Chromosome Botany (2006) 1: 7-11 © Copyright 2006 by the International Society of Chromosome Botany

Fluorescence in situ hybridization and genomic in situ hybridization to identify the parental genomes in the intergeneric hybrid between japonicum and Nipponanthemum nipponicum

Magdy Hussein Abd El-Twab1, 2 and Katsuhiko kondo1, 3

1Laboratory of Chromosome and Gene Stock, Graduate School of Science, Hiroshima University, 1-4-3 Kagamiyama, Higashi-Hiroshima City 739-8524, Japan; 2Department of Botany, Faculty of Science, El-Minia University, El-Minia City, Egypt

3Author for correspondence ([email protected]) Received February 6, 2006; accepted September 5, 2006

ABSTRACT. Chrysanthemum japonicum (2n=18;♀) and Nipponanthemum nipponicum (2n=18;♂) were artificially crossed and produced healthy F1 hybrid by aids of the series of embryo rescue, axenic culture and acclimatization. FISH biotin- labeled probes of 5S rDNA and pTa71 for 45S rDNA distinguished 5S rDNA and NOR regions of the chromosomes in the genome of respective species in the chromosome complement of the F1 hybrid. One 5S rDNA-signal and two 45S rDNA-signals were shown in the nine chromosomes of C. japonicum genome in the F1 hybrid chromosome complement (2n=18), while no 5S rDNA-signal and one 45S rDNA-signal was shown in the nine chromosomes of N. nipponicum genome. GISH differentiated the genome of C. japonicum and that of N. nipponicum in the F1 hybrid chromosome complement using the probe of total genomic DNA of N. nipponicum and showed that the parental genomes in the chromosome complement of the hybrid were generally in separate domains during mitosis.

KEYWORDS: Chrysanthemum japonicum, FISH, GISH, Nipponanthemum nipponicum

The has two Japanese endemic species such as bearing chromosomes (Ørgaard and Hesloph-Harrison Chrysanthemum japonicum in the subtribe Artemisiinae, 1994). Analyses of NOR distribution and organization in the tribe and Nipponanthemum nipponicum rDNA-carrying chromosomes are important for under- (Franchet ex Maxim.) Kitamura in the subtribe Leucan- standing the events of divergence and hybridization of theminae, the tribe Anthemideae Cass (Nakata et al. 1987). plant species. FISH methods have been applied to chro- Chromosomes of C. japonicum and N. nipponicum mosomes in various plants (e.g., Alonso-Balanco et al. have been studied by using various cytological methods 1994; Xu and Earle 1996; Schmidt et al. 1997). Molecular (Tanaka and Shimotomai 1961; Tanaka 1966, 1969; cytogenetic approaches to elucidate the species relation- Taniguchi et al. 1975; Honda et al. 1997; Ogura and ships among the Japanese Chrysanthemum (=Dendran- Kondo 1998; Kondo et al. 1999; Abd El-Twab and Kondo thema) species were firstly reported by Kondo et al. 1999, 2001b). (1996). FISH physical mapping of 5S rDNA was reported In higher eukaryotes, rDNAs are organized into two for the first time in Chrysanthemum (=Dendranthema) distinct gene classes of the major rDNA cluster encoding species by Abd El-Twab and Kondo (2002). The rDNA 45S rDNAs; and the minor rDNA cluster encoding 5S probe of pTa71 has been used to mark the NORs of rDNA. The minor rDNAs are usually found in loci that Japanese and Chinese species of Chrysanthemum (=Den- are separated from those of the major rDNAs and, unlike dranthema) and other closely related genera (Khaung et al. major rDNA, the minor rDNA is not involved in the 1997; Honda et al. 1997; Abd El-Twab and Kondo 1999; nucleolus formation (Inafuku 2000; Abd El-Twab and Abd El-Twab et al. 1999). The technique of genomic in Kondo 2002, 2004a). situ hybridization (GISH) has been useful in identification In situ hybridization (ISH) is a valuable method to of parental genomes and determination of levels and clarify distribution of DNA sequences and copy numbers incorporation positions of alien chromatin in interspecific at different sites in chromosomes and to detect evolutio- and intergeneric plant hybrids (e.g., Schwarzacher et al. nary changes in their physical organization in the genome 1989; Anamthawat-Jónsson et al. 1990; Leich et al. (Harrison and Heslop-Harrison 1995). Fluorescence in 1991; Bennett et al. 1992; Mukai et al. 1993; Parokonny situ hybridization (FISH) makes it possible to provide et al. 1994; Chen et al. 1995; Takahashi et al. 1997; information on molecular characters of nucleolar orga- Ogura and Kondo 1998; Kondo et al. 1999; Abd El-Twab nizing region (NORs) in chromosomes of plant species et al. 1999; Abd El-Twab and Kondo 1999, 2001a, b, (Heslop-Harrison et al. 1991). NORs have been most 2004b; Abd El-Twab et al. 2004 ). intensively studied in terms of structure and function. It Nipponanthemum nipponicum has been taxonomically has been indicated that rRNA genes are located on NOR- placed in unclear position in the subtribe Leucantheminae

7 8 ABD EL-TWAB AND KONDO FISH AND GISH TO CHRYSANTHEMUM JAPONICUM X NIPPONANTHEMUM NIPPONICUM 9

(Bremer and Humphries 1993), although it has been blocking DNA. The probe mixtures were denatured by sometimes treated into the subtribe Chrysantheminae boiling for 5 min, centrifuged briefly, and transferred (Shih and Fu 1983). Thus, the present research is the series immediately to ice for 10 min. which has the following objectives: (1) to investigate the The slides were denatured in a preheated formamide intergeneric crossability of N. nipponicum to the closely solution (70% in 2X SSC) at 70°C for 2 min, dehydrated related genera that placed in different subtribes in the in an ice-cold ethanol series (70, 80, and 100% for 3 min tribe Anthemideae (=Chrysanthemum in broad sense), each) and then, allowed to air-dry. (2) intergenomic characterization and the type of changes The probe mixtures were applied to the appropriate in the rDNA loci on the chromosome complement of the slides and covered with coverslips without air bubbles. hybrids that resulted after hybridization of the species in The slides were, then, incubated in a preheated humid Chrysanthemum in broad sense and (3) to determine chamber at 63°C for 2h of hybridization. After gently whether the prevalence of genomic changes could start removing the coverslips, the slides were washed for 2 X within the first generation. In this study, FISH and GISH 5min in 2X SSC at 63°C, transferred to an incubator at were applied to characterize the parental chromosomes in 37°C for 20 min, and allowed to cool at room temperature. the F1 hybrid between C. japonicum and N. nipponicum Visualization of the hybridized probes followed during mitosis. Kondo et al. (1999) and Abd El-Twab and Kondo (1999).

MATERIALS AND METHODS RESULTS AND DISCUSSION

Plant material A plant of Chrysanthemum japonicum The maternal Chrysanthemum japonicum (2n=18) and Makino was collected in Japan: Ehime Prefecture, Uwa- the paternal Nipponanthemum nipponicum (2n=18) were

Cho (2n=18; ID No. 0411104) and used as the maternal artificially crossed and produced healthy 1F hybrid plants. parent, while a plant of Nipponanthemum nipponicum The hybrids were easily obtained by germination in vitro (Franchet ex Maxim.) Kitamura was collected in Japan: and growth acclimatization to be strong and vigorous in Kanto District (2n=18; ID No. 0410201) and used as the the greenhouse indicating the two species could be paternal parent. They were cultivated in the cultivation genetically as well as taxonomically closely related to field of Laboratory of Plant Chromosome and Gene each other. Stock, Graduate School of Science, Hiroshima University. FISH biotin-labeled probes of 5S rDNA and 45S The artificial intergeneric hybridization between them rDNA (NORs; pTa71) distinguished the 5S rDNA and was made by hand pollination and the embryo rescue NOR regions separately in certain chromosomes in the followed Abd El-Twab and Kondo (2001a). The artificial genome of respective species in the chromosome intergeneric F1 hybrid (Accession No.: j-2-4-1) was complement of the F1 hybrid. One interstitial FISH- cultivated and conserved in the Laboratory of Plant signal of 5S rDNA was hybridized with the probe in one Chromosome and Gene Stock, Graduate School of Sci- chromosome that might be relating to C. japonicum, ence, Hiroshima University. while no signal of 5S rDNA in the chromosomes of N.

nipponicum in the chromosome complement in their F1 Labeling of the DNA probes Biotinylated nick translation hybrid was hybridized. Among the chromosomes in the labeling of the genomic DNA and pTa71 rDNA probes chromosome complement of the F1 hybrid, one of the were carried out according to the manufacturer’s protocol largest sat-chromosomes which showed one 45S rDNA (Gibco BRL) with biotin-14-dATP nick translation kit. terminal signal could be come from the genome of N. nipponicum, while two smallest sat-chromosomes which FISH and GISH For FISH, the PCR amplified 5S rDNA showed 45S rDNA signals could be come from the and pTa71 of 45S rDNA probes were used following genome of C. japonicum. Abd El-Twab and Kondo (2004a). For GISH, the total The genome of C. japonicum and that of N. nipponicum genomic DNA was isolated from 0.5-1.0 g of fresh in the chromosome complement of the intergeneric F1 young leaf material of the two parental species following hybrid were differentially stained and fluoresced as the protocol of Abd El-Twab et al. (1999). Total genomic distinct domains of two different colors following GISH DNA of N. nipponicum was mechanically sheared and without using any blocking DNA. This was documented labelled with biotin-14-dATP for use as the in situ probe. in Fig. 1. Thus, the two genomes may be distantly related Chromosome preparations using meristems of root tips to each other: This phenomenon has already been were made according to Kondo et al. (1996). Probe dena- reported in these and other related species (Ogura and turation and hybridization mixture followed Anamthawat- Kondo 1998; Kondo et al. 1999; Abd El-Twab and Kondo Jónsson and Reader (1995) and Abd El-Twab et al. (2004). BD EL-TWAB 1999, 2001b, 2004b). The probe hybridization mixture 40μl per slide contained GISH using the biotin-labeled total DNA probe of N. 10% dextran sulphate, 2X SSC, 0.25% SDS (lauryl nipponicum to mitotic chromosomes of the hybrid showed sulphate), and 100 ng of the biotinylated probe without that nine yellow (FITC)-colored chromosomes of N. 8 ABD EL-TWAB AND KONDO FISH AND GISH TO CHRYSANTHEMUM JAPONICUM X NIPPONANTHEMUM NIPPONICUM 9

Fig. 1. FISH and GISH applied on mitotic metaphase chromosomes of the F1 hybrid between Chrysanthemum japonicum and Nipponanthemum nipponicum. A, C and E: Interphase nuclei; F: prophase; B, D, G and H: Metaphase chromosomes. E and F: Yellow chromosomes of N. nipponicum hybridized with the biotin-labelled probe of N. nipponicum and red chromosomes of C. japonicum. Yellow-color was fluoresced and visualized by FITC and red-color by PI. A and B: one yellow signal of 5S rDNA detected with FISH and hybridized with the biotin labeled probe of PCR amplified 5S rDNA. C and D: Three yellow signals of 45S rDNA detected with FISH and hybridized with the biotin labeled probe of pTa71. E: A nucleus at interphase with distinct red C. japonicum and yellow N. nipponicum (with distinct yellow-GISH-bands) domains, representing the parental genomes. F: Yellow chromosomes of N. nipponicum hybridized with the biotin-labeled probe of N. nipponicum showing terminal bands and nine red PI-counterstained chromosomes of C. japonicum. G and H. Nine yellow chromosomes of N. nipponicum hybridized with the biotin-labeled probe of N. nipponicum showing terminal bands and nine red PI-counterstained chromosomes of C. japonicum. Bar=10 μm.

nipponicum hybridized with the probe and exhibited chromosomes after GISH using the total genomic DNA prominent fluorescent bands at the terminal region of of N. nipponicum as a probe showed that the nine chro- most of the chromosomes, while the nine chromosomes mosomes of N. nipponicum that hybridized with the probe of C. japonicum were red-colored when counterstained were larger than the nine chromosomes of C. japonicum, by propidium iodide (PI) (Fig. 1I). The somatic metaphase which did not hybridize. The terminal bands might be 10 ABD EL-TWAB AND KONDO FISH AND GISH TO CHRYSANTHEMUM JAPONICUM X NIPPONANTHEMUM NIPPONICUM 11 correlated with the A-T rich heterochromatin regions, ACKNOWLEDGMENTS. This study was supported by the Grant- in-Aid for Scientific Research Program (A) No. 14255014 of the Japan since application of DAPI to the same mitotic plate after Society for the Promotion of Science (JSPS; Representative: Katsuhiko GISH showed that all positive GISH-band sites appeared Kondo), the Postdoctoral Fellowship for Foreign Research of JSPS, as DAPI-positive bands (Abd El-Twab and Kondo 2000, Grant-in-Aid for JSPS Fellow 15・03313 of the Japanese Ministry of Education, Science, Culture, Sports and Technology (Representative: K. 2001). The terminal bands could also be correlated with Kondo), and the National Bioresource Project “Chrysanthemum sensu the C-banded regions of the chromosomes in Chrysan- lato” by Japanese Ministry of Education, Science, Culture, Sports and themum sensu lato (Taniguchi et al. 1975). Technology. This paper was contributed from Laboratory of Plant Chromosome and Gene Stock, Graduate School of Science, Hiroshima Leich et al. (1991) demonstrated that the parental University (Contribution No. 91). genomes lay in various non-intermixed configurations, including lateral and concentric arrangement in interphase LITERATURE CITED nuclei in intergeneric hybrid. In contrast, Abd El-Twab and Kondo (2001a) discovered that one of the two paren- Abd El-Twab, M. H. and Kondo, K. 2004a. Identification of muta- tion and homologous chromosomes in four cultivars of tal genomes of an intergeneric hybrid was separated as Dendranthema grandiflora by physical mapping of 5S and nine chromatin domains condensed and separated in 45S rDNAs using fluorescence in situ hybridization. Chrom. interphase nucleus while the two genomes were separated Sci., 8: 81-86. Abd El-Twab, M. H. and Kondo, K. 2004b. Identification of parental in two domains without intermix in meiotic interphase chromosomes of artificial intergeneric F1 hybrid between nuclei in another intergeneric hybrid (Abd El-Twab and Dendranthema horaimontana and Nipponanthemum nipponi- Kondo 2001b). The present study showed that the inter- cum by fluorescence in situ hybridization (GISH) and fluores- cence in situ hybridization (FISH). Chrom. Sci. 8: 71-79. phase nuclei showed the yellow colored chromosomes of Abd El-Twab, M. H., Shinoyama, H. and Kondo, K. 2004. Evi- N. nipponicum were observed as individual chromosome dences of intergeneric somatic hybrids between Dendranthema territories in domains without intermingled with the red grandiflora cv. Shuho-no-chikara and Artemisia sieversiana and their chromosomal mutations by using fluorescent in situ colored chromosomes of C. japonicum (Fig. 1E). The hybridization and genomic in situ hybridization. Chrom. Sci. mitotic chromosomes at prophase and metaphase showed 8: 29-34 that the yellow- and red-colored chromosomes of the two Abd El-Twab, M.H. and Kondo, K. 2002. Physical mapping of 5S rDNA in chromosomes of Dendranthema by fluorescence in parents were keeping the same interphase position of situ hybridization. Chrom. Sci. 6: 13-16. attachment without intermix (Fig. 1F-H). Abd El-Twab, M. H. and Kondo, K. 2001a. Genome territories of If N. nipponicum was used as the paternal parent for Dendranthema horaimontana in mitotic nuclei of F1 hybrid between D. horaimontana and Tanacetum parthenium. Chrom. artificial cross, the chromosomal mutations such as Sci., 5: 63-71. deletion and translocation could be invistegated in the Abd El-Twab, M. H. and Kondo, K. 2001b. Molecular cytogenetic chromosome complements of the intergeneric hybrids in identification of the parental genomes in the intergeneric hybrid between Leucanthemella linearis and Nipponanthemum both meiotic pollen mother cells (Abd El Twab and Kondo nipponicum during meiosis and mitosis. Caryologia 54: 2001b) as well as somatic cells (Abd El Twab and Kondo 109-114. 2004b). In the present study chromosomal mutations Abd El-Twab, M. H. and Kondo, K. 2000. Discrimination and isolation of terminal regions of Dendranthema occidentali- could not be detected, indicating chromosomal stability japonense in the chromosomes of F1 hybrid between D. in the present intergeneric hybrid. occidentali-japonense and D. boreale by using GISH. Chrom. The highlight in the present result the potential of the Sci. 4: 87-93. Abd El-Twab, M. H. and Kondo, K. 1999. Identification of nucleolar artificial cross, rDNA markers, in compared to the organizing regions and parental chromosomes in F1 hybrid of genomic probing, as they can differentiate individual Dendranthema japonica and Tanacetum vulgare simultane- NOR-bearing chromosomes in each parental chromosome- ously by fluorescence in situ hybridization. Chrom. Sci., 3: 59-62. set in the somatic hybrid; the two genera readily produced Abd El-Twab, M. H., Kondo, K. and Hong, D. Y. 1999. Isolation of the artificial hybridization, the FISH signals of the 45S a particular chromosome of Ajania remotipinna in a chromo- rDNA in the chromosome complement of the hybrids some complement of an artificial F1 hybrid of Dendranthema lavandulifolia X Ajania remotipinna by use of genomic in were the average of both parents, the chromosome set of situ hybridization. Chrom. Sci. 3: 21- 28. N. nipponicum had quite larger chromosomes than those Alonso-Blanco, C., Pendás, A. M., Gracia-Suarez, R., Roca, A., of C. japonicum, the two parental genomes were separated Goicoechea, P. G. and Giraldez, R. 1994. Physical mapping of 5S rDNA reveals a new locus on 3R and unexpected com- in two domains without intermix during the mitotic divi- plexity in a rye translocation used in chromosome mapping. sion, and there was not any detectable somatic aberration Chromosoma (Berl.) 103: 331-337. in the hybrids. Thus, FISH and GISH in this study were Anamthawat-Jónsson, K. and Reader, S. M. 1995. Pre-annealing of total genomic DNA probes for simultaneous genomic in situ considered key markers in identification of parental hybridization. Genome 38: 814-816. chromosomes. The combination of genomic and rDNA Anamthawat-Jónsson, K., Schwarzacher, T., Leitch, A. R., Bennett, FISH, would be a simple and effective way to characterize M. D. and Heslop-Harrison, J. S. 1990. Discrimination between closely related Triticeae species using genomic DNA as a diverse genome collections of germplasm materials and probe. Theor. Appl. Genet. 79: 721-728. breeding lines correctly. It is undoubtedly important to Bennett, S. T., Kenton, A. Y. and Bennett, M. D. 1992. Genomic in understand how the different genomes interact to the situ hybridization reveals the allopolyploid nature of Milium montanum (Gramineae). Chromosoma (Berl.) 101: 420-424. benefit of the plants themselves, if they are to be domes- Bremer, K. and Humphries, C. J. 1993. The generic monograph of ticated further. the Asteraceae-Anthemideae. Bull. Nat. Hist. Mus. London 10 ABD EL-TWAB AND KONDO FISH AND GISH TO CHRYSANTHEMUM JAPONICUM X NIPPONANTHEMUM NIPPONICUM 11

(Bot.) 23: 71-177. hybridization to the chromosome complement of the interge- Chen, Q., Conner, R. L. and Laroche, A. 1995. Identification of the neric hybrid between Leucanthemella linearis (Matsum.) parental chromosomes of the wheat-alien amphidiploid Tzuvelev and Nipponanthemum nipponicum (Franch. et Agrotana by genomic in situ hybridization. Genome 38: Maxim.) Kitamura. Chrom. Sci. 2: 91-93. 1163-1169. Ørgaard, M. and Heslop-Harrison, J. S. 1994. Investigations of Harrison, G. E. and Heslop-Harrison, J. S. 1995. Centromeric genome relationships between Leymus, Psathyrostachys and repetitive DNA sequences in the genus Brassica. Theor. Appl. Hordium inferred by genomic DNA: DNA in situ hybridiza- Genet. 90: 157-165. tion. Ann. Bot. 73: 195-203. Heslop-Harrison, J. S., Schwarzacher, T., Anamthawat-Jónsson, Parokonny, A. S., Kenton, A., Gleba, Y. Y. and Bennett, M. D. K., Leich, A. R., Shi, M. and Leich, I. J. 1991. In situ hybrid- 1994. The fate of recombinant chromosomes and genome ization with automated chromosome denaturation. Technique interaction in Nicotiana asymmetric somatic hybrids and their 3: 109-116. sexual progeny. Theor. Appl. Genet. 89: 488-497. Honda, Y., Abd El-Twab, M. H., Ogura, H., Kondo, K., Tanaka, R. Schmidt, T., Jung, C., Heslop-Harrison, J. S. and Kleine, M. 1997. and Shidahara, T. 1997. Counting sat-chromosome numbers Detection of alien chromatin conferring resistance to the beet and species characterization in wild species of Chrysanthemum cyst nematode (Heterodera schachii Schm.) in cultivated beet sensu lato by fluorescence in situ hybridization using pTa71 (Beta vulgaris L.) using in situ hybridization. Chrom. Res. 5: probe. Chrom. Sci., 1: 77-81. 186-193. Inafuku, K., Nabeyama, M., Kikuma, Y.,Saitoh, J., Kubota, S. and Schwarzacher, T., Leitch, A.R., Bennett, M. D. and Heslop- Kohno, S. 2000. Chromosomal location and nucleotide Harrison, J. S. 1989. In situ localization of parental genomes sequence of 5S ribosomal DNA of two cyprinid species in a wild hybrid. Ann. Bot. 64: 315-324. (Osteichthyes, Pisces). Chrom. Res. 8: 193-199. Shih, C. and Fu, G. 1983. Angiospermae, Dicotyledoneae, Khaung, K. K., Kondo, K., Tanaka, R. 1997. Physical mapping of Compositae (3) Anthemideae, Angiospermae, Dicotyledoneae rDNA by fluorescent in situ hybridization using pTa71 probe 76 (1), Science Press, Peking, pp. 149. in three tetraploid species of Dendranthema. Chrom. Sci. 1: Takahashi, C., Leitch, I. J., Ryan, A., Bennett, M. D., Brandham, 25-30. P. E. 1997. The use of genomic in situ hybridization (GISH) Kondo, K., Abd El-Twab, M. H. and Tanaka, R. 1999. Fluorescence to show transmission of recombinant chromosomes by a in situ hybridization identifies reciprocal translocation of partially fertile bigeneric hybrid, Gasteria lutzii X Aloe somatic chromosomes and origin of extra chromosome by an aristata (Aloaceae), to its progeny. Chromosoma (Berl.) 105: artificial, intergeneric hybrid betweenDendranthema japonica 342-348. X Tanacetum vulgare. Chrom. Sci. 3: 15-19. Tanaka, R. 1966. DNA replication in Chrysanthemum lineare, Ch.

Kondo, K., Honda, Y. and Tanaka, R. 1996. Chromosome marking nipponicum and their F1 hybrid. Bot. Mag. Tokyo, 79: 447-456. in Dendranthema japonica var. wakasaense and its closely Tanaka, R.1969. A comparative study on the distribution of related species by fluorescence in situ hybridization using labelled DNA in the chromosomes of Chrysanthemum lineare rDNA probe. La Kromosomo II-81: 2785-2791. and Ch. nipponicum. Cytologia 34: 353-510. Leitch, A. R., Schwarzacher, T., Mosgöler, W., Bennett, M. D. and Tanaka, R. and Shimotomai, N. 1961. Karyotypes in four diploid Heslop-Harrison, J. S. 1991. Parental genomes are separated species of Chrysanthemum. Cytologia 26: 309-319. throughout the cell cycle in a plant hybrid. Chromosoma Taniguchi, K., Tanaka, R., Yonezawa, Y. and Komatsu, H. 1975. (Berl.) 101: 206-213. Types of banding patterns of plant chromosomes by modified Mukai, Y., Friebe, B., Hatchett, J. H., Yamamoto, M. and Gill, B. S. BSG method. La Kromosomo 100: 3123-3153 (in Japanese). 1993. Molecular cytogenetic analysis of radiation-induced Xu, J. and Earle. D. 1996. High resolution physical mapping of wheat-rye terminal recombinant chromosomes and genome 45S (5.8, 18S and 25S) rDNA gene loci in the tomato genome interaction in Nicotiana asymmetric somatic hybrids and their using a combination of karytyping and FISH of pachytene sexual progeny. Theor.Appl.Genet. 89: 488-497. chromosomes. Chromosoma (Berl.) 104: 545-550. Ogura, H. and Kondo, K. 1998. Application of genomic in situ