Fluorescent Band Pattern of Chromosomes in Ephedra Americana Var

Fluorescent band pattern of chromosomes in Ephedra americana var. andina, Ephedraceae

Masahiro Hizume1 and Kazuo Tominaga

Faculty of Education, Ehime University, Matsuyama 790-8577, Japan

1Author for correspondence: ([email protected]) Received June 15, 2016; accepted July 7, 2016

ABSTRACT: Somatic chromosome number of Ephedra americana var. andina was 2n = 14 and its karyotype was composed of ten long metacentric chromosomes and four short subtelocentric chromosomes. Chromomycin A3 (CMA) bands appeared on small satellites of six metacentric chromosomes indicating CG-rich nucleolar organizer or rRNA locus. Satellites were DAPI-negative and weak DAPI-dots were detected in the interstitial regions of four metacentric chromosomes.

KEYWORDS: Chromomycin A3, Chromosome, DAPI, Ephedra americana, Fluorescent banding, Karyotype, Satellite

In old taxonomic system of seed plants (Chamberlain 1935; and basic chromosome number was x=7. The karyotype by Eames 1952) Ephedraceae, Gnetaceae and Welwitschieae a conventional staining was very similar among species and were combined into Gnetopsida and extensive studies have a haploid karyotype composing of five long metacentric conducted on the basis of morphological observations of chromosomes and two short subetelocentric chromosomes extant plants and fossils (Meyen 1984; Crane 1986; Doyle is accepted for Ephedra. In several species satellites were and Donoghue 1986; Loconte and Stevenson 1990). Recent frequently observed at the terminal region of long molecular phylogenetic studies also revealed that three metacentric chromosome. The satellites were varied in monotypic families or orders were put into same clade number and location among species or reports (Table 1, separating other gymnosperms, and support the previous Hunziker 1955a, b; Mehra and Khitha 1981). The satellite taxonomic treatments (Hasebe et al. 1992a, b; Huang and seems a good cytological marker for chromosome analysis Price 2003; Rydin et al. 2004). In cytological studies of in Ephedra, but their appearance is somewhat uncertain Gnetopsida, Ephedra (n=7), Gnetum (n=11) and depending on size of satellite and chromosome Welwitschia (n=21), their karyotypes were quite different condensation. The modern technique for chromosome in basic number, chromosome morphology and genome analysis is not applied to Ephedra species until now. In size (Khoshoo and Ahuja 1963; Chouhdry 1984; Hizume et other groups of gymnosperm the fluorescent banding al. 1993; Leitch et al. 2001). Their chromosomes were technique using the fluorochrome specifically binding studied only by conventional staining and modern certain DNA bases showed clear fluorescent banding techniques were not applied at all. pattern and the banding pattern varied among species Ephedra is a large genus composing about 40-60 (Hizume et al. 1983). In the report one Ephedra species, E. species and widely distributes in Eurasia, North Africa, and americana var. andina was investigated on its North and South America (Kubitzki 1990; Price 1996). chromosomes especially to the satellite by the fluorescent Several Ephedra species are used as a traditional medical banding technique in order to determine the fluorescent herb and contain several important chemicals effective for banding is effective for chromosome analysis of Ephedra certain symptoms (Abourashed et al. 2003). Therefore, or not. Ephedra species are investigated in various aspects such as pharmacology, anatomy, embryology, taxonomy and MATERIALS AND METHODS cytology. Molecular phylogenetic analysis using rbcL, Seeds of Ephedra amaricana var. andina (Poepp. ex C. A. ITS1, and rps4-tmS sequences was applied to Ephedra Mey.) Stapf. were collected and supplied from The species (Huang and Price 2003; Ickert-Bond and University Botanic Garden, Cambridge, England. The Wojciechowski 2004; Rydin et al. 2004) and the seeds were germinated on a wet filter paper in Petri dishes phylogenetic trees suggest correlation between molecular at 25℃. Primary roots were taken and treated with 0.05% phylogenetic group and geographic distribution, but colchicine at 20 ℃ for 8 h. The roots were fixed in additional data of other sequences in more species is ethanol-acetic acid mixture (3:1) and stored in a freezer. needed for understand of reliable phylogeny in Ephedra. The fixed root tip was macerated in 45% acetic acid at 60℃ The chromosome number and karyotype of Ephedra for 10 min and dissected its meristematic tissue on a glass were reported in 25 species and 5 varieties (Table 1). slide, and the tissue was squashed in 45% acetic acid under Previous studies revealed that Ephedra species were a cover glass. After frozen on dry ice, the cover glass was diploid (2n=14) and tetraploid (2n=28) (Chouhdry 1984) removed from the glass slide and the preparation was 28 HIZUME AND TOMINAGA

Table 1 Karyotype and chromosomal chracteristics in Ephedra.

Chromosome No. of sat*, sec**, Species of Ephedra Karyotyoe Reference number nucleoli E. altissima 2n=28 20m+8st 1 sat, 4 nucleoli Chouhdry (1984) E. altissima var. algerica n=14 10m+4st 3 sec, 4 sat: 1sat Mehra (1946) E. andina n=14 10m+4st 3 sat Hunziker (1955a) E. americana n=7 5m+2st 3 sec 1+2 Hunziker (1955a) E. americana 2n=14 10m+4st - Nakata and Ogimuma (1989) E. americana 2n=28 20m+8st - Chouhdry (1984) E. amaricana var. andina 2n=28 20m+8st - Chouhdry (1984) E. amaricana var. andina 2n=14 10m+4st 6 sat (CMA-bands) present study E. breana n=7 5m+2st 2 sec Hunziker (1955a) E. chilensis 2n=28 20m+8st 2 sat Mehra (1946) E. chilensis n=14 10m+4st 2 sat: 1 sat Chouhdry (1984) E. ciliata 2n=14 10m+4st - Chouhdry (1984) E. distachya 2n=28 20m+8st - Chouhdry (1984) E. foliata n=7, 2n=14 1sm+ 4m+2st ♀6 sat，♂ 5 sat Mehra and Khitha (1981) E. foliata 2n=14 10m+4st 1 sat Chouhdry (1984) E. foliata var. cilata 2n=14 5m+2st - Sarma (1968, 1969) E. foliata var. cilata n=7 5m+2st 1 sec, 1sec Mehra (1946) E. fragilis 2n=14,28 10m+4st - Chouhdry (1984) E. fragilis var. campylopoda n=14 10m+4st 1 sec, 1 sat Mehra (1946) E. fragilis var. campylopoda 2n=14 10m+4st 1 sat, 6 nucleoli Chouhdry (1984) E. frustillata n=7 5m+2st 1 sec Hunziker (1955a) E. geradiana 2n=28 20m+8st - Mehra (1946) E. geradiana n=7 5m+2st 2 sat; 1sat Chouhdry (1984) E. geradiana var. saxatilis 2n=28 20m+8st - Chouhdry (1984) E. internedia var. tibetica 2n=14 10m+4st 2 sat Chouhdry and Tanaka (1981) E. likiangensis n=14 10m+4st 2 sat Mehra (1946) E. major 2n=14+2B 10m+4st - Chouhdry (1984) E. minuta 2n=14 10m+4st - Chouhdry (1984) E. multiflora n=7 5m+2st 3 sec 1+2 Hunziker (1955) E. nebrodensis 2n=14 10m+4st - Chouhdry (1984) E. nevadensis 2n=28 20m+8st - Chouhdry (1984) E. ochreata n=7 5m+2st 3 sec Hunziker (1955b) E. rupestris n=7 5m+2st 2 sec Hunziker (1955b) E. sinica 2n=28 20m+8st - Mehra (1946) E. sinica 2n=28 20m+8st 2 sec Chouhdry (1984) E. saxatilis n=14 10m+4st 4 sat: 1 sat Mehra (1946) E. triandra n=7 5m+2st 3 sec Hunziker (1955a) E. tweediana n=7 5m+2st 1 sec Hunziker (1955a) E. tweediana 2n=14 10m+4st - Chouhdry (1984) E. viridis 2n=28 20m+8st - Hunziker (1955b), Chouhdry (1984) *: sattelite, **: secondary constriction.

FLUORESCENT CHROMOSOME BANDING IN EPHEDRA AMERICANA 29

Fig. 1. Fluorescent banded chromosomes in EphedraFLUORESCENT americana CHROMOSOME. A: CMA-banded, BANDING B: DAPI IN EPHEDRA-banded. Bar=10μm.AMERICANA 00

subtelocentric chromosomes with about 10μm length (Figs. 1 and 2). Lengths of metacentric chromosomes and subetelocentric chromosomes were discontinuous and then the karyotype was considered to bimodal. After CMA-banding, six small CMA-bands appeared on satellite at the end of one arm of six metacentric chromosomes (Figs. 1A and 2A). Clear DAPI-band was not observed but four weak DAPI-dots appeared at the interstitial region of four metacentric chromosomes (Figs. 1B and 2B). The region of satellite was observed in dark also supposing that the satellite region contains GC-rich DNA sequence. The karyotype of E. americana is coincident with a consensus karyotype of Ephedra species (Table 1) and confirms a constancy in karyotype of Ephedra. Satellite or secondary constriction appeared at the terminal region of metacentric chromosomes in several species and its number varied among species (Table 1). The small satellite was observed as CMA-band in the species but any interstitial or proximal secondary constriction was not observed. Detailed chromosome studies of Hunziker (1955a, b) reported a variation in number of satellites in a chromosome complement among species and a presence of variation of satellites seems reliable in some Ephedra Fig. 2. Schematic karyotype of E. americana banded with species. The number and location of satellites seems an CMA-staining (A) and DAPI-staining (B). important chromosomal character in karyotype analysis of Ephedra having conservative karyotype. Most other reports of karyotype with no satellite might miss to observe the air-dried overnight. Fluorescent banding technique using satellite causing too small or fusing to chromosome arm by GC-specific fluorochrome, chromomycin A3 (CMA) and chromosome condensation. The CMA-staining can AT-specific fluorochrome, 4',6-diamidino-2-phenylindole visualize clearly a satellite as bright CMA-band in any (DAPI) was followed in Hizume et al. (1983) . conditions. Feature of CMA-bands on the satellite indicated that whole satellite was GC-rich. The RESULTS AND DISCUSSION CMA-banding is the most simple and easy technique for Ephedra americana var. andina had somatic chromosomes detection of GC-rich satellite or secondary constriction. In of 2n=14 and was diploid condition. The chromosome plant chromosomes the secondary constriction or nucleolar complement was composed of ten long metacentric organizing region is CMA-positive and contains tandem chromosomes with 15-12 μm length and four short repeats of rRNA gene (Hizume 1992). When the 30 HIZUME AND TOMINAGA

CMA-banding or fluorescence in situ hybridization (FISH) in P. nigra by fluorescent banding method. Bot. Mag. Tokyo using rDNA probe are applied to chromosomes of many 96: 273-276. Ephedra species, a deposit of information on satellite will Hizume, M., Gu, Z., Yue, Z. and Kondo, K., 1993. be expected to reveal inter- and/or intra-specific variation Karyomorphological study of Gnetum montanum collected in and relationships among species. Comparing of the Yunnan, China. Chromosome Inform. Serv. 54: 23–25. grouping of species on the basis of satellite with molecular Huang, J. and Price, R. A. 2003. Estimation of the age of extant phylogenetic trees (Huang and Price 2003; Ickert-Bond and Ephedra using chloroplast rbcL sequence data. Mol. Biol. Wojciechowski 2004; Rydin et al. 2004) will revel the Evol. 20: 435–440. phylogenetical significance of satellites in Ephedra. Hunziker, J. H. 1955a. 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