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 comparative 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 chromosome 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. veitchii. After DAPI-banding, a clear DAPI-band Eight CMA-bands appeared in the chromosome com- was not observed and the short chromosome 11 had a plement of this species. Four long metacentric chromo- thin DAPI-band at the centromeric region (Fig. 3B). somes pairs (chromosomes 2, 4–6) have a CMA-band at the interstitial region of one arm of each pair (Fig. 4A). 64 M. Hizume et al. Cytologia 81(1)

of chromosomes is applicable among Abies species, and after identifying each chromosome pair, a detailed comparative karyotype analysis is allowed. The relationships among one North American species and five Asian species, examined in this study, and four Euro- pean species, studied previously by fluorescent banding and FISH using rDNA (Roth et al. 1997, Shibata et al. 2004, Besendorfer et al. 2005, Puizina et al. 2008), were jointly compared and discussed. Somatic cells of the North American and Asian species had eight CMA- bands at the interstitial region of long metacentric chromosomes, whereas the European species had 10 CMA- bands or FISH signals of 45S rDNA. The American and Asian species excepting A. mariesii belong to section Balsameae, and the European species to section Abies & Piceaster. The two sections seem closely related and are put into nearby clades in molecular phylogeny Fig. 5. Fluorescent-banded chromosomes in Abies faxoniana. (Farjon and Rushforth 1989, Isoda et al. 2000, Xiang A: CMA-banding, B: DAPI-banding. Bar=5 µm. et al. 2015). Abies mariesii, having many DAPI-bands, is distant from other species examined in molecular phylogeny, belonging to section Amabilis, and had eight The CMA-banding pattern of this species was simi- CMA-bands in diploid genome. Other members of lar to those of other species. On the other hand, many Amabilis should be analyzed for their DAPI-banding. DAPI-bands appeared at the interstitial and centromeric These results might suggest that Abies species have four regions (Fig. 4B). Interstitial DAPI-bands appeared on CMA-bands in haploid in common, which increase to both arms of chromosomes 1 and 5. Chromosome 5 had five in European species, Section Abies & Piceaster. the DAPI-band at a closely proximal adjacent site of the Intraspecific variation of CMA-band appearing on interstitial CMA-band, and chromosomes 2 and 3 at one chromosome 4 was observed in A. veitchii and A. arm without CMA-bands. Chromosomes 3, 6 and 11 had sachalinensis, distributed separately on high mountain centromeric DAPI-bands. The DAPI-banding pattern regions of Honshu, and plane and mountain regions of was quite different from those of the other five species. Hokkaido to Sakhalin, respectively. These species are considered to be differentiated by geographic isola- Abies faxoniana tion caused by repeating glacial–interglacial events in Eight CMA-bands appeared at the interstitial regions the Pleistocene of the Quaternary Period. The species of long metacentric chromosomes (chromosomes 2, were put in the same section Balsamea and form the 4–6), and their locations were somewhat different in po- same clade in molecular phylogeny (Farjon and Rush- sition (Fig. 5A). The CMA-banding of this species was forth 1989, Xiang et al. 2015). The possibility that the very similar to or the same as those of the other species. variation of CMA-bands on the chromosome occur in The clear DAPI-band was not observed and negative ancestors and is shared between the species will be clari- DAPI-bands appeared on each CMA-band (Fig. 5B), fied by analyzing the CMA-banding pattern in another similar to other species except for A. mariesii. related speciesm such as A. sibirica of the same section. In most probably all genera of Pinaceae, the second- Abies georgei ary constriction showed CMA-band and FISH signal Eight CMA-bands appeared at the interstitial regions of 45S rDNA indicating the presence of rDNA repeats of the long metacentric chromosomes (chromosomes (Pinus: Hizume et al. 1992, 2001, 2002b; Picea: Hizume 2, 4–6), and their locations were very similar to or and Kuzukawa 1995, Brown and Carlson 1997, Shibata the same as those of other species (Fig. 6A). The clear et al. 2004; Larix: Hizume et al. 1988, Liu et al. 2007; DAPI-band was not observed and negative DAPI-bands Pseudotsuga: Hizume and Akiyama 1992, Amarasinghe appeared on each CMA-band (Fig. 6B), similar to other and Carlson 1998; Cedrus: Dagher-Kharrat et al. 2001; species except for A. mariesii. Abies: Puizina et al. 2008) as in other plant species (Sch- weizer 1976, Hizume et al. 2013, Kuroki et al. 2013). In In Abies, karyotypes are very similar among the the species of Larix, Pseudotsuga and Picea, one CMA- species (Hizume 1988), and each chromosome pair in band is consistent with the FISH signal of 5S rDNA karyotypes of different species was identified by chro- (Hizume et al. 1996, Amarasinghe and Carlson 1998, mosome shape, fluorescent band pattern and FISH signal Liu et al. 2007). Abies species have DAPI-bands at the pattern (Shibata et al. 2004). The identification system proximal or centromeric region of chromosome 11, and 2016 Fluorescent Chromosome Banding Patterns in Six Species of Abies, Pinaceae 65

et al. 1988, Hizume and Akiyama 1992). Neverthe- less, Larix species have clear DAPI-bands on nearly all chromosomes at their proximal region of one arm, but Pseudotsuga have no proximal DAPI-bands at all (Hizume et al. 1988, Hizume and Kondo 1992, Hizume et al. 1993, 1994). AT-rich repetitive sequences cloned by Hizume et al. (2002a) located at the proximal region of most chromosomes and/or genome in the examined Larix species were not found in the Pseudotsuga genome. These results suggest that AT-rich sequences might appear even in certain closely related genera or species groups of the genus. The Abies DAPI-banding pattern is desired to be studied by fluorescent banding, especially DAPI-banding, in many other species to reveal their phylogenetic significance in taxonomy and phylogeny. This report shows that the pattern of fluorescent bands and FISH signals supply important information on taxonomic and phylogenetic relationships among Abies species. In order to draw whole taxonomical and phylo- Fig. 6. Fluorescent-banded chromosomes in Abies georgei. genetical pictures of Abies, more of the species should be A: CMA-banding, B: DAPI-banding. Bar=5 µm. analyzed for their chromosomes by fluorescent banding and FISH with not only rDNA but also other probes such as sequences cloned in Pinus densiflora (Hizume et al. FISH signals of 5S rDNA are also present at the proxi- 2001), Larix leptolepis (Hizume et al. 2002a) and Picea mal region of the short arm (Brown and Carlson 1997, species (Brown et al.1998, Vischi et al. 2003, Shibata Shibata et al. 2004, Besendorfer et al. 2005). Six se- and Hizume 2008). quences of 5S rDNA repeat units of A. alba registered in the NCBI nucleotide database (Besendorfer et al. 2005) References are 50–56% CG-contents and not AT-rich. This evidence suggests that the centromeric DAPI-band of the short Amarasinghe, V. and Carlson, J. E. 1998. Physical mapping and chromosome does not contain 5S rDNA repeats. characterization of 5S rRNA genes in Douglas-fir. J. Hered. 89: 495–500. In A. mariesii, the DAPI-band pattern had many Begum, R., Sultana, S. S. and Alam, S. S. 2010. Comparison of CMA- DAPI-bands at the interstitial and/or centromeric regions karyotypes in three Dendrobium spp. Cytologia 75: 185–188. of most chromosomes, which was complex and quite Besendorfer, V., Krajacic-Sokol, I., Jelenic, S., Puizina, J., Mlinarec, different from those of the other seven species reported, J., Sviben, T. and Papes, D. 2005. Two classes of 5S rDNA unit which have very few or no DAPI-bands (Figs. 1–3, 5, 6; arrays of the silver fir, Abies alba Mill.: Structure, localization and evolution. Theor. Appl. Genet. 110: 730–741. Shibata et al. 2004, Puizina et al. 2008). Several species Brown, G. R. and Carlson, J. E. 1997. Molecular cytogenetics of the of Pinus (Hizume et al. 1983, 1989, 1990, Doudrick genes encoding 18s–5.8s–26s rRNA and the 5s rRNA in two spe- et al. 1995) and of Picea (Brown and Carlson 1997, cies of spruce (Picea). Theor. Appl. Genet. 95: 1–9. Siljak-Yakovlev et al. 2002) also have complex DAPI- Brown, G. R., Newton, C. H. and Carlson, J. E. 1998. Organization banding patterns; these variations in DAPI-banding pat- and distribution of Sau3A tandem repeated DNA sequence in tern among species might occur independently in each Picea (Pinaceae) species. Genome 41: 560–565. Dagher-Kharrat, M. B., Grenier, G., Bariteau, M., Brown, S., Siljak- genus and indicate that DAPI-banding patterns should Yakovlev, S. and Savouré, A. 2001. Karyotype analysis reveals be evaluated within the genus. The phenomenon might interspecific differentiation in the genus Cedrus despite genome be coincident with taxonomic and phylogenetic treat- size and base composition constancy. Theor. Appl. Genet. 103: ment in each genus. Genera Larix and Pseudotsuga are 846–854. considered clearly to be closely related in systematics Doudrick, R. L., Heslop-Harrison, J. S., Nelson, C. D., Schmidt, T., Nance, W. L. and Schwarzacher, T. 1995. Karyotype of Slash and molecular phylogeny (Gernandt and Liston 1999, pine (Pinus elliottii var. elliottii) using patterns of fluorescence Farjon 2010, Xiang et al. 2015), and in cytology, they in situ hybridization and fluorochrome banding. J. Hered. 86: share a similar bimodal karyotype (Hizume 1988), pat- 289–296. terns of CMA fluorescent bands and ISH/FISH signals Farjon, A. 2010. A Handbook of the World’s Conifers, Vols. 1–2. E. J. of rDNA (Hizume et al. 1988, Hizume and Kondo 1992, Brill, Leiden. Farjon, A. and Rushforth, K. D. 1989. A classification of Abies Miller Brown and Carlson 1997, Liu et al. 2007, Goryachkina (Pinaceae). Notes Roy. Bot. Gard. Edinburgh 46: 59–77. et al. 2013). Between the two genera, a major difference Fawzia, R. and Alam, S. S. 2011. Fluorescent karyotype analysis in was reported in the DAPI-banding pattern (Hizume four varieties of Solanum melongena L. Cytologia 76: 345–351. 66 M. Hizume et al. Cytologia 81(1)

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