© 2016 The Japan Mendel Society Cytologia 81(1): 61–67 Fluorescent Chromosome Banding Patterns in Six Species of Abies, Pinaceae Masahiro Hizume*, Yoko Yamasaki and Mayumi Kan Faculty of Education, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790–8577, Japan Received April 16, 2015; accepted November 12, 2015 Summary The six Abies species, A. laciocarpa, A. veitchii, A. sachalinensis, A. mariesii, A. faxoniana and A. georgei, were investigated for their chromosomes by the fluorescent banding method using fluorochromes chro- momycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI). All six species have 2n=24 chromosomes and a similar karyotype consisting of seven pairs of long metacentric chromosomes and five pairs of shorter submeta- and subtelocentric chromosomes, supporting previous studies. Eight clear CMA-bands appeared at the interstitial region of one arm of long metacentric chromosomes in all species, and in A. veitchii and A. sachalinensis, their number varied from six to eight among plants and/or populations. A weak CMA-band appeared at the interstitial region of one chromosome pair, while a weak CMA-band appeared at the proximal region in some species. In most species DAPI did not produce clear bands, and sites of clear CMA-bands were DAPI negative. Only A. mariesii showed many DAPI-bands at the interstitial and/or centromeric regions of several chromosomes. A few weak DAPI-bands appeared in some other species. The fluorescent banding and FISH patterns reported in Abies species were compared and discussed with taxonomic treatment and molecular phylogeny of Abies. Key words Abies, Chromosome, CMA, DAPI, Fluorescent banding, Karyotype. Abies is one of the genera in Pinaceae and includes karyotype was composed of seven long metacentric over 40 species distributed in mountains of cold temper- chromosome pairs and five short submeta- or subtelo- atures, such as at high altitudes and in boreal regions of centric pairs. The secondary constrictions were reported the Northern Hemisphere. Abies has 25 species in North- in many species, but the exact number and location were ern and Eastern Asia (including the Western Himalayas), different among reports, probably caused by difficulty 16 species in Central and North America and 7 species in detection of secondary constrictions by conventional in the Mediterranean area of Europe and Southwestern chromosome analysis. The detailed karyotype analysis Asia (Liu 1971, Farjon 2010). The genus Abies was by banding and FISH to clearly detect secondary con- divided into two subgenera and either 15 sections (Liu strictions and/or segmental differentiation is desired to 1971) or 10 sections (Farjon and Rushforth 1989). be applied to Abies species. Until now, fluorescent chro- All cytological studies on Abies species showed a mosome banding and/or FISH with rDNA probes was chromosome number of 2n=24 that is a common chro- applied to few Abies species (Roth et al. 1997, Shibata mosome number in Pinaceae. The karyotype of Abies et al. 2004, Besendorfer et al. 2005, Puizina et al. 2008). species were reported in about 15 species (Sax and Sax This study aimed to reveal fluorescent banding pattern 1933, Mehra and Khoshoo 1956, Mergen and Bubley in six species of Abies for comparison with taxonomic 1964, Kormutak 1985, Hizume 1988, Kvitko et al. treatment (Liu 1971, Farjon and Rushforth 1989) and 2011). The karyotype is stable among the species in molecular phylogeny (Isoda et al. 2000, Xiang et al. Abies as shown in Hizume (1988), and the constancy 2004, 2015). in karyotype among species is beneficial to compara- tive karyotype analysis of banding and signal pattern of Materials and methods fluorescence in situ hybridization (FISH). To compare karyotypes among species, chromosome identification in Seeds of A. laciocarpa (Hooker) Nutt. were supplied the karyotype of each species is important. Fortunately, from the Hortus Botanicus Academiae Scientiarum Lat- in Abies species, most chromosome pairs were identi- viensis, Latvia and those of A. sachalinensis F. Schmidt, fied by chromomycin A3 (CMA)-banding and FISH with A. veitchii Lindl. and A. mariesii Mast. from the seed 45S rDNA (Shibata et al. 2004, Puizina et al. 2008). bank of Forestry and Forest Products Research Institute, With some differences among reports, the consensus Tsukuba, Japan. The seeds of A. faxoniana Rehd. & Wils. and A. georgei Orr. were collected in Ruoergai, Si- * Corresponding author, e-mail: [email protected] chuan and Lijiang Ganheba, Yunnan, China, respective- DOI: 10.1508/cytologia.81.61 ly. The seeds were germinated on sterilized fine sand in 62 M. Hizume et al. Cytologia 81(1) a pot or wet filter paper in Petri dishes. After about 10 d, the primary roots growing straight were collected and treated with 0.05% colchicine for 10 h, fixed in a fixa- tive mixed with ethanol–acetic acid–chloroform (2 : 1 : 1) and then stored in a deep freezer until use. Fluorescent banding using CMA and 4′,6-diamidino-2-phenylindole (DAPI) was adapted to plant chromosomes by Sch- weizer (1976) and is effective for karyotype analysis in other various plant species (Hoshi et al. 2008, Urdampil- leta et al. 2008, Zaman and Alam 2009, Begum et al. 2010, Fawzia and Alam 2011, Hoshi et al. 2011, Shahla and Alam 2011, Shirakawa et al. 2012, Kuroki et al. 2013). The fluorescent banding procedure simplified and Fig. 1. Fluorescent-banded chromosomes in Abies laciocarpa. A: adapted for conifer chromosome analysis by Kondo and CMA-banding, B: DAPI-banding. Bar=5 µm. Hizume (1982) was used. The numbering and order of chromosomes in a chro- mosome complement of Abies were followed according of the long arm of the long metacentric chromosomes to Shibata et al. (2004). Chromosomes 1–7 were long (chromosome 2), four CMA-bands at the terminal in- metacentric, and chromosomes 8–12 were shorter and terstitial region of the long arm of the long metacentric submeta- and subtelocentric. Chromosomes 2, 4, 5, 6, chromosomes (chromosomes 4 and 5), and two addition- 7 have an interstitial 45S rDNA site or CMA-band on al CMA-bands at the terminally interstitial region of the their single arm, and these chromosomes were identified short arm of the long metacentric chromosomes (chro- by chromosome length and position of the FISH signal/ mosome 7) (Fig. 1A). Six long metacentric chromosomes CMA-band on the chromosome arm. Two short chromo- (chromosomes 1, 3, 7) and 10 submeta- or subtelocentric somes 8 and 10 were distinguished in length and exhib- chromosomes (chromosomes 8–12) had no clear CMA- ited the most asymmetric centromere position compared bands. A weak, thin CMA-band frequently appeared at to other short chromosomes. the interstitial region of one arm of chromosome 3. After DAPI-banding, negative DAPI-bands appeared clearly Results and discussion at the position of all clear CMA-bands, a weak DAPI- band appeared at the proximal region of the short arm All six Abies species examined had 2n=24 somatic or centromeric region of chromosome 11, and several chromosomes and a similar karyotype composed of very weak interstitial DAPI-bands also appeared in some seven pairs of long metacentric and five pairs of short chromosomes (Fig. 1B). submeta- and subtelocentric chromosomes including a somewhat long submetacentric chromosome pair (Figs. Abies veitchii 1–6). These karyotypic features are similar to those Several CMA-bands appeared at interstitial regions reported previously by conventional stain techniques of the long metacentric chromosomes, and their num- (Sax and Sax 1933, Mehra and Khoshoo 1956, Kormu- ber varied from eight to six depending on plants and/or tak 1985, Hizume 1988, Muratova and Matveeva 1996), populations (Fig. 2A, C, E). The locations of eight CMA- fluorescent banding (Roth et al. 1997, Puizina et al. bands were very similar to that of A. laciocarpa. One 2008) and FISH (Shibata et al. 2004, Besendorfer et al. chromosome pair carrying interstitial CMA-band (chro- 2005, Puizina et al. 2008). Secondary constrictions were mosome 2) had a weak and thin CMA-band at the inter- observed at the interstitial region of some chromosomes stitial region of the chromosome arm without the CMA- and different in number and position depending on re- band. In chromosome 5, the appearance of CMA-bands ports, whereas in FISH and fluorescent banding the sec- varied among plants in number between eight (Fig. 2A), ondary constrictions appeared clearly. The fluorescent seven (Fig. 2C) and six (Fig. 2E). A clear DAPI-band banding pattern of the chromosomes was described in was not observed, and sites of CMA-bands were DAPI- each species. negative (Fig. 2B, D, F), similar to A. laciocarpa. Abies laciocarpa Abies sachalinensis The species had eight CMA-bands at the interstitial Six to eight CMA-bands appeared on the interstitial region of the long metacentric chromosomes. The exact region of the long metacentric chromosomes (Fig. 3A location of the CMA-bands on the chromosome arms shows chromosomes with eight CMA-bands). A thin was somewhat different from each other chromosome CMA-band also appeared on the interstitial region of the pair and distinguished each homologous chromosome. arm of chromosome 4 with no large CMA-bands. The Two CMA-bands were located at the middle region observed intraspecific numeral variation of the interstitial 2016 Fluorescent Chromosome Banding Patterns in Six Species of Abies, Pinaceae 63 Fig. 2. Fluorescent-banded chromosomes in Abies veitchi. A, C, E: CMA-banding; B, D, F: DAPI-banding. Chromosomes of plants showed eight (A), seven (C) and six CMA-bands. Bar=5 µm. Fig. 3. Fluorescent-banded chromosomes in Abies sachalinensis. Fig. 4. Fluorescent-banded chromosomes in Abies mariesii. A: CMA-banding, B: DAPI-banding. Bar=5 µm. A: CMA-banding, B: DAPI-banding. Bar=5 µm. CMA-band of chromosome 5 was the same phenomenon Abies mariesii as in A.