Fluorescent Banding Pattern in Chromosomes of Tsuga Forrestii and T

Fluorescent banding pattern in chromosomes of Tsuga forrestii and T. sieboldii, Pinaceae

Masahiro Hizume

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

Author for correspondence: [email protected] Received January 21, 2015; accepted April 4, 2015

ABSTRACT: In two East Asian species of Tsuga, T. forrestii and T. sieboldii their somatic chromosomes were investigated by fluorescent banding technique using DNA-base specifically binding fluorochromes of chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI). Their chromosome numbers were commonly 2n=24. Their karyotypes were very similar to each other and consisted of eight pairs of long metacentric chromosomes and four pairs of short chromosomes. The two pairs of the short chromosomes were submetacentric, one pair was metacentric chromosomes, and the shortest chromosome pair was submeta-subtelocentric. After CMA-staining six CMA-bands appeared at interstitial region of each one arm of six long metacentric chromosomes. Positive DAPI-band was not observed and DAPI-negative regions appeared coincident with all CMA-bands. Centromeric regions of DAPI-stained chromosomes were darker than chromosome arms. Karyotypes and their fluorescent banding patterns of the two Tsuga species were very similar to each other. The fluorescent banded karyotypes of the Tsuga species are compared with those of other Pinaceae genera reported.

KEYWORDS: Chromosome, CMA, DAPI, Fluorescent banding, Karyotype, Pinaceae, Tsuga

Tsuga is one of the 11 genera of the Pinaceae. The chromosomes of other Pinaceae genera up to the present Pinaceae consists of two to four subfamilies and the most was in Larix (Hizume et al. 1988; Hizume and Tanaka of taxonomic treatments pleased Tsuga into the subfamily 1990; Hizume et al. 1993a, 1994, 1998), Picea (Hizume et Abietoideae. Tsuga consists of nine species, one al. 1989b, 1991; Hizume and Kuzukawa 1995), Abies subspecies and three varieties. They grow in Northern (Shibata et al. 2004; Puizina et al. 2008), Keteleeria hemisphere and restricted to regions with oceanic to (Hizume et al. 1993b), Pseudotsuga (Hizume and subcontinental climate in North America and East Asia, Akiyama 1992; Hizume and Kondo 1992), Cedrus where precipitation is available during their growing (Dagher-Kharrat et al. 2001) and Pseudolarix (Hizume season (Farjon 1990) and the species are divided into two 2015). The comparative study on the fluorescent banding groups (Melchior and Werdermann 1954). Tsuga patterns of these genera is expected to supply information longibracteata was separated and established into revealing intraspecific, interspecific and intergeneric monotypic genus of Nothotsuga (Hu 1951). relationships in the Pinaceae. Fluorescent banding Since Sax and Sax (1933) reported the chromosome technique has not yet applied to chromosomes of the number and the simple idiogram of T. caroliniana, many species of Cathaya, Nothotsuga and Tsuga. more karyotypes in eight species of Tsuga such as T. The present report aims to reveal fluorescent banded chinensis var. formosana (Kuo et al. 1972), T. canadensis, karyotype of Tsuga species. The fluorescent banded T. caroliniana, T. chinensis, T. deiversifolia, T. karyotypes of Tsuga species were compared and discussed heterophylla and T. sieboldii (Hizume 1988), T. chinensis with those of other genera of the Pinaceae. ver. tchekiangensis (Li 1988), T. longibracteata (Li 1991) and T. mertensiana (Li et al. 2000) have been reported. MATERIALS AND METHODS The karyotypes of Tsuga species are basically similar to Seeds of Tsuga forrestii Downie and T. sieboldii Carr. each other in having four pairs of shorter chromosomes were collected in natural forests in Yunshaping, Lijiang, and some differences in number of secondary constrictions Yunnan Province, the People’s Republic of China and in and their location in their karyotype. Odamiyama, Uchiko-cho, Kita-Gun, Ehime Prefecture, The appearance of secondary construction tends Japan, respectively. Their seeds were sown and germinated frequently to be different among the reports depending on on wet filter paper in petridishes at 20℃ for 7-10 days. observation condition. Conventional karyotype of Tsuga The primary root-tips were collected and treated in 0.05% was similar to those of Picea, Cedrus, Abies and colchicine for 8 h. Then, those root-tips were fixed in Keteleeria. mixture of ethanol, acetic acid and chloroform (volume In the Pinaceae, especially the genus Pinus, the ratio=2:1:1) and stored in the fixative in a freezer. Fixed fluorescent CMA- and DAPI-banding patterns supply very root-tips were washed in water after removing fixative useful information for chromosome identification and with 70% ethanol. Then, root-tips were transferred in 45% comparative karyotype analysis among the species acetic acid for 5 min, and treated with 45% acetic acid at (Hizume et al. 1983, 1989a, 1990; Doudrick et al. 1995). 60℃ for 10 min. Then, meristematic tissue was dissected Application of the fluorescent banding technique on from the root-tip and put on a glass slides. An aliquot of 96 HIZUME

Fig. 1. Fluorescent banded chromosomes of Tsuga forrestii. A: CMA staining, B: DAPI staining. Bar=10μm.

45% acetic acid was dropped on the tissue and cover slip glycerin, rinsed briefly with distilled water and then was put on it. Meristematic cells were squashed to spread. air-dried. The preparation was put in the buffer without The preparation was put on a dry ice for a few minutes and MgSO4 for 10 min, treated with 0.1 mg/mL actinomycin D then, ripped off cover slip. The preparation was air-dried for 10 min and the wash again with the buffer for 10 min. overnight. Sequential fluorescent banding technique with The preparation was stained with 0.1 μg/mL DAPI for 5 CMA and DAPI were described early by Kondo and min then washed with the buffer for 5 min. After mounting Hizume (1982). The dried preparation immersed into with the buffer-glycerin (v/v=1:1) mixture the same McIlvaine buffer pH 7.0 for 30 min. The slide glass was chromosomes observed CMA fluorescence were observed treated with 0.1 mg/mL distamycin A for 10 min, then under the fluorescence microscope with UV filter block. washed with the buffer containing 5 mM MgSO4 for 10 Fluorescence images of same chromosomes stained CMA min and stained with 0.1 mg/mL CMA in the buffer for 10 and DAPI were taken on the film (TMAX, Kodak) and min. After washing with the buffer for 10 min the developed with double diluted D-76. preparation was mounted with non-fluorescence glycerin and stored in a refrigerator at 4℃ overnight or more. RESULTS AND DISCUSSION After storage the CMA-stained preparation was observed Tsuga forrestii and T. sieboldii had the somatic under a fluorescence microscope equipped with B filter chromosome number of 2n=24 supporting previous count block. Then preparation was dipped in distilled water until in T. sieboldii (Hizume 1988) and same chromosome drop off the cover glass. Then the preparations were number of other species of Tsuga reported. Karyotypes of treated with acetic-alcohol (3:1) to remove CMA and these two species were consisted of eight pairs of long FLUORESCENT BANDING PATTERN OF CHROMOSOMES IN TSUGA 97

Fig. 2. Fluorescent banded chromosomes of Tsuga sieboldii. A: CMA staining. B: DAPI staining. Bar=10μm

metacentric chromosomes and four somewhat short pairs the other species did not show any secondary constriction, of chromosomes. Two short pairs were metacentric to and then, exact number or location of secondary submetacentric chromosomes, the 11th pair of chromo- constrictions were unclear caused by different conditions somes were metacentric and the shortest pair of in conventional chromosome preparation. The karyotype chromosomes were submeta-subtelocentric. These of T. longibracteata had species-specific, elongated karyotypes were also similar to the description of centromere at the pair of the shortest chromosomes (Li conventional karyotypes reported in eight species of Tsuga 1991). Tsuga longibracteata described by Cheng (1932) (Kuo et al. 1972; Hizume 1988; Li 1988, 1991; Li et al. was proposed for a new genus, Nothotsuga longibracteata 2000). Tsuga karyotypes in the previous reports showed by Hu (1951) and Page (1988). This might indicate some species had a few secondary constrictions at the generic differentiation in karyotype level between Tsuga interstitial zone of the long metacentric chromosomes and and Nothotsuga. In order to confirm this speculation, 98 HIZUME analyses on chromosomes of these species by fluorescent chromosomes. In this report on the chromosomes of two banding and in situ hybridization using rDNA or other Tsuga species, the fluorescent banding karyotypes had six probes are desired to reveal the phylogenetic relationships. interstitial CMA-bands and no centromeric DAPI-band If fluorescent banding technique with DNA base (Figs. 1 and 2). The banding pattern of Tsuga species specific fluorochromes was applied to chromosomes of the suggested that Tsuga was not considered to be relative of two Tsuga species, bright CMA-bands were observed at Pseudolarix in location and number of CMA-bands and no the interstitial region of six long metacentric chromosomes appearance of any DAPI-band. In point of view of in both species (Figs. 1A and 2A). Bright DAPI-band was centromeric DAPI-bands Cedrus would probable not observed at all and the region of CMA-band showed candidate of close relative of Pseudolarix, but CMA-band DAPI-negative (Figs. 1B and 2B). Chromosome arms pattern was different distinctly from that of Pseudolarix without CMA-band region displayed homogeneous and Cedrus. In the closely related genera Larix and fluorescence of CMA and DAPI. The centromeric regions Pseudotsuga had nearly the same bimodal karyotype showed homogeneous CMA fluorescence and several (Hizume et al. 1988; Hizume and Akiyama 1992; Hizume chromosomes showed dull DAPI fluorescence. The and Kondo 1992) and putting into the same clade in fluorescent feature of centromeric regions might suggest molecular phylogenetic trees (Chaw et al. 1997; Gernandt that the centromeric region of chromosomes of T. forrestii et al. 2008), in Larix AT-rich repetitive sequence (Hizume and T. sieboldii have less affinity to DAPI than et al. 2002) might amplify very quickly and form proximal chromosome arms or contained specific and somewhat DAPI-bands of most of the chromosomes since more GC-rich DNA sequence than that of chromosome differentiation of these genera have started chromosome arms. differentiation, while on the other hand, Pseudotsuga has The fluorescent banding pattern of two Tsuga species not occurred this phenomenon at all. CMA-bands was compared with those of the genera of the Pinaceae containing 45S rDNA repeats frequently appear or reported. The banding pattern of Tsuga chromosomes was disappear by deletion, transposition or translocation in different from those of Pinus with many interstitial plant chromosomes. These changes of CMA- and/or DAPI-bands (Hizume et al. 1983, 1989b, 1990; Doudrick DAPI-bands should be recognized to occur in et al. 1995), Larix and Cedrus with many proximal chromosomes of species, genera in the Pinaceae. The DAPI-bands (Larix: Hizume et al. 1988, 1993b, 1998; fluorescent banding patterns with CMA and DAPI of all Hizume and Tanaka 1990; Cedrus: Dagher-Kharrat et al. genera of the subfamily Abietoideae excepting Nothotsuga 2001), Picea with a pair of chromosomes with two seem difficult to indicate the genus that is the most closely CMA-bands located very close to each other (Hizume and related with Pseudolarix. To reveal karyotype differenti- Kuzukawa 1995; Hizume et al. 1989b, 1991), Larix and ation in the Pinaceae, in addition to fluorescent banding Pseudotsuga which have weak CMA-band composed of karyotype, many more analyses of the repetitive DNA 5S rDNA at the terminal interstitial region of one long pair located on CMA-band, DAPI-band and C-band in their of the metacentric chromosome (Hizume et al. 1996; sequence and distribution on chromosomes of species Amarasinghe et al. 1998), and Pseudolarix with many and/or genera are expected. Recently, genome projects proximal DAPI-bands and CMA-bands (Hizume 2015). progress in several conifers species of Pinus, Picea, Fluorescent banding pattern of Tsuga seemed similar to Pseudotsuga and Larix and are constructing saturated those of Keteleeria (Hizume et al. 1993b) and Abies (Roth genome maps. 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