA sequences (Table 1). As expected, Radka1 showed signi¢cant homology with 26S rRNA gene of many plants, while Radka2 showed 97% homology with the 5S ribosomal RNA gene of rice and many other

93 plant species. A 61-bp segment of Radka5 showed 93% homology with M. acuminata repetitive element (GenBank Y10144, Baurens et al. 2000). However, no homology was found for its £anking region. Radka7 was homologous with the 26S rRNA gene of rice but was not homologous with Radka1. A 543-bp fragment of Radka7 corresponds to the 30 end of 26S rRNA gene of rice, and was found to be homologous (96%) to 26S rDNA of many plant species. The 543-bp fragment of Radka7 contains two regions (142-bp and 156-bp) that were not homologous to rDNA of other plant species. High homology (92%) was found between Radka8 and Radka9, the only difference was cloning in the opposite direction. Both sequences are homologous (85%) to the 30 non-coding part of a 4.0-kb EcoRI fragment of monkey retrotransposon (Balint-Kurti et al. 2000) with the exception of a 57-bp fragment of Radka8 and a 82-bp fragment of Radka9, which have no homology with known sequences. Radka10 was found to be homologous (90%) to a non-coding part of the MUSA1 clone (Ndowora et al. 1999). Remaining clones showed no signi¢cant homology to DNA sequences deposited in GenBank. Long-range genomic distribution Physical distribution of all twelve newly isolated repetitive DNA sequences was analysed on the metaphase chromosomes of M. acuminata ‘Calcutta 4’ and M. balbisiana ‘Tani’ using FISH. Based on the genomic distribution, the sequences could be divided into four types (Table 1). The ¢rst type involved Radka2 sequence, which was localized on eight chromosomes of ‘Clacutta 4’ and on six chromosomes of ‘Tani’ with a strong cluster on one pair of chromosomes in both species (Figure 2a). The second group of DNA sequences involved three clones (Radka1, 7, 14), which localized to the nucleolus organizer region (NOR) on one pair of chromosomes in ‘Calcutta 4’ and ‘Tani’ (Figure 2b). All probes prepared from clones homologous to rDNA sequences described in this paper gave stronger signals compared with FISH with heterologous probes for 45S and 5S rDNA. The third type of genomic distribution was characteristic for six clones (Radka3, 5, 6, 8, 9,

M. Vala¤rik et al.

94 and 12), which preferentially localized to centromeric regions of all chromosomes. In addition, they exhibited dispersed distribution throughout the genome with the exception of four pairs of chromosomes, where only one arm was labelled. This pattern of distribution was more pronounced for sequences with lower copy numbers (Figure 2c, d). All sequences localized to M. acuminata and M. balbisiana, the only difference being weaker signals for the latter species. The largest difference in signal strength was found for Radka5, which gave signi¢cantly weaker signals on M. balbisiana chromosomes. Two clones, Radka4 and Radka10, which preferentially localized to centromeric regions of all chromosomes, represented the fourth type of genomic distribution. Other parts of chromosomes showed only weak dispersed signals (Figure 2e, f). Compared with Radka4, Radka10 exhibited weaker signals. FISH signals of Radka4 and Radka10 were comparable in both Musa species, the only difference being weaker signals in M. balbisiana. Copy number Copy number of the repetitive sequences was determined for M. acuminata represented by ‘Calcutta 4’ and M. balbisiana represented by ‘Tani’ (Table 2). DNA clones carrying ribosomal DNA sequences showed the highest abundance. The sequence Radka7 shows similar copy number to sequence Radka1. These ¢ndings predict similarity of the pairs of sequences as con¢rmed by sequence analysis described above (Table 1). The number of copies of Radka1 and Radka7 was similar for M. acuminata and M. balbisiana. On the other hand, the Radka2 clone (carrying

5S rDNA) was almost two times more abundant in M. acuminata. The sequence Radka14 shows a similar copy number to Radka2 but was localized in the NOR region only. In general, the copy number of repetitive sequences was higher for M. acuminata genome, where it ranged from several hundred to more than 104 per unreplicated haploid chromosome set. While the abundance of some repeated sequences was similar in both species (Radka4, 8 9, 10, 12), other sequences showed larger differences (Radka3, 6). The maximal difference in copy number between M. acuminata and M. balbisiana was found for Radka5, which contains part of the M. acuminata repetitive element (GenBank Y10144, Baurens et al. 2000).

Discussion Genome size in angiosperms is known to vary over several orders (Bennet & Leitch 1995). It is now believed that the proportion of coding DNA sequences is similar in different plant species and that the variation in genome size is mainly due to repetitive DNA (SanMiguel et al. 1996, Pearce et al. 1996). Although the debate continues (Bennetzen & Kellogg 1997, Petrov 1997), an increasing amount of evidence has been accumulated proving that ancestral angiosperms had small genomes and that repetitive DNA sequences accumulated during evolution (Leitch et al. 1998). Characterization of repetitive DNA sequences revealed two major classes: tandem repeated sequences and interspersed repetitive sequences; the latter represented mainly by various types of mobile elements (Flavel & Moore 1996, Bennetzen

(Opposite) Figure 2. Localization of newly isolated repetitive DNA sequences on metaphase chromosomes of Musa acuminata ‘Calcutta 4’ by £uorescence in-situ hybridization with FITC-labelled probes (yellow colour). Chromosomes were counterstained by DAPI whose £uorescence is shown in red. (a) Eight loci (marked by arrowheads) were observed for clone Radka2 carrying 5S rDNA; (b) Clone Radka1 (carrying part of 26S rDNA) hybridized to secondary constriction on one pair of chromosomes (arrowheads); (c) DNA clone Radka3 represents type 3 of genomic distribution described in this work. The distribution is characterized by preferential localization to centromeric regions of all chromosomes and dispersed distribution over all chromosome arms with the exception of four pairs of chromosomes where only one arm is labelled (arrowheads). Secondary constrictions are marked by arrows; (d) The same type of distribution observed after FISH with a probe for less abundant DNA clone Radka8. Unlabelled arms are marked by arrowheads, secondary constrictions are marked by arrows; (e ^ f) Two DNA clones displaying type 4 of genomic distribution. Radka4 (e) and Radka10 (f) hybridized preferentially to centromeric regions of all chromosomes. Other parts of chromosomes showed only weak dispersed signals. Scale bars ¼ 5 mm.

Repetitive DNA sequences in bananas

95

96

Table 1. Genomic distribution and results of homology search of newly isolated repetitive DNA sequences. Genomic distribution

Homology to known DNA sequences

Name of the clone

Type of distribution

Localization after FISH

DNA sequence

Radka2

1

8 sites in M. acuminata ‘Calcutta 4’ 6 sites in M. balbisiana ‘Tani’

Radka1 Radka7 Radka14

2

Radka5

3

Radka8 Radka9

GenBank accession number

Smallest sum probability

Hordeum murinum 5S ribosomal RNA gene

AF096721

3  1007

Secondary constriction

Rice 26S ribosomal RNA gene Rice 26S ribosomal RNA gene S

M11585 M11585 S

0.0 0.0 S

Strong signals in centromeric regions of all chromosomes, other signals scattered over the most of chromosome arms with the exception of four pairs of chromosomes where the sequences could be detected only on the one arm.

M. acuminata repetitive element

Y10144

6  1016

M. acuminata retrotransposon monkey

AF143332

3  1021

M. acuminata retrotransposon monkey

AF143332

3  1021

S S S

S S S

S S S

MUSA1 clone carrying cacao swollen shoot badnavirus S

AF106947

10108

S

S

Radka3 Radka6 Radka12 Radka10 Radka4

4

Signals clustered preferentially in centromeric regions of all chromosomes.

M. Vala¤rik et al.

Repetitive DNA sequences in bananas

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Table 2. Basic characteristics of newly isolated repetitive sequences. Copy number per unreplicated haploid genome Clone name

Insert length (bp)

GC content (%)

M. acuminata ‘Calcutta 4’

M. balbisiana ‘Tani’

Copy number ratio acuminata/balbisiana

Accession number

Radka1 Radka2 Radka3 Radka4 Radka5 Radka6 Radka7 Radka8 Radka9 Radka10 Radka12 Radka14

685 409 808 605 742 193 596 337 334 689 298 411

51 60.4 54.1 50.6 42.8 45.3 56.9 46.9 45.2 40.8 41.8 57.4

1:6  104 4:6  103 1:9  103 3:1  103 3:4  103 4:1  102 1:6  104 3:2  102 3:2  102 5:8  102 6:3  102 7:3  103

1:3  104 2:7  103 5  102 2:3  103 1:2  102 1:4  102 1:3  104 2:4  102 2:5  102 3:6  102 4:1  102 3:5  103

1.2 1.7 3.8 1.35 28.3 2.9 1.2 1.3 1.28 1.6 1.5 2

AF399949 AF399942 AF399943 AF399944 AF399945 AF399946 AF399947 AF399948 AF399938 AF399939 AF399940 AF399941

1998). Repetitive DNA sequences and retroelements in particular, once believed to be parasitic (Doolittle & Sapienza 1980, Orgel & Crick 1980), probably played a major role in genome evolution and speciation, modulation of genes and gene expression, and in maintaining important chromosomal domains, such as paracentromeric heterochromatin, subtelomeric regions and telomeres (Kubis et al. 1998, Schmidt & Heslop-Harrison 1998). Various types of repetitive DNA sequences were found to be useful molecular cytogenetics markers for chromosome identi¢cation and orientation (Vershinin et al. 1994, Ueng et al. 2000). In contrast to other important crops, virtually nothing is known about the large-scale organization of Musa chromosomes. Only one pair of chromosomes has been identi¢ed unambiguously using FISH with heterologous probes for 18S^26S rDNA (Dolez›elova¤ et al. 1998, Osuji et al. 1998). In the search for repetitive DNA sequences in Musa, we have screened two partial genomic libraries. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for Radka1 and Radka7 (carrying different fragments of Musa 26S rDNA) resulted in clear signals at NORs, thus con¢rming previous observations (Dolez›elova¤ et al. 1998, Osuji et al. 1998). Speci¢c labelling of NOR was also observed after FISH with a probe for Radka14,

for which no homology with known DNA sequences was found. Further study will be needed to unravel the molecular structure of Musa NOR and the nature of Radka14 sequence. Only one clone homologous to 5S rDNA, named Radka2, has been recovered by screening the two genomic libraries. FISH showed differences in the number of 5S rDNA loci per genome between M. acuminata ‘Calcutta 4’ and M. balbisiana ‘Tani’ con¢rming previous results (Dolez›elova¤ et al. 1998, Osuji et al. 1998). Compared with earlier studies, the use of homologous probes for rDNA sequences resulted in stronger £uorescent signals. All remaining repetitive DNA sequences exhibited more or less pronounced clustering at centromeric regions of all Musa chromosomes. Six of the sequences were also found to be dispersed over most of the chromosome arms with the exception of four pairs of chromosomes, where the sequences were scattered over only one of the arms. These chromosomes included a chromosome pair carrying NOR. The fact that, with the exclusion of Radka8 and Radka9, the sequences were not related indicates a preferential accumulation of repetitive sequences in speci¢c regions of the Musa genome. A 61-bp fragment of Radka5 was found to be homologous to a M. acuminataspeci¢c repetitive element described by Baurens et al. (1996). As the authors did not study the

98 genomic distribution, our results provide further characterization of the sequence. The observation of weak FISH signals of Radka5 on chromosomes of M. balbisiana and strong labelling of M. acuminata chromosomes is on a line with an almost 30-fold difference in the copy number. Two sequences belonging to this group, Radka8 and Radka9, showed homology to the 30 £anking region of monkey retrotransposon. Their genomic distribution corresponded fairly well to that observed previously after FISH with a probe for the 4-kb subclone of monkey (Balint-Kurti et al. 2000) with the notable exception of the absence of signals at NOR. The reason for the difference is not clear. As Radka8 and 9 do not contain coding parts of monkey, it is possible that their sequences are absent in NOR. This assumption is supported by the presence of variable parts of Radka8 and 9, which would indicate structural differentiation of the sequences during evolution independently of coding sequences of monkey. The most speci¢c clustering at the centromeric region of all chromosomes was observed for Radka4. This clone, which is not homologous to any of the known DNA sequences, may represent a speci¢c component of Musa centromere. Altogether, our results indicate the presence of several different types of repetitive DNA sequences in the centromere. However, due to the limit of resolution of conventional FISH, it was not possible to discriminate between centromeric and pericentromeric locations of individual sequences. Weaker and less-speci¢c clustering at centromeres of all chromosomes was characteristic for Radka10, which was found to be homologous to the non-coding part of the MUSA1 clone. This clone carries one of the two badnavirus sequences, which have been found integrated into the Musa genome, namely the ‘dead’ badnavirus sequence closely related to cacao swollen shoot badnavirus (Ndowora et al. 1999). It should be noted that the genomic distribution of Radka10 described in this work may not re£ect the distribution of the badnavirus as its sequence and the £anking sequence Radka10 may not always be present simultaneously. In contrast to many plant species where mobile genetic elements and their remnants contribute most of the nucleotide content (SanMiguel et al.

M. Vala¤rik et al. 1996, Pearce et al. 1996), our results indicate that these elements do not represent a major fraction of the Musa genome. The discrepancy may be due to small genome size, which ranges from 591 to 615 Mbp for M. acuminata and from 534 to 552 Mbp for M. balbisiana (Dolez›el et al. 1994, Lysa¤k et al. 1999). A relatively small proportion of mobile elements was also found in the small genome of Arabidopsis thaliana (The Arabidopsis Genome Initiative 2000). With the exception of rDNA, most repetitive DNA sequences localized preferentially to centromeric regions. This seems to be typical for species with a small genome size, where intercalary heterochromatin blocks are rare and most of the heterochromatin is localized proximally (Kamisugi et al. 1993, Nakayama & Fukui 1997). A lack of repeated sequences in the distal parts of Musa chromosomes, which limits the potential of GISH to detect interspeci¢c chromosomal exchanges was noted by D’Hont et al. (2000). Our study indicated small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana. On the other hand, all repetitive sequences were more abundant in M. acuminata. While for some sequences the differences were relatively small, for others, e.g. Radka5, the difference between species was almost thirty-fold. Variation in the copy number of repetitive sequences between Musa species and subspecies was also reported by Baurens et al. (1996, 1997b). Our previous studies showed that the nuclear genome of M. acuminata is larger by about 12% (68 Mbp) than that of M. balbisiana (Dolez›el et al. 1994, Lysa¤k et al. 1999). Based on the insert length and difference in copy number between M. acuminta and M. balbisiana (Table 2), it may be calculated that the repetitive sequences described here contribute 10.5 Mbp to the difference in genome size. The fact that repetitive sequences of various natures are more abundant in M. acuminata, indicates that they were subject to a unidirectional change in copy number. Similar observations were made by Ohmido et al. (2000) for repetitive DNA sequences in indica and japonica rice. The reason for a directed increase and/or decrease in copy number, which accompanied phylogenetic differentiation of M. acuminata and M. balbisiana remains

Repetitive DNA sequences in bananas unclear. Although only a limited set of repetitive DNA sequences has been isolated and analysed, the results presented in this study provide the ¢rst insights into the long-range organization of chromosomes and karyotype evolution in Musa. The use of homologous probes obtained in this study will permit more speci¢c analysis of genomic distribution of rDNA sequences in Musa. On the whole, our results indicate similar organization of chromosomes of M. acuminata and M. balbisiana. However, it should be stressed that this type of analysis does not detect small intra- or inter-chromosomal exchanges. Work is in progress to analyse genomic distribution of repetitive DNA sequences in other Musa species and to characterize other components of their genomes.

Acknowledgements We are grateful to INIBAP (International Network for Banana and Plantain Improvement, Montpellier, France), Prof. R. Swenen and Ir. I. Van den Houwe (Katholieke Universiteit, Leuven, Belgium) for providing plant material. We thank Dr. J. Macas, Dr. P. Neuman and Dr. M. Nouzova¤ for helpful advice concerning construction and screening of genomic libraries, and Ms. R. Tus› kova¤ for excellent technical assistance. We are grateful to Dr. O. Alkhimova for critical reading of the manuscript. The study was undertaken as a part of the Global Programme for Musa Improvement (PROMUSA) and was supported by the International Atomic Energy Agency (Research Contract No. 8145/RB).

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