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

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 A 3 (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. In old taxonomic system of seed plants (Chamberlain 1935; Eames 1952) Ephedraceae, Gnetaceae and Welwitschieae were combined into Gnetopsida and extensive studies have conducted on the basis of morphological observations of extant plants and fossils (Meyen 1984; Crane 1986; Doyle and Donoghue 1986; Loconte and Stevenson 1990). Recent molecular phylogenetic studies also revealed that three monotypic families or orders were put into same clade separating other gymnosperms, and support the previous taxonomic treatments (Hasebe et al. 1992a, b; Huang and Price 2003; Rydin et al. 2004). In cytological studies of Gnetopsida, Ephedra (n=7), Gnetum (n=11) and Welwitschia (n=21), their karyotypes were quite different in basic number, chromosome morphology and genome size (Khoshoo and Ahuja 1963; Chouhdry 1984; Hizume et al. 1993; Leitch et al. 2001). Their chromosomes were studied only by conventional staining and modern techniques were not applied at all. Ephedra is a large genus composing about 40-60 species and widely distributes in Eurasia, North Africa, and North and South America (Kubitzki 1990; Price 1996). Several Ephedra species are used as a traditional medical herb and contain several important chemicals effective for certain symptoms (Abourashed et al. 2003). Therefore, Ephedra species are investigated in various aspects such as pharmacology, anatomy, embryology, taxonomy and cytology. Molecular phylogenetic analysis using rbcL, ITS1, and rps4-tmS sequences was applied to Ephedra species (Huang and Price 2003; Ickert-Bond and Wojciechowski 2004; Rydin et al. 2004) and the phylogenetic trees suggest correlation between molecular phylogenetic group and geographic distribution, but additional data of other sequences in more species is needed for understand of reliable phylogeny in Ephedra. The chromosome number and karyotype of Ephedra were reported in 25 species and 5 varieties (Table 1). Previous studies revealed that Ephedra species were diploid (2n=14) and tetraploid (2n=28) (Chouhdry 1984) and basic chromosome number was x=7. The karyotype by a conventional staining was very similar among species and a haploid karyotype composing of five long metacentric chromosomes and two short subetelocentric chromosomes is accepted for Ephedra. In several species satellites were frequently observed at the terminal region of long metacentric chromosome. The satellites were varied in number and location among species or reports (Table 1, Hunziker 1955a, b; Mehra and Khitha 1981). The satellite seems a good cytological marker for chromosome analysis in Ephedra, but their appearance is somewhat uncertain depending on size of satellite and chromosome condensation. The modern technique for chromosome analysis is not applied to Ephedra species until now. In other groups of gymnosperm the fluorescent banding technique using the fluorochrome specifically binding certain DNA bases showed clear fluorescent banding pattern and the banding pattern varied among species (Hizume et al. 1983). In the report one Ephedra species, E. americana var. andina was investigated on its chromosomes especially to the satellite by the fluorescent banding technique in order to determine the fluorescent banding is effective for chromosome analysis of Ephedra or not. MATERIALS AND METHODS Seeds of Ephedra amaricana var. andina (Poepp. ex C. A. Mey.) Stapf. were collected and supplied from The University Botanic Garden, Cambridge, England. The seeds were germinated on a wet filter paper in Petri dishes at 25℃. Primary roots were taken and treated with 0.05% colchicine at 20 ℃ for 8 h. The roots were fixed in ethanol-acetic acid mixture (3:1) and stored in a freezer. The fixed root tip was macerated in 45% acetic acid at 60℃ for 10 min and dissected its meristematic tissue on a glass slide, and the tissue was squashed in 45% acetic acid under a cover glass. After frozen on dry ice, the cover glass was removed from the glass slide and the preparation was Chromosome Botany (2016) 11(2): 27-30