c Indian Academy of Sciences

RESEARCH NOTE

Physical mapping of 5S and 45S rDNA in and related genera of the by FISH, and species relationships

MAGDY HUSSEIN ABD EL-TWAB1∗ and KATSUHIKO KONDO2

1Department of Botany and Microbiology, Faculty of Science, Minia University 61519, El-Minia City, Egypt 2Laboratory of Genetics and Breeding Research, Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, Funako 1737, Atsugi City, Kanagawa Prefecture,

[Abd El-Twab M. H. and Kondo K. 2012 Physical mapping of 5S and 45S rDNA in Chrysanthemum and related genera of the Anthemideae by FISH, and species relationships. J. Genet. 91, 245–249]

Introduction repeated clusters: the major family encodes for 18S, 5.8S and 26S rRNA genes (45S rDNA) and the minor family encodes The present study has shown that fluorescence in situ rRNA genes (5S rDNA). Their localization and reciprocal hybridization (FISH) physical mapping of rDNA sites is a relationships are an important source of information for kary- valuable method to gain insight into the genome in Chrysan- ological analysis (Abd El-Twab and Kondo 2003a, 2006). themum and related genera of the Anthemideae. FISH signals The major rDNA gene family corresponds to the nucleolar revealed that 45S rDNA sites were localized on the terminal organizer region (NOR), which is one of the most studied regions of the sat-chromosomes, except in one case where the portions of eukaryotic genomes in terms of both their struc- signals were detected on the subterminal region. However, ture and function. The minor rDNA is usually found in loci 5S rDNA was distributed among the terminal, subterminal that are separate from those of the major rDNA and is not and / or interstitial regions. The results show the colocaliza- involved in nucleolus formation (Hanson et al. 1996; Inafuku tion of the 5S and 45S rDNA in case of Argyranthemum, et al. 2000). FISH makes chromosomes of Chrysanthemum Artemisia, Leucanthemmilla, Nipponanthemum and Tanace- species providing information to know molecular characters tum. The interesting outcome of this study is the overlap- of NORs, as well as, physical mapping of the 5S and 45S ping of 5S and 45S rDNA loci. Therefore, the species were rDNA approach to elucidate the relationships among closely meaningfully distinctive and divided into four groups from related genera (Kondo and Abd El-Twab 2002;AbdEl-Twab the perspective of molecular cytogenetics, which could be a and Kondo 2002, 2003a,b, 2006). valuable tool for generic genome identification. This study aims to i) present evidence for various physical Chrysanthemum and 26 other genera, has been taxonom- forms of the 5S rDNA loci and colocalization of the 5S and ically placed in the subtribe Chrysantheminae O. Hoffm. 45S rDNA on chromosomes of Chrysanthemum and related (Shih and Fu 1983) or distributed in different subtribes in genera; and ii) discuss possible congeneric relationships to the tribe Anthemideae Cass (family , subfamily Chrysanthemum based on organization of the 5S and 45S ) (Bremer and Humphries 1993). The principal rDNA loci. taxonomic problems within the tribe are entirely of relation- ships and circumscription among the genera (Bremer and Humphries 1993). In plant evolutionary studies and breeding Materials and methods there is often a need to discriminate between the genomes of closely related genera or species and to find the ancestors, The of the present study were Argyranthemum especially polyploids (Abd El-Twab and Kondo 2003a). frutescens (L.) Sch. Bip., Artemisia manshurica (Kom.) In land plants studied so far, ribosomal genes are organized Kom., C. horaimontanum Masam., C. latifolium DC. Nip- into two highly repeated gene families that occur in tandemly ponanthemum nipponicum Kitam., Tanacetum achilleifolium (M. Bieb.) Sch. Bip. and T. parthenium (L.) Schultz- Bip. The plants have been preserved in the greenhouse of the Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, Funako 1737, Atsugi City, ∗ For correspondence. E-mail: [email protected]. Japan. Keywords. Chrysanthemum; 5S rDNA; 45S rDNA; FISH.

Journal of Genetics, Vol. 91, No. 2, August 2012 245 Magdy Hussein Abd El-Twab and Katsuhiko Kondo

Chromosome preparations method with arithmetic mean (UPGMA) to generate a den- drogram and to describe relationships among genotypes. Chromosome preparations were performed following Abd El-Twab and Kondo (2006).

Results and discussion Ribosomal DNA probes A number of 5S and 45S rDNA sites and colocalization 5S rDNA probe: The probe was produced by the method sites were detected after FISH on the chromosomes of the described by Abd El-Twab and Kondo (2002). Chrysanthemum and other species tested (table 1). Chro- mosomal localization of 5S and 45S rDNA was exam- ined by single-colour and multi-colour FISH using biotin- 45S rDNA probe: The probe, pTa71 (45S rDNA), consisted of labelled and digoxigenin-labelled probes. In simultaneous a9-kbEcoRI fragment of rDNA derived from Triticum aes- hybridization biotin-labelled 5S rDNA probe was detected tivum L. (Gerlach and Bedbrook 1979), which was recloned as yellow signal (figure 1a, c, e, g & h; colour of FITC) into pUC19 plasmid. and digoxigenin-labelled pTa71 homologous to 45S rDNA was detected as red signal (figure 1b, d & f; colour of antidigoxigenin rhodamine). FISH signals revealed that the Labelling of the probes 45S rDNA sites were localized on the terminal regions of The probes were labelled either with biotin-14-dATP nick the sat-chromosomes, except in one case where the sig- translation kit (Gibco BRL, USA) or digoxigenin-dUTP by nals were detected on the subterminal region. However, 5S Dig DNA labelling kit (Boehringer Mannheim, Germany). rDNA sites were distributed among the terminal, subtermi- Labelling of each probe was carried out according to the nal and/or interstitial regions. The 5S rDNA localization on manufacturer’s protocols. the chromosomes has been described for 13 Chrysanthe- The FISH procedure, washing and detection of the probe mum species (table 1). It is localized at an interstitial site followed the methods described by Abd El-Twab and Kondo in chromosomes that do not bear NORs. Two 5S rDNA (2006). PAST computer program was used for a hierarchi- and two 45S rDNA sites were detected on the chromo- cal clustering analysis based on the unweighted pair group somes of C. horaimontanum (figure 1a). On the other hand,

Table 1. Numbers of 5S rDNA, 45S rDNA and colocalization sites after FISH on chromosomes of Chrysanthemum species.

Chromosome No. of 5S No. of 45S No. of Reference Species number rDNA sites rDNA sites colocalized sites for 5S rDNA

Artemisia manshurica (Kom.) Kom. 36 6 6 6 Present study Achillea millefolium L. 54 17 11 11 Abd El-Twab and Kondo (2009) Argyranthemum frutescens (L.) Sch. Bip. 18 4 4 4 Present study Chrysanthemum makinoi Matsum. et Nakai 18 2 4 0 Abd El-Twab and Kondo (2002) C. seticuspe (Maxim.) Hand.-Mazz. 18 2 6 0 Abd El-Twab and Kondo (2002) f. boreale (Makino) H.Ohashi et Yonek. C. horaimontanum Masam 18 2 2 0 Present study, and Abd El-Twab and Kondo (2002) C. lavandulifolium 18 2 8 0 Abd El-Twab and Kondo (2002) (Fisch. ex Trautv.) Makino C. remotipinnum Hand.-Mazz. 18 4 4 0 Abd El-Twab and Kondo (2007a) C. indicum L. 36 4 8 & 12 0 Abd El-Twab and Kondo (2002) C. latifolium DC. 36 4 8 0 Present study C. wakasaense Shimot. ex Kitam. 36 4 8 0 Abd El-Twab and Kondo (2002) C. yoshinaganthum Makino ex Kitam. 36 4 10 0 Abd El-Twab and Kondo (2002) C. morifolium Ramat. 54 8 30 0 Abd El-Twab et al. (2004) C. japonense (Makino) Nakai 54 8 10 &18 0 Abd El-Twab and Kondo (2002) C. vestitum (Hemsl.) Stapf 54 8 10 0 Abd El-Twab and Kondo (2002) C. zawadskii Herbich 54 8 14 7 Abd El-Twab and Kondo (2006) Nipponanthemum nipponicum 18 34 2 2 Present study, and (Franchet ex Maxim.) Kitam. Abd El-Twab and Kondo (2007b) Leucanthemella linearis 18 8 6 6 Abd El-Twab and Kondo (2007b) (Matsum. ex Nakai) Tzvelev Tanacetum achilleifolium Sch. Bip 18 2 4 2 Present study T. parthenium (L.) Sch. Bip. 36 8 8 8 Present study

246 Journal of Genetics, Vol. 91, No. 2, August 2012 Linkage of 5S and 45S rDNA in Chrysanthemum

Figure 1. a–h, FISH signals of 5S and 45S rDNA loci on metaphase chromosomes simultaneously probed with 5S rDNA labelled with biotin (a, c, e, g and h; yellow signals of FITC) and pTa71 (45S rDNA) labelled with digoxigenin (b, d and f; red signals of rhodamine); fluorescence signals of each probe were detected at chromosomal locations and the counterstains are blue colour of DAPI (a–f) and red of PI (g–h). a, Chrysanthemum horaimontanum;b,Tanacetum achilleifolium; c–d, Artemisia manshurica; e–f, Argyranthemum frutescens;g, T. parthenium; h, Nipponanthemum nipponicum (2 n = 17 one chromosome missed). Scale bar = 10 μm.

C. latifolium showed four 5S and eight 45S rDNA sites (data (Siroky et al. 2001; Muravenko et al. 2004;AbdEl-Twab not shown; table 1). FISH signals on the chromosomes of and Kondo 2006; Yoshikazu et al. 2006). Recently, colocal- Tanacetum achilleifolium showed that two of the 5S rDNA ization was reported in Artemisia, a closely related to sites were colocalized with two of the four 45S rDNA sites Chrysanthemum (Matoba and Uchiyama 2009). The present (figure 1b). Colocalization of all 5S rDNA and 45S rDNA results show colocalization of 5S and 45S rDNA in Argy- sites was noticed in the case of Artemisia manshurica (six ranthemum, Artemisia, Leucanthemmilla, Nipponanthemum sites, figure 1,c&d),Argyranthemum frutescens (four sites, and Tanacetum. Colocalization at some but not all loci has figure 1,e&f)andT. parthenium (eight sites, figure 1g; data been described in Chrysanthemum (Abd El-Twab and Kondo not shown for 45S rDNA). However, only two out of 34 5S 2006), Silene, (Siroky et al. 2001), Vicia (Raina et al. 2001), rDNA sites were colocalized with two of the 45S rDNA sites Linum (Muravenko et al. 2004)andArtemisia (Torrell et al. on the chromosomes of N. nipponicum (figure 1h). 2003). Our observation of 34 signals for 5S rDNA sites in N. nipponicum is consistent with that reported by Abd El- Twab and Kondo (2007b). These observations of colocal- Distribution patterns of 5S and 45S rDNAs in Chrysanthemum ization may be interpreted as (i) intermingling of the clus- and species relationships ters of 5S and 45S rDNA tandem arrays within a locus Studies of the origin and chromosome constitution of the and/or (ii) presence of the genes encoding both classes of native species of Chrysanthemum can provide understand- rRNA in a single rDNA unit. Matoba and Uchiyama (2009) ing of phylogeny and species relationships (Kondo and Abd explained the colocalization of 5S and 45S rDNA as acci- El-Twab 2002). FISH studies have shown colocalization of dental insertion of the 5S rDNA into the 45S rDNA repeat the 5S and 45S rDNA in some gymnosperm (Murray et al. unit by transposon-like, DNA-mediated or retrotransposon- 2002) and angiosperm species including Chrysanthemum like RNA-mediated transposition. It was reported that rDNA

Journal of Genetics, Vol. 91, No. 2, August 2012 247 Magdy Hussein Abd El-Twab and Katsuhiko Kondo

Figure 2. Dendrogram drawn from the UPGMA cluster analysis based on FISH physical mapping of 5S and 45S rDNA sites on metaphase chromosomes showed four types of species relationships: 1, Chrysanthemum type (separated sites); 2, Artemisia type (colocalized sites); 3, Leucanthemella type (both sep- arated and colocalized sites); 4, Nipponanthemum type (separated, colocalized and terminally dispersed sites). loci can rapidly change places on chromosomes (Dubcovsky hand, Chrysanthemum species from showed both and Dvorak 1995), as rapidly as in hybrids of the first gener- colocalization and separation of the two gene clusters (Abd ation in Chrysanthemum (Abd El-Twab and Kondo 2007a). El Twab and Kondo 2006). This indicates that these species Further, the rDNA loci can undergo genetic exchange with resulted from natural hybridization between distantly related the rest of the genome (Lim et al. 2004; Abd El-Twab and species with different patterns of physical localization of the Kondo 2007a). two genes. The endemic Japanese species of Nipponanthe- NORs containing the major rDNAs in Chrysanthemum mum and Leucanthemilla showed peculiar localization very have a particular evolutionary significance due to the distinctive from the other species (Abd El-Twab and Kondo frequent interspecific and intraspecific polymorphisms in 2007b). many species (Kondo and Abd El-Twab 2002;AbdEl- Anthemideae shows four cytotypes of FISH physical map- Twab and Kondo 2003a). NOR transposition to new chro- ping of 5S and 45S rDNA sites (figure 2): i) Chrysan- mosome sites could be detected through translocation of themum type (separated genes), ii) Artemisia type (colo- rDNA loci between homologous or nonhomologous chro- calized genes), iii) Leucanthemella type (both separated mosomes and/or duplication of these loci after direct artifi- and colocalized genes), iv) Nipponanthemum type (sepa- cial hybridization in F1 hybrids (Abd El-Twab and Kondo rated, colocalized and terminally dispersed genes). These 2003b, 2007a;AbdEl-Twabet al. 2004). Such chromosome four cytotypes might suggest that Artemisia, Chrysanthe- rearrangements may be facilitated by transposable elements, mum, Leucanthemilla and Nipponanthemum have different which offer the substrates for recombination. The presence genomes and are genetically/taxonomically distantly related. of transposons in the genome of different Chrysanthemum However, in case of Achilea mellifolium, C. zawadskii and species has been demonstrated (unpublished data). Tanacetum achilifolium, the results (table 1) indicate the Based on geographical considerations of the species dis- existence of natural intergeneric hybridization between two tribution; the isolated taxa of Chrysanthemum species from different cytotypes: Breeding between Chrysanthemum and and Japan share similar configurations for the sepa- closely related genera is possible and the production rate of ration of the 5S and 45S rDNA sites in each chromosome the F1 hybrids depends on the species relationships (Abd complement (Abd El-Twab and Kondo 2002). On the other El-Twab and Kondo 2003b, 2007a).

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The present work demonstrates that studying the organi- rich regions in Achillea millefolium showing intrachromosomal zation of the 5S and 45S rDNAs provides a valuable tool variation by FISH and DAPI. Chrom. Bot. 4, 37–45. for genome identification and breeding in Chrysanthemum. Bremer K. and Humphries C. J. 1993 The generic monograph of the Asteraceae-Anthemideae. Bull. Nat. Hist. Mus., London (Bot.) In addition, the finding of extensive colocalization in genera 23, 71–177. closely related to Chrysanthemum,suchasArtemisia, Argy- Dubcovsky J. and Dvorak J. 1995 Ribosomal-RNA multigene loci ranthemum, Leucanthemilla, Nipponanthemum and Tanace- nomads of the Triticeae genomes. Genet. 140, 1367–1377. tum, can provide more insights about ribosomal DNA evolu- Gerlach W. L. and Bedbrook J. R. 1979 Cloning and characteri- tion and species interrelationships. In conclusion, using FISH zation of ribosomal RNA genes from wheat and barley. Nucleic Acid Res. 7, 1869–1885. for physical mapping of the two gene families in all the sub- Hanson R. E., Nurul Islam-Faridi M., Percival E. A., Crane C. F., tribes of Anthemideae might shed light on the evolution of Ji Y., McKnight T. D. et al. 1996 Distribution of 5S and 18S-28S and relationships among the taxa in the tribe Anthemideae. rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors. Chromosoma 105, 55–61. Inafuku K., Nabeyama M., Kikuma Y., Saitoh J., Kubota S. and Acknowledgements Kohno S. 2000 Chromosomal location and nucleotide sequence of 5S ribosomal DNA of two cyprinid species (Osteichthyes, This study was supported by the Grant-in-Aid for Scientific Pisces). Chrom. Res. 8, 193–199. Research Programme (A) no. 10044209 of Japan Society for the Kondo K. and Abd El-Twab M. 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H. and Kondo K. 2003a Physical mapping of Artemisia (Asteraceae) with distinct Basic chromosome numbers. 45S rDNA loci by fluorescent in situ hybridization and Evo- Cytologia 74, 115–123. lution among polyploid Dendranthema species. Chrom. Sci. 7, Muravenko O. V., Amosova A.V., Samatadze T. E., Semenova O., 71–76. Nosova I. V., Popov K. V. et al. 2004 Chromosome localization of Abd El-Twab M. H. and Kondo K. 2003b Rapid genome changes 5S and 45S ribosomal DNA in the genomes of Linum L. species after inter specific hybridization between Dendranthema indica of the section Linum (syn. Protolinum and Adenolinum). Russ. J. × D. vestita identified by fluorescent in situ hybridization and 4, Genet. 40, 193–196. 6-diamidino-2-phenylindole. Chrom. Sci. 7, 77–81. Murray B. G., Friesen N. and Heslop-Harrison J. S. 2002 Molecular Abd El-Twab M. H., Shinoyama H. and Kondo K. 2004 Evi- cytogenetic analysis of Podocarpus and comparison with other dences of intergeneric somatic-hybrids between Dendranthema gymnosperm species. Ann. Bot. (London) 89, 483–489. grandiflora cv. Shuho-no-chikara and Artemisia sieversiana and Raina S. N., Mukai Y., Kawaguchi K., Goel S. and Jain A. 2001 their chromosomal mutations by using fluorescence in situ Physical mapping of 18S-5.8S-26S ribosomal RNA gene families hybridization and genomic in situ hybridization. Chrom. Sci. 8, in three important vetches (Vicia species) and their allied taxa 29–34. constituting three species complex. Theor. Appl. Genet. 103, 839– Abd El-Twab M. H. and Kondo K. 2006 FISH physical mapping 845. of 5S, 45S and Arabidopsis-type telomere sequence repeats in Shih C. and Fu G. 1983 Angiospermae, Dicotyledoneae, Com- Chrysanthemum zawadskii showing intra-chromosomal variation positae (3) Anthemideae, Angiospermae, Dicotyledoneae 76 (1). and complexity in nature. Chrom. 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Received 1 October 2011, in final revised form 23 April 2012; accepted 28 May 2012 Published on the Web: 9 August 2012

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