Plant Systematics and Evolution Molecular cytogenetic analysis of Siberian Larix species by fluorescence in situ hybridization --Manuscript Draft-Manuscript Number:
PLSY-D-12-00098
Full Title:
Molecular cytogenetic analysis of Siberian Larix species by fluorescence in situ hybridization
Article Type:
Short Communication
Keywords:
Chromosome; karyotype; FISH; 45S rDNA; 5S rDNA; Larix
Corresponding Author:
Olga V. Goryachkina, Ph.D. V.N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch Krasnoyarsk, RUSSIAN FEDERATION
Corresponding Author Secondary Information: Corresponding Author's Institution:
V.N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch
Corresponding Author's Secondary Institution: First Author:
Olga V. Goryachkina, Ph.D.
First Author Secondary Information: Order of Authors:
Olga V. Goryachkina, Ph.D. Ekaterina D. Badaeva, Ph.D., Prof. Elena N. Muratova, Ph.D., Prof. Alexandr V. Zelenin, Ph.D., Prof.
Order of Authors Secondary Information: Abstract:
Data on cytogenetic study of three Larix species (L. sibirica, L. gmelinii and L. cajanderi) most widely spread in Siberian forests are presented. Fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allows to identify of homologous chromosome pairs in the larch karyotypes and to facilitate the comparative karyotype analysis. Two major 45S rDNA loci (per haploid genome) were observed in the intercalary regions of two metacentric chromosomes of L. sibirica; additionally, minor NORs were mapped in the pericentromeric regions of four chromosomes. Two closely related species, L. gmelinii and L. cajanderi, showed similar hybridization patterns. Both species possessed an additional major 45S rDNA locus in the distal region of the long arm of submetacentric chromosome that is absent in L. sibirica. Only one 5S rDNA locus was commonly observed in each species studied. The differences at FISH signal locations were compared and the interspecific relationship in the genus Larix was discussed.
Suggested Reviewers:
Konstantin V. Krutovsky, Ph.D. Associate Professor, Texas A&M University
[email protected] Konstantin V. Krutovsky is a famous scientist in the field of population genetics and genomics of forest trees. He collaborates on the Douglas-fir Genome Project (DFGP) and the Loblolly Pine Genome Project. His competence in the matters of plant genetics is beyond any doubt. Masahiro Hizume, Doctor of Science Professor, Ehime University
[email protected] Masahiro Hizume is a principal scientist in the field of gymnosperms karyology and karyosystematics. He has study conifers chromosomes by conventional karyotype analysis, fluorescent banding and hybridization in situ in the last several decades.
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Berthold Heinze, Dr. Federal Research Centre for Forests
[email protected] Dr. Berthold Heinze is a head of Unit of Genome Research. He is a famous researcher in the genetics of forest trees.
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Molecular cytogenetic analysis of Siberian Larix species by fluorescence in situ hybridization 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Olga V. Goryachkina1*, Ekaterina D. Badaeva2, Elena N. Muratova1, Alexandr V. Zelenin2 1
V.N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch,
Academgorodok Street 50/28, Krasnoyarsk 660036, Russia 2
V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street
32, Moscow 119991, Russia * Corresponding author (e-mail:
[email protected]; fax +7 391 2433686) Abstract Data on cytogenetic study of three Larix species (L. sibirica, L. gmelinii and L. cajanderi) most widely spread in Siberian forests are presented. Fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allows to identify of homologous chromosome pairs in the larch karyotypes and to facilitate the comparative karyotype analysis. Two major 45S rDNA loci (per haploid genome) were observed in the intercalary regions of two metacentric chromosomes of L. sibirica. Two closely related species, L. gmelinii and L. cajanderi, showed similar hybridization patterns. Both species possessed an additional major 45S rDNA locus in the distal region of the long arm of submetacentric chromosome that is absent in L. sibirica. The minor 45S rDNA loci were mapped in the pericentromeric regions of four chromosomes; it is the first discovery of the minor rDNA sites in the larch chromosomes. Only one 5S rDNA locus was commonly observed in each species studied. The differences at FISH signal locations were compared and the intra- and interspecific karyotype variation in the genus Larix were discussed.
Keywords: Chromosome; karyotype; FISH; 45S rDNA; 5S rDNA; Larix.
Abbreviations FISH fluorescence in situ hybridization DAPI 4,6-diamidino-2-phenylindole SSC saline-sodium citrate PBS phosphate buffered saline NOR nucleolus organizing region rDNA ribosomal DNA
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Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
The genus Larix Mill. is one of the most widely spread and economically important genera of conifers in the world. Larix species are dominant over large areas of temperate and boreal zones in the Northern Hemisphere; they grow in the mountains of East Asia and North America, often forming the northern timberline. According to different taxonomic systems, the genus Larix contains between 10 and 25 species (Patscke 1913; Sukachev 1924; Dylis 1961; Bobrov 1972; Farjon 2001). However, because the distribution areas of some larch species overlap and because of the species are able to hybridize easily in natural habitats, the development of a taxonomic system for Larix is significantly complicated. It specifically concerns the larches growing in East Asia. Species of the genus Larix are the most abundant trees in Russian Federation. Siberian larch species—L. sibirica Ledeb., L. gmelinii (Rupr.) Rupr., and L. cajanderi Mayr—form more than 90 % of the larch forest area in the country. Having very high adaptability, these larch species and their natural hybrid complexes form large tracts of monodominant and open forests at high latitudes (Abaimov 2010). Of them, L. cajanderi is the most interesting because the taxonomic status of this larch is not completely solved. Some Russian scientists support L. cajanderi as a separate species (Bobrov 1972; Abaimov 2010), while others consider it as the eastern subspecies of L. gmelinii (Dylis 1961) or do not recognize it at all (Farjon 2001). The foreign scientists often make use of the species epithet ―gmelinii‖ for the areas of L. cajanderi that is likely cause by the lack of using the materials from the researches performed by Russian scientists and published only in Russian language in the last several decades. L. cajanderi grows under severe conditions of the Siberian North-East, has definite habitat, morphologically isolated, and is characterized by a series of biological and ecological peculiarities (Abaimov 2010). Recently, molecular studies on mtDNA and cpDNA revealed weak but visible differentiation between L. gmelinii and L. cajanderi, suggesting independent glacial histories for these species (Polezhaeva et al. 2010). Cytogenetic approaches are widely used for the analysis of intra- and interspecific diversity and the structure and genotypic composition of the natural populations of angiosperms. However, this technique has very limited applications in gymnosperms, including Larix species. This is due to the difficulties of chromosome analysis in conifers because of their karyotype similarity and the impossibility of applying standard banding techniques. Karyotypes of all larch species possess 12 chromosome pairs: 6 pairs are long metacentric chromosomes and 6 pairs are short submeta- or subacrocentric chromosomes (Hizume 1988; Muratova 1991, 1994; Muratova et al. 2007). Only a few publications are currently available on the molecular-cytogenetic analysis of larch genomes. The 5S and 45S rRNA genes have been mapped on the chromosomes of L. potaninii var. macrocarpa (Hizume et al. 1995), L. decidua 2
(Lubaretz et al. 1996), L. kaempfeeri, L. olgensis, L. principis-rupprechtii (Liu et al. 2006, 2007), 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
L. leptolepis (Zhang et al. 2010). Additionally, the genus-specific family of tandem repeats, the LPD (Larix proximal DAPI band), was isolated and localized to the proximal regions of many larch chromosomes (Hizume et al. 2002). Here, we present the results of a comparative cytogenetic analysis of three Siberian larch species (L. sibirica, L. gmelinii and L. cajanderi) using fluorescence in situ hybridization (FISH) with the 5S and 45S ribosomal RNA gene probes and DAPI staining. Data on chromosomal locations of rDNA loci for L. sibirica and L. cajanderi were obtained in this work for the first time. In addition, some new features supplemented the results of the previous studies were revealed on L. gmelinii karyotype (Liu et al. 2006, 2007; Zhang et al. 2010).
Materials and methods Seed collection and slide preparation L. sibirica, L. gmelinii and L. cajanderi seeds were collected from open-pollinating natural stands and artificial plantation (Table 1). Between three to five plants were analyzed for each population.
Table 1. Geographic origins of Larix populations sampled for the present study
The seeds were germinated in Petri dishes on a moist filter paper at room temperature. Root tips were grown to a length of 0.5-1.0 cm, pretreated with 0.2 % colchicine for 15-18 h at 20°С, submerged in ice water for 1 h, and then fixed in 3 : 1 ethanol : acetic acid for a minimum of 3 h. Materials were washed in distilled water for 1 h, macerated in 0.6 % cellulase Cellulysine (Calbiochem, Switzerland) water solution for 14-16 h at 24° С or in an enzyme mix consisting of 2 % cellulase Onozuka R-10 (Sigma-Aldrich, USA) and 2 % Pectolyase Y-23 (Fisher Scientific, USA) in citrate buffer (pH 4.0) at 37° С for 30 min. The root tips were squashed in a drop of 45 % acetic acid, and the coverslips were removed after freezing in liquid nitrogen. The slides were dehydrated in ethanol and stored at 4°С before use. Probe DNA labeling Two wheat DNA probes, pTa794 (5S rDNA) and pTa71 (45S rRNA), were used for in situ hybridization. The pTa794 clone is a 410-bp BamHI fragment of wheat 5S rDNA, cloned into the pBR322 plasmid (Gerlach and Dyer 1980). The pTa71 clone is a 9-kb Eco RI fragment of wheat 45S rRNA cloned into the pUC19 plasmid (Gerlach and Bedbrook 1979). The probes were labeled with biotin or digoxigenin by nick translation, according to manufacturer’s protocol (Roche, Germany). 3
Fluorescence in situ hybridization 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
The slides were treated with 100 μL of RNAse solution (100 μg mL–1 in 2 × SSC) at 37° С for 1 h and then washed three times in 2 × SSC (saline-sodium citrate, pH 7.0) for 10 min each. The slides were dehydrated in an ethanol series and then air-dried. The slides were denatured at 74° С for 2 min in a 70 % formamide solution in 2 × SSC and then dehydrated in a series of 70 %, 85 % and 100 % ethanol at -20° С. A hybridization mix containing 50 % formamide (V/V), 10 % salmon sperm DNA, 20 % dextran sulfate and 5 % of each labeled DNA probe in 2 × SSC was denatured at 85° С for 5 min and then chilled on ice. Hybridization was carried out overnight at 37° С in a moisture chamber. After hybridization, the slides were washed twice in 0.1 × SSC for 10 min at 37° C, twice in 2 × SSC for 10 min at 37° C, and once in 2 × SSC for 5 min at room temperature. The slides were then washed in 1 × PBS (phosphate buffered saline, pH 7.4) for 5 min at room temperature. DIG-labeled probes were detected using anti-digoxigenin-FITC (Roche Biochemicals, Sussex, UK) and anti-fluorescein/Oregon Green, Alexa Fluor-488 conjugates (Invitrogen, USA). Biotin-labeled probes were detected using Streptavidin-Cy3 (Amersham Pharmacia Biotech, USA). The slides were counterstained with 1 μg/ml DAPI (4’, 6diamidino-2-phenylindole, Sigma, USA) in the Vectashield mounting media (Vector Laboratory, Peterborough, UK) Microscopic evaluation, image processing and karyotype analysis The slides were analyzed on an Imager D1 microscope (Carl Zeiss, Germany) coupled with an AxioCam HRm black-and-white camera. The selected metaphase cells were captured using AvioVision software, release 4.6. Images were processed using Adobe Photoshop CS4 (Adobe Systems, Edinburgh, UK). Pairs of homologous chromosomes were determined on the basis of chromosome morphologies, size and position of 5S and 45S rDNA loci, and DAPI bands, when available. Chromosome classification was based on chromosome morphology (according to decreasing length). The results of cytogenetic analyses on other larch species were also taken into consideration (Hizume et al. 1995; Lubaretz et al. 1996; Liu et al. 2006, 2007; Zhang et al. 2010). Chromosome measurements were carried out in 12 to 24 well-spread metaphase plates. The following parameters were determined for each chromosome: total length, relative length of each chromosome pair, arm ratio, position of secondary constrictions (SC = length from centromere to secondary constriction / length of the arm ×100), the presence or absence of rDNA loci and / or DAPI bands, the location of FISH signal (length from centromere to the middle of FISH signal / length of the arm ×100).
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Results 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
The chromosome identification in conifers by conventional karyotype analysis is very difficult because the chromosomes are similar in their shape and size. The karyotypes of Larix species are similar to each other and consist of six pairs (I-VI) of long metacentric chromosomes and six pairs (VII-XII) of short submetacentric chromosomes (Tables 2-4). Two of the metacentric chromosome pairs (III and IV) carry secondary constrictions in the intercalary regions of one of the arms. It is also possible to distinguish chromosome pair VI because they are the smallest and more asymmetric among the metacentrics. The morphologies of chromosome pair VII are close to subacrocentric, which allows for their discrimination. The secondary constriction in the long chromosome arm was found to be diagnostic for L. gmelinii and L. cajanderi chromosome VII.
Table 2. Morphometric data from 15 metaphase cells of L. sibirica (population 2) Table 3. Morphometric data from 17 metaphase cells of L. gmelinii Table 4. Morphometric data from 24 metaphase cells of L. cajanderi
FISH analysis showed that the number of major sites for 45S rRNA genes corresponds to the number of the constant secondary constrictions in all species studied (Tables 2-4). In L. sibirica karyotype, the NOR regions were positioned in the intercalary regions of two metacentric chromosome pairs (Fig. 1). The FISH signal in the long arm of chromosome III usually were more intensive whereas the short arm of chromosome IV contained a rather smaller signal. Hybridization patterns of the рТа71 DNA probe on L. gmelinii and L. cajanderi chromosomes were identical, and they differed from that of L. sibirica in the presence of additional, third major 45S rDNA locus in the long arm of chromosome pair VII (Fig. 1). Hybridization in situ with the рТа794 probe showed that the 5S rDNA loci were positioned in the subtelomeric region of the short arm of chromosome III that also harbored the major 45S rDNA site on the long arm in all species studied (Fig. 1).
Fig. 1. Physical mapping of 45S rDNA (red) and 5S rDNA (green) using bicolor FISH on metaphase chromosomes of three Larix species from five populations (numbers of populations are shown according to the Table 1). Chromosome pairs are identified by the combined use of morphometric data and distribution of the probes. Scale bar 10 μm.
All major 45S rDNA sites observed in the Larix chromosomes are functionally active, as determined by counting of the AgNO3-stained nucleoli in the interphase nuclei (Table 5). The mean number of nucleoli per nucleus in L. sibirica was 2.88±0.03, while in L. gmelinii and L. 5
cajanderi; the mean numbers of nucleoli were 3.72±0.03 and 3.23±0.04, respectively. In many 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
cells, the nucleoli of different sizes were observed. In particular, the NOR locus located on the submetacentric chromosome VII formed a smaller nucleoli than the NORs of the metacentric chromosomes III and IV (Fig. 2a), which can be attributed to the different number of rDNA repeats at the respective loci. Additionally, a common feature of conifers is the frequent fusion of nucleoli (Fig. 2b), which occurs when several NOR-bearing chromosomes are involved in the formation of a large nucleolus during interphase (Karvonen et al. 1993).
Table 5. The number of nucleoli per interphase nucleus in Larix species studied.
Fig. 2. Nucleoli in the interphase nuclei of L. gmelinii (a) and L. sibirica (b-d). Additional nucleoli are shown by arrows.
The minor NOR sites were detected on four chromosome pairs in all larch species studied here. These sites were usually located in the pericentromeric regions of the short arms; however, the minor 45S rDNA site on the chromosome II was spatially separated from the centromere and was positioned at the edge of a DAPI band (Fig. 3). Chromosomes I and II bore the weak signals; in different species, their incidence varied between 34.6 and 79.3 % for chromosome I and between 27.3 and 70.0 % for chromosome II. The minor 45S rDNA site on the chromosome VI was the more intensive, and it was clear visible in 76.5-87.5 % metaphase plates. Weak 45S rDNA signal could also be found on the smallest submetacentric chromosome XII (frequency of occurrence 48.8-88.1 %).
Fig. 3. Comparative idiogram of L. sibirica (A), L. gmelinii (B) and L. cajanderi (C) chromosomes showing the chromosomal locations of 45S rDNA (black bands), 5S rDNA (black circles) and DAPI bands (gray bands). Scale bar 10 μm.
When the chromosomes of Larix species were counterstained with DAPI after in situ hybridization, DAPI bands appeared in the pericentromeric regions of chromosomes. The pattern of DAPI staining of the three larch species was similar, although the banding was most distinct on L. gmelinii chromosomes. The location of DAPI bands over the chromosomes was different. The bands were clearly observed in the long arms of chromosomes I, IV, VI and in the short arms of chromosomes III and V (Fig. 3). Chromosome II possessed wide DAPI bands in the proximal regions of both arms. Small DAPI bands were also observed in the pericentromeric regions of the short arms of the most submetacentric chromosomes; however, these bands 6
demonstrated less contrast than the bands on the metacentric chromosomes. Only chromosome X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
lacked the DAPI bands, although in L. cajanderi, we also failed to see the bands on the chromosome VII (Fig. 3). Thus, fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allowed for identification of all homologous chromosome pairs in the karyotypes of three Larix species. Our results showed high similarity of closely related species L. gmelinii and L. cajanderi in hybridization patterns; they both contained an additional, third major 45S rDNA locus that was absent in L. sibirica. Comparative idiogram of the Larix chromosomes shown in Fig. 3 was constructed based on the results of chromosome measurements. The karyotypes of studied Larix species were very similar, and most of the differences were in the total length of the chromosomal set. Our results agreed well with the genome sizes of the respective conifer species, which were estimated to be 13692 Mbp in L. gmelinii and 12029 Mbp in L. sibirica (Ohri and Khoshoo 1986). Intraspecific variation of the genome size in larch species had not been documented. Although we analyzed three populations of Siberian larch from different origins, we failed to find any differences between them, either by hybridization patterns of rDNA gene families, or by DAPI staining (Fig. 1).
Discussion The genes encoding the 5S and 45S ribosomal RNA are widely used as molecular cytogenetic markers in evolutionary and genomic studies of forest trees because of their highly conserved structure and tandem organization (Ribeiro et al. 2008). The rDNA loci have been mapped onto the chromosomes in a number of gymnosperm species, including those belonging to the Pinaceae family. To date, the Pinus genus is the best studied; a comparison of 20 pine species revealed high interspecific karyotype divergence as a result of variation in the distribution of ribosomal RNA gene loci, especially the 45S rRNA genes (Cai et al. 2006). In the genus Larix, the 5S and 45S rRNA genes have been mapped onto the chromosomes of 9 species, including our results. Larch species have showed the low interspecific karyotype variation and the lowest number of major 45S rDNA loci among the Pinaceae family, with only 2 to 6 loci per diploid genome (Hizume et al. 1995; Lubaretz et al. 1996; Liu et al. 2006, 2007; Zhang et al. 2010). Between 4 and 14 rDNA sites were recorded in the different Picea species (Shibata and Hizume 2008), whereas the highest number of 45S rDNA sites was found within the genus Pinus. Thus, the karyotypes of some pines may contain up to 20 chromosomes with the 45S rDNA sites of different size (Cai et al. 2006). Low numbers of rDNA loci in the larch genomes may be
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indicative of the relatively recent origin of this genus, which is considered one of the most 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
advanced genera in the Pinaceae family. Our study is the first discovery of minor 45S rDNA loci in the pericentromeric regions of the larch chromosomes. These signals are commonly observed in most Pinus species; their incidence varied from 2 to 18 per diploid set in different species (Liu et al. 2003; Islam-Faridi et al. 2003; Bogunic et al. 2011). It was shown that the centromeric regions of Pinus densiflora chromosomes may contain the 18S and 26S rDNA sequences (Hizume et al. 2001). The authors proposed that these sequences are nonfunctional because the number of nucleoli in the interphase nuclei of Pinus densiflora does not exceed the number of major NORs. Another situation seems to be observed in Larix species. Probably, at least one of the minor NORs observed in our study displays functional activity, as determined by nucleolar count. Several cells in each species were found to possess more nucleoli than the total number of major NORs on their chromosomes (Fig. 2c, d). As long as the most intensive minor 45S rDNA site is located on the chromosome VI, it is likely to be connected with additional nucleolus formation in the Larix cells. In connection with discussed above, interesting becomes the fact that one of the North American larch species, L. laricina, contains a bright CMA band in the proximal region of the smallest pair of metacentric chromosomes, which coincides with the position of the secondary constriction (Hizume and Tanaka 1990). Two other CMA bands were localized in the intercalary regions of the two pairs of long metacentric chromosomes, which are also characteristic for most Larix species. The maximum number of nucleoli in L. laricina corresponds to the number of CMA bands, namely, 6. Unfortunately, the karyotype of this species has not been examined by FISH. However, considering these data, we can assume that in the three larch species we studied here, chromosome VI contains a small number of 45S rDNA repeats that may reflect a common ancestry of Eurasian and North American Larix species (Wei and Wang 2003). Gymnosperms are known to possess between 1 and 2 loci of 5S rDNA per haploid genome (Ribeiro et al. 2008). Interspecific variation in the number and position of 5S rDNA loci has only been described in the Pinus genus (Liu et al. 2003; Cai et al. 2006). The only 5S rDNA site in the larch karyotypes is located on the metacentric chromosome III, which also carries the major 45S rDNA locus in the opposite arm in the most larch species. Currently, this chromosome contained loci of both ribosomal RNA gene families have been detected in all larch species studied, excepting L. potaninii var. macrocarpa (Hizume et al. 1995). A homologous chromosome was also found in Picea karyotypes; however, spruce species contain an additional major 5S rDNA locus in the long arm of this chromosome that is adjacent to the 45S rDNA site (Shibata and Hizume 2008).
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Based on the number of secondary constrictions and major 45S rDNA sites genus Larix can be 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
subdivided into some karyotypic groups. Taxa with one pair of satellite chromosomes in karyotype compose the first group formed by L. griffithiana with small area in the East Himalayas, and L. potaninii var. macrocarpa growing in Central and South China (Simak 1966; Hizume et al. 1995). These species belong to the more primitive section Multiseriales (Bobrov 1972). Karyological data confirm V.N. Sukachev’s hypothesis of old aged larch species growing in China (Sukachev 1924). It is suggested that Larix taxa with two pairs of satellite chromosomes are likely to be more specialized. This group includes two species from section Multiseriales (L. potaninii var. potaninii and L. occidentalis) and some species from section Pauciseriales (Larix): L. leptolepis, L. sibirica, L. kaempfeeri, L. olgensis, L. chinensis (Hizume and Tanaka 1990; Muratova 1991; Hizume et al. 1993, 1998; Liu et al. 2006; Muratova et al. 2007; Zhang et al. 2010). Most species from section Pauciseriales (Larix) have three pairs of chromosomes with secondary constrictions. This group includes East Siberian and East Asian species (L. gmelinii, L. cajanderi, L. ochotensis, L. principis-rupprechtii) and European species L. decidua and L. polonica and Northern American species L. laricina (Hizume and Tanaka 1990; Hizume et al. 1993; Lubaretz et al. 1996; Liu et al. 2006, 2007; Muratova et al. 2007). According to botanical and palaeontological data these species are young and phylogenetically progressive (Sukachev 1924; Dylis 1961; Bobrov 1972). An analysis of the evolution of Siberian larch species showed that L. sibirica probably emerged in north-eastern Siberia at the end of the Pliocene while L. gmelinii was established as a new species in the Pleistocene. During subsequent spreading L. gmelinii forced out L. sibirica from its previous area of distribution (Sukachev 1924; Dylis 1961; Bobrov 1972). L. cajanderi is considered to be the most recent species, and it arose at the end of Pleistocene. Differences between L. gmelinii and L. cajanderi were also confirmed by molecular and genetic analysis (Semerikov and Polezhaeva 2007; Polezhaeva et al. 2010). Geographic distribution of mtDNA and cpDNA reflects the independent development of larch populations on both sides of the Verkhoyansk mountain range, which led to the emergence of L. cajanderi. Our data agree with estimations of the evolutionary age of L. gmelinii and L. сajanderi, although we failed to find any differences between these species at the karyotype level.
Acknowledgement The authors thank Alexey P. Isaev, Ph.D. (Institute for Biological Problems of Cryolithozone SB RAS, Yakutsk): Alexey P. Barchenkov, Ph.D. (V.N. Sukachev Institute of Forest SB RAS, Krasnoyarsk) and Candagdorj Jamiyansuren, Ph.D. (Institute of Botany, Mongolian Academy of 9
Sciences, Ulaanbaatar) for supplying the seed samples. This work was supported by grant No. 76 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
from the Integration program of Siberian Branch of Russian Academy of Sciences and the Russian Foundation for Basic Research (projects No. 11-04-00063 and 10-04-90780).
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References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1. Abaimov AP (2010) Geographical Distribution and Genetics of Siberian Larch Species. In: Osawa A, Zyryanova OA, Matsuura Y, Kajimoto T, Wein RW (eds.) Permafrost Ecosystems: Siberian Larch Forests. Ser.: Ecological Studies, 209, pp. 41–58. 2. Bobrov EG (1972) History and systematics of Larix. Nauka, Leningrad (in Russian). 3. Bogunic F, Siljak-Yakovlev S, Muratovic E, Ballian D (2011) Different karyotype patterns among allopatric Pinus nigra (Pinaceae) populations revealed by molecular cytogenetics. Plant Biology 13: 194–200. 4. Cai Q, Zhang DM, Liu ZL, Wang XR (2006) Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus. Ann Bot 97 (5): 715–722. 5. Dylis NV (1961) Larch of East Siberia and Far East. USSR Academy of Sciences, Moscow (in Russian). 6. Farjon A (2001) World checklist and bibliography of Conifers. Kew: Royal Botanic Gardens. 7. Gerlach WL, Bedbrook JR (1979) Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res 7 (7): 1869–1885. 8. Gerlach WL, Dyer TA (1980) Sequence organization of the repeated units in the nucleus of wheat which contains 5S-rRNA genes. Nucleic Acids Res 8 (21): 4851–4865. 9. Hizume M (1988) Karyomorphological studies in the family Pinaceae. Mem Fac Educ Ehime University. Ser III. Natural Sci 8 (2): 1–108. 10. Hizume M, Tanaka A (1990) Fluorescent chromosome bandings in two American larches, Larix occidentalis and L. laricina. Kromosomo II (58): 1979–1987. 11. Hizume M, Tominaga K, Kondo K, Gu Z, Yue Z (1993) Fluorescent chromosome banding in six taxa of Eurasian Larix, Pinaceae. Kromosomo II (69): 2342–2354. 12. Hizume M, KuzukawaY, Kondo K, Yang Q, Hong D, Tanaka R (1995) Localization of rDNAs and fluorescent bandings in chromosomes of Larix potaninii var. macrocarpa collected in Sichuan, China. Kromosomo II (78): 2689–2694. 13. Hizume M, Kondo K, Zhang S, Hong D (1998) Fluorescence chromosome banding in a Chinese larch, Larix chinensis Beissn. Chromosome Sci 2 (2): 95–98. 14. Hizume M, Shibata F, Maruyama Y, Kondo T (2001) Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc. Chromosoma 110: 345–351. 15. Hizume M, Shibata F, Matsumoto A et al (2002) Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae. Genome 45: 777–783. 11
16. Islam-Faridi NM, Nelson CD, Kubisiak TL et al (2003) Loblolly pine Karyotype using 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
FISH and DAPI positive banding. Proc. of the 27-th Southern Forest Tree Improv. Conf. Stillwater, Oklahoma, pp. 184–188. 17. Karvonen P, Karjalainen M, Savolainen O (1993) Ribosomal RNA genes in Scots pine (Pinus sylvestris L): chromosomal organization and structure. Genetica 88 (1): 59–68. 18. Liu Z, Zhang D, Hong D, Wang X (2003) Chromosomal localization of 5S and 18S-5.8S25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization. Theor Appl Genet 106: 198–204. 19. Liu B, Zhang SG, Zhang Y, Lan TY, Qi LW, Song WQ (2006) Molecular cytogenetic analysis of four Larix species by bicolor fluorescence in situ hybridization and DAPI banding. Int J Plant Sci 167: 367–372. 20. Liu B, Qi L, Chen R, Song W (2007) Multicolor fluorescence in situ hybridization with combinatorial labeling probes enables a detailed karyotype analysis of Larix principisrupprechtii. Biol Res 40: 23–28. 21. Lubaretz O, Fuchs J, Ahne R, Meister A, Schubert I (1996) Karyotyping of three Pinaceae species via fluorescent in situ hybridization and computer-aided chromosome analysis. Theor Appl Genet 92: 411–416. 22. Muratova EN (1991) Karyological studies in Larix sibirica (Pinaceae) from different parts of its area. Botanicheskyi zhurnal 76: 1586–1595 (in Russian). 23. Muratova EN (1994) Chromosome polymorphism in the natural populations of Gmelin larch, Larix gmelinii (Rupr.) Rupr. Cytologija i Genetika 28: 14–22 (in Russian). 24. Muratova EN, Sedelnikova TS, Pimenov AV, Karpjuk TV, Sizikh OA, Kvitko (Goryachkina) OV (2007) Karyological analysis of larch species from Siberia and the Far East of Russia. Forest Sci Tech 3: 89–94. 25. Ohri D, Khoshoo TN (1986) Genome size in gymnosperms. Plant Syst Evol 153: 119– 132. 26. Patschke W (1913) Uber die extra-tropischen ostasi-atischen Coniferen und ihre Bedeutung fur die pflanzengeographische Gliederung von Ostasiens. Bot Jahrb 48: 651– 655. 27. Polezhaeva MA, Lascoux M, Semerikov VL (2010) Cytoplasmic DNA variation and biogeography of Larix Mill. in Northeast Asia. Mol Ecol 19: 1239–1252 28. Ribeiro T, Barão A, Viegas W, Morais-Cecíli L (2008) Molecular cytogenetics of forest trees. Cytogenet Genome Res 120 (3-4): 220–227. 29. Semerikov VL, Polezhaeva MA (2007) Mitochondrial DNA variation pattern in larches of eastern Siberia and the Far East. Russ J Genet 43: 646–652. 12
30. Shibata F, Hizume M (2008) Comparative FISH karyotype analysis of 11 Picea species. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Cytologia 73 (2): 203–211. 31. Simak M (1966) Karyotype analysis of Larix griffithiana Carr. Hereditas 56: 137–141. 32. Sukachev VN (1924) To the history of larch development. In: The forest issues. MoscowLeningrad: Novaya derevnya: 12–44 (in Russian). 33. Wei X, Wang X (2003) Phylogenetic split of Larix: evidence from paternally inherited cpDNA trnT-trnF region. Plant Syst Evol 239: 67–77 34. Zhang SG, Yang WH, Han SY, Han BT, Li MX, Qi LW (2010) Cytogenetic analysis of reciprocal hybrids and their parents between Larix leptolepis and Larix gmelinii: implications for identifying hybrids. Tree Genet Genomes 6 (3): 405–412.
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List of figure legends 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Fig. 1. Physical mapping of 45S rDNA (red) and 5S rDNA (green) using bicolor FISH on metaphase chromosomes of three Larix species from five populations (numbers of populations are shown according to the Table 1). Chromosome pairs are identified by the combined use of morphometric data and distribution of the probes. Scale bar 10 μm.
Fig. 2. Nucleoli in the interphase nuclei of L. gmelinii (a) and L. sibirica (b-d). Additional nucleoli are shown by arrows.
Fig. 3. Comparative idiogram of L. sibirica (A), L. gmelinii (B) and L. cajanderi (C) chromosomes showing the chromosomal locations of 45S rDNA (black bands), 5S rDNA (black circles) and DAPI bands (gray bands). Scale bar 10 μm.
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Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Table
Table 1. Geographic origins of Larix populations sampled for the present study Species L. sibirica
L. gmelinii L. cajanderi
Population 1 Khakasia, Russia 2 Krasnoyarsk region, Russia 3 Mungun Mort, NE Mongolia 4 Western Yakutia, Russia 5 Central Yakutia, Russia
Lat. °N 54°23’ N 56°22’ N 48°20’ N 58°06’ N 62°25' N
Long. °W 89°45’ E 92°57’ E 108°39’ E 119°09’ E 130°40' E
Alt. (m) 950 250 1600 1000 170
1
Table
Table 2. Morphometric data from 15 metaphase cells of L. sibirica (population 2) Chromosome Total length, μm pair (Mean±SD)
Relative length, %
Arm ratio, %
SC, %
FISH signal location, % 45S 5S
I 14.05±0.25 11.50 1.10±0.02 II 12.44±0.21 10.18 1.06±0.01 III 11.78±0.20 9.64 1.03±0.01 59.66 (l) 60.14 (l) 85.83 (s) IV 12.55±0.22 10.27 1.18±0.01 62.19 (s) 62.51 (s) V 11.87±0.17 9.71 1.07±0.02 VI 10.86±0.16 8.89 1.24±0.03 VII 9.07±0.13 7.42 2.34±0.07 VIII 8.82±0.16 7.22 2.81±0.04 IX 8.21±0.12 6.72 2.04±0.06 X 7.78±0.14 6.37 2.08±0.03 XI 7.47±0.12 6.11 2.20±0.05 XII 7.29±0.07 5.97 2.18±0.02 SD – standard deviation, SC – location of secondary constrictions, s – short arm of chromosome, l – long arm of chromosome
Table
Table 3. Morphometric data from 17 metaphase cells of L. gmelinii Chromosome Total length, μm pair (Mean±SD)
Relative length, %
Arm ratio, %
SC, %
FISH signal location, % 45S 5S
I 13.92±0.31 11.18 1.09±0.01 II 12.72±0.31 1.05±0.01 10.21 III 12.20±0.24 1.01±0.01 58.94 (l) 59.84 (l) 81.88 (s) 9.80 IV 12.39±0.29 1.23±0.01 60.98 (s) 61.54 (s) 9.95 V 12.17±0.29 1.03±0.01 9.77 VI 11.15±0.26 1.21±0.02 8.95 VII 9.18±0.27 7.37 2.38±0.05 60.91 (s) 60.99 (l) VIII 9.08±0.19 2.66±0.04 7.29 IX 8.24±0.23 1.94±0.04 6.62 X 8.04±0.18 2.05±0.03 6.46 XI 7.89±0.16 2.23±0.03 6.33 XII 7.57±0.18 2.11±0.03 6.08 SD – standard deviation, SC – location of secondary constrictions, s – short arm of chromosome, l – long arm of chromosome
Table
Table 4. Morphometric data from 24 metaphase cells of L. cajanderi Chromosome Total length, μm pair (Mean±SD)
Relative length, %
Arm ratio, %
SC, %
FISH signal location, % 45S 5S
I 14.65±0.14 11.03 1.08±0.01 II 13.81±0.15 10.40 1.05±0.01 III 12.68±0.14 9.55 1.07±0.01 59.19 58.92 (l) 85.43 (s) IV 13.31±0.13 10.02 1.19±0.01 61.26 61.42 (s) V 12.59±0.16 9.48 0.03±0.01 VI 11.59±0.13 8.73 1.22±0.01 VII 9.81±0.09 7.39 2.29±0.02 62.19 62.06 (l) VIII 9.92±0.10 7.47 2.67±0.02 IX 9.31±0.10 7.01 1.90±0.02 X 8.67±0.09 6.53 1.99±0.03 XI 8.26±0.11 6.22 2.15±0.03 XII 8.20±0.08 6.17 2.06±0.02 SD – standard deviation, SC – location of secondary constrictions, s – short arm of chromosome, l – long arm of chromosome
Table
Table 5. The number of nucleoli per interphase nucleus in Larix species studied. Species
Scored
L. sibirica L. gmelinii L. cajanderi
1072 1178 609
Frequency of cells with different number of nucleoli, % 1
2
3
4
5
6
7
8
5.60 27.99 40.49 25.09 0.84 2.89 10.87 27.33 36.33 17.23 4.16 0.59 0.59 3.78 22.82 32.02 31.36 8.87 0.66 0.49 -
