Chromosome Differentiation and Genome Organization in Carnivorous Plant Family Droseraceae

Chromosome Botany (2011) 6: 111-119 © Copyright 2011 by the International Society of Chromosome Botany Chromosome differentiation and genome organization in carnivorous plant family Droseraceae Junichi Shirakawa1, Yoshikazu Hoshi2,4 and Katsuhiko Kondo3 1Graduate School of Bioscience, Tokai University, Kawayou, Minamiaso-mura, Aso-gun, Kumamoto 869-1404, Japan; 2Department of Plant Science, School of Agriculture, Tokai University, Kawayou, Minamiaso-mura, Aso-gun, Kumamoto 869-1404, Japan; 3Laboratory of Plant Genetics and Breeding Science, Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, Funako 1737, Atsugi City, Kanagawa 243-0034, Japan 4Author for correspondence: ([email protected]) Received October 5, 2011; accepted November 28, 2011 ABSTRACT: A carnivorous plant lineage including the Droseraceae and its closely related family Drosophyllaceae shows wide range of chromosome size or genome size, whereas all other carnivorous families possess rather small in the genomes sizes. This study gives an overview of the genome size diversity in carnivorous plant families, and evolutional trend of chromosome differentiation especially in the Doroseraceae. KEYWORDS: Chromosome size, Doroseraceae, FISH, Genome The diversities of the chromosome and genome sizes in fl ow cytometry and microscopic chromosome data to our angiosperms have generated considerable interest over limited knowledge of genome size in carnivorous plants. the years (Bennett and Leitch 2005; Leitch et al. 2007; Concerning high genome diversity, special chromo- Leitch and Bennett 2007). Nevertheless, little work has somal feature called ‘diffuse centromeric type’ found in focused specifi cally on carnivorous plant families. There Drosera should be noted. The chromosomes of most are still a lack of C-value data and an uneven eukaryotes have ‘localized centromere’ which presents as karyomorphological knowledge in the carnivorous plant a primary constriction. Whereas, in some plant genera families. For example, the fi rst C-value record for such as Luzula (Juncaceae) (Castro et al. 1949) and carnivorous plant was originally given by Rothfels and Eleocharis (Cyperaceae) (Håkansson 1958), ‘non- Heimburger (1968), nearly 20 years after the fi rst plant localized centromere’ or ‘diffuse centromere’ are known. genome size estimation (Ogur et al. 1951). In contrast, the It does not show any constriction or localized centromere chromosome numbers of the plants, especially Drosera position on chromosome. In Drosera, distinct primary species, were known in the early 1900s (Rosenberg 1903, constriction, localized centromere or clear chromosomal 1904, 1909). Up to the present date, the Plant DNA gap between sister chromatid has not been observed in the C-values Database and the Index to Plant Chromosome past, supporting the diffuse centromere hypothesis (Kondo Number, which are most available database sites, have 1976; Kondo et al. 1976; Kondo and Lavarack 1984; contained only 7.2% and 22.2% of the informative data in Kondo and Segawa 1988). Since the diffused type all carnivorous plant species, respectively. The carnivorous possesses centromere function dispersed along the whole plant family Droseraceae historically includes four chromosome length, in theory all fragments of this type of genera, Aldrovanda, Dionaea, Drosera and Drosophyllum chromosomes are stably transmitted after cell division. (Diels 1906; Cronquist 1981), although some Due to existing intraspecifi c aneuploid cytotypes with morphological studies (Takahashi and Sohma 1982; several fragmented chromosomes, chromosome Juniper et al. 1989; Conran et al. 1997) and molecular differentiation event such as section Bryastrum and phylogenetic analyses (Albert et al. 1992; Williams et al. section Lasiocephala of Drosera is able to explain using 1994; Rivadavia et al. 2003) suggest that Drosophyllum diffuse centromeric hypothesis, but not section Drosera does not belong to the Droseraceae. Three genera are due to showing strict about rules of poliploidization. A monotypic plants, and only genus Drosera comprises well demonstration of the diffuse centromeric chromosome more than 100 species distributed mainly in the Southern has done to test the ability of regular cell division and Hemisphere, with some in the Northern Hemisphere accurate segregation of each chromosome fragment, (Juniper et al. 1989; Lowrie 1998). First, thus, C-values induced by Gamma- or X-ray irradiations in Luzura and chromosome sizes for 48 and 134 out of 582 species (Castro et al. 1949). In Drosera, Gamma-radiated plants in 12 families were used to provide an overview of the of Austrarian anuploidal species have also shown evidence genome diversity and chromosome differentiation in these that typical segregations of fragments and minute highly specialized plants, especially in the Doroseraceae. chromosomes at mitotic anaphase stages are observed in The present work also adds new C-value data obtained by mitotic cell division (Sheikh et al. 1995; Furuita and 112 SHIRAKAWA ET AL. Kondo 1999), confi rming the validity of the hypothesis buffer (pH 7.0) containing 5 mM MgSO4 and 50% proposed by earlier workers (Kondo et al. 1976; Kondo glycerol. These chromosome preparations stained with and Lavarack 1984). However, there is no experiment CMA were observed with a BV fi lter. Then, the slides using polyploid materials in Drosera species. To discuss were used for sequential 4’,6-diamidino-2-phenylindole the chromosome differentiation, this work also gives the (DAPI) (Nacalai Tesque, Inc., Kyoto, Japan) staining. The data of Gamma-irradiated chromosome behaviors both of slides were destained in 45% acetic acid for 30 min, D. petioralis as Austrarian aneuploid group and D. dehydrated in a series of ethanol, and air-dried for 30 min. rothundifolia, representative species in the Northern They were stained with 1 µg/ml DAPI in McIlvaine’s Hemisphere, as polyploid group. buffer containing 50% glycerol. The chromosomes stained with DAPI were observed with a U fi lter. Chromosome MATERIALS AND METHODS sizes (lengths) on digitally-recorded chromosome images Plant materials Plant materials in most Drosera species were determined using ImageJ 1.33u (National Institutes used in our experimental study are cultivated in greenhouse of Health, MD, USA). and some strains are cultured on hormone-free 1/2 Murashige and Skoog basal medium (Murashige and Gamma radiation treatment Plants of Drosera petiolaris Skoog 1962) supplemented with 0.35% gellan gum and and D. rotundifolia in vitro were exposed to Gamma 3% sucrose for in vitro culture. radiation in the Facility of Faculty of Engineering, Hiroshima University. Doses were determined by time Flow cytometry Performance validation of the EPICS and distance from the source around a circular disc in the XL ADC fl ow cytometer was done using fl uorescent bead ‘Gamma shine’ (Co unit). Soon after exposed to Gamma mixtures provided by the manufacturer, and according to radiation, the fl asks were returned to the culture room of their instructions (Beckman Coulter). Analysis was based the Laboratory for continuous in vitro culture. on light-scatter and fl uorescence signals produced from 20 mW laser illumination at 488 nm. Signals corresponding RESULTS AND DISCUSSION to forward angle- and 90o-side scatter (FALS, SS) and Genome diversities of the Droseraceae and other fl uorescence were accumulated, the fl uorescence signals carnivorous plant families Table 1 shows all genome (pulse area measurements) being screened by the size information in carnivorous plants with our new data. following fi lter confi gurations: (a) FL-2: a 585/40 nm Even including our new experimental data, 1 C-values in band-pass fi lter, and (b) FL-3: a 670 nm longpass fi lter. Drosera are known only for nine species ranged from Threshold levels were empirically set (10,000 for FALS, 0.30 pg in D. capensis to 0.95 pg in D. intermedia (if the with a second threshold of 1,000 for FL-2) to eliminate related genera are included, in this case, max value is from detection the large amounts of irrelevant debris that Drosophyllum lusitanicum with 15 pg) (Table 1). All are found in plant homogenates. Templates for uni- and information of the chromosome numbers of 66 out of 142 bi-parametric frequency distributions were established, species in the Droseraceae, including Drosophyllum show and on identifi cation of the region corresponding to nuclei, that each carnivorous family posses rather small genome data was collected to a total count of 5,000-10,000 nuclei. size less than 1 pg, but not the Droseraceae. In case of The fl ow cytometer was routinely operated at the Slow including Drosophyllum, they are quite variable Flow Rate setting (14 lL sample/minute), and data angiosperm lineage with values ranging 50 fold from 1C, acquisition for a single sample typically occupied 3-5 and 20 fold from average chromosome size. Given this, it min. suggests that genome evolution in the Droseraceae and its related lineage is dynamic. Chromosome slide preparation After root tips were pretreated with 0.2 mM 8-hydroxyquinoline for 2 h at 18 Superimposing chromosome information and the genome oC, they were fi xed in 70% ethanol for 1 h on ice, washed size data onto the molecular phylogenic tree of the with distilled water for 60 min, and then macerated in an Droseraceae The intrageneric classifi cation of Drosera enzymatic mixture containing 4% Cellulase Onozuka RS had been problematic for a long time (Rivadavia et al. (Yakult Pharmaceutical Industry Co., Ltd., Tokyo,

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