Annals of Botany 89: 483-489, 2002
© 2002 Annals of Botany Company
Molecular Cytogenetic Analysis of Podocarpus and Comparison with Other Gymnosperm Species
1School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand, 2Botanical Garden of the University of Osnabrück, Albrechtstrasse 29, 49076 Osnabrück, Germany and 3Department of Biology, University of Leicester, Leicester LE1 7RH, UK
* For correspondence. Fax: +64 9 373 7416, e-mail b.murray{at}auckland.ac.nz
Received: 3 January 2001 Returned for revision: 24 February 2001; Accepted: 11 December 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DNA sequences have been mapped to the chromosomes of Podocarpus species from New Zealand and Australia by fluorescent in situ hybridization. Unlike other conifers, these species show only one pair of major sites of 45S rDNA genes, and two additional minor sites were seen in the Australian P. lawrencei. Unusually, 45S sequences collocalize to the same chromosomal region as the 5S rDNA. The telomere probe (TTTAGGG)n hybridizes to the ends of all chromosomes as well as to a large number of small sites distributed along the length of all chromosomes. Two other simple sequence repeats, (AAC)5 and (GATA)4, show a diffuse pattern of hybridization sites distributed along chromosomes. Southern blots using a variety of probes obtained from the reverse transcriptase of retroelements (gypsy, copia and LINE) from P. totara, P. nivalis and Dacrycarpus dacrydioides show that these retroelements are abundant and widespread in Podocarpaceae and also in others conifers. Some retroelements such as copia pPonty3 and gypsy pPot1li are more abundant in the genome of Picea abies and Ginkgo biloba than in the species from which they were amplified.
Key words: In situ hybridization, karyotypes, Podocarpus, rDNA, Pinaceae, retroelements, genome evolution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Modern gymnosperms are representatives of an ancient group of plants first recorded as fossils in the Upper Devonian (350 million years ago; Ma) (Biswas and Johri, 1997). They consist of two major groups, conifers and cycads. The monophyletic origin of the gymnosperms has been questioned (see Page, 1990) but recent molecular phylogenetic studies provide strong support for monophyly (Qiu et al., 1999; Soltis and Soltis, 2000). The gymnosperms are characterized by large genomes and relatively low chromosome numbers; polyploidy is rare (Khoshoo, 1959; Murray, 1998). Enigmatic features of the chromosomes of the gymnosperms include their characteristically large size and the remarkable constancy of number and karyotype amongst the species of most genera. Conspicuous examples of this are found, for example, in Pinaceae. Hizume (1988) has summarized much of the available information and has shown that 178 out of 180 species in nine genera have the chromosome number 2n = 24. This uniformity must have been maintained for a very long period of time since in Pinus the same karyotype is found in different sections of the genus that were already present in the Cretaceous (90 Ma) (Stockey and Nishida, 1986). Support for the ancient nature of the sectional divisions of the genus also comes from the work of Krupkin et al. (1996) and Wang et al. (2000) using ‘molecular clocks’.
When more detailed studies are made using differential chromosome banding (MacPherson and Filion, 1981; Hizume et al., 1989, 1993, 1995; Davies et al., 1997) and in situ hybridization of repeat sequence DNA (Hizume et al., 1992; Brown et al., 1993; Doudrick et al., 1995; Lubaretz et al., 1996), a greater diversity of chromosome organization becomes apparent. Chromosome banding reveals that species differ in the number of banded chromosomes, size of the bands and their molecular composition (MacPherson and Filion, 1981; Hizume et al., 1989, 1993, 1995; Davies et al., 1997). Most of the in situ studies have investigated the distribution of ribosomal DNA (rDNA) genes. In Pinus, for example, the number of major sites of 45S rDNA ranges from ten pairs in Pinus radiata D. Don and Pinus taeda L. (Jacobs et al., 2000) to 12 pairs in Pinus sylvestris L. (Lubaretz et al., 1996). The 5S sites are less numerous but range in number from one to three pairs (Doudrick et al., 1995; Hizume et al., 1995) and, in relation to the 45S sites, may be either on different chromosomes (Hizume et al., 1995), on the same chromosome but on different arms (Doudrick et al., 1995) or closely adjacent (Lubaretz et al., 1996). Other highly repeated DNA sequences, such as retrotransposons, have been localized to the chromosomes of Pinus elliottii Engelm. (Doudrick et al., 1995; Kamm et al., 1996), and a satellite DNA family cloned from Picea species has been located on different subsets of chromosomes of Picea glauca (Moench) Voss and Picea sitchensis (Bong.) Carr. (Brown et al., 1998). The telomere repeat (TTTAGGG)n found in most angiosperms has been localized at the ends of gymnosperm chromosomes. Picea abies (L.) Karsten and Larix decidua Mill. showed only terminal sites, but hybridization at centromeric sites was also seen in Pinus sylvestris (Fuchs et al., 1995; Lubaretz et al., 1996), and at a number of intercalary sites in Pinus elliottii (Schmidt et al., 2000).
In contrast to the uniformity of chromosome number and karyotype in most conifer families, Podocarpaceae are chromosomally variable (Hair and Beuzenberg, 1958; Hizume et al., 1988; Davies et al., 1997). Chromosome numbers range from 2n = 18 to 2n = 38 and the karyotypes consist of a mixture of different numbers of metacentric, acrocentric and telocentric chromosomes. There have been no previous molecular cytogenetic studies on the family, and the aim of the present work was to investigate the number and distribution of the repeat sequences in Podocarpaceae so that comparisons can be made with the other gymnosperm families.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For in situ hybridization, five species of Podocarpus and a number of natural and artificially produced interspecific hybrids were studied (Table 1). Voucher specimens of all the plants are deposited in the herbarium of the University of Auckland (AKU). DNA from a range of gymnosperms (Table 2) was also used for analysis by Southern hybridization.
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Chromosome preparations were made from root tips that were pretreated with 0·05 % colchicine for 24 h at 4 °C followed by a further 24 h in water at 4 °C before fixation in acetic acid : ethanol, 1 : 3. The fixed roots were digested for 30–40 min at 37 °C in a mixture of 2 % cellulase (Calbiochem, La Jolla, CA, USA), 4 % cellulase (Onozuka R10, Merck, Darmstadt, Germany), 2 % hemicellulase (Sigma, St Louis, MO, USA), 0·5 % pectolyase (Seishin, Tokyo, Japan) and 20 % liquid pectinase (Sigma) in 0·01M citrate buffer, pH 4·6, and squashed without heating on a slide.
The slides were hybridized with the following probes that were labelled with biotin and digoxigenin by nick translation, PCR or random priming as described in Schwarzacher and Heslop-Harrison (2000): (1) pTa71, which contains the 18S-26S rDNA (frequently called 45S rDNA) from Triticum aestivum L. (Gerlach and Bedbrook, 1979) labelled by nick translation; (2) pTa794, which contains a 410 bp BamH1 fragment of the 5S rDNA from T. aestivum (Gerlach and Dyer, 1980) labelled by PCR; (3) pPot1li and pPongy2 which contain 416 and 419 bp fragments of reverse transcriptase of gypsy-like retroelements from Podocarpus totara and P. nivalis, respectively (Friesen et al., 2001; EMBL nucleotide database accession numbers AJ290646-AJ290647); and (4) synthetic oligonucleotides (TTTAGGG)5, (ACG)7, (AAC)5 and (GATA)4 labelled by random priming.
The hybridization mixture was prepared as outlined in Schwarzacher and Heslop-Harrison (2000) and the chromosomes were denatured at 90 °C for 8 min in a Hybaid Omnislide thermal cycler, hybridized overnight at 37 °C and washed, with the most stringent washes in 20 % formamide in 0·1 x SSC at 42 °C (allowing sequences with approx. 85 % identity to remain hybridized). Digoxigenin-labelled probes were detected with an FITC conjugated sheep anti-digoxigenin antibody (Roche, Basel, Switzerland) while biotin-labelled probes were detected with streptavidin conjugated to Cy3 (Sigma). Slides were counterstained with DAPI (4',6-diamidino-2-phenylindole), mounted in Citifluor AF1 (Agar Scientific, Stansted, UK) and observed with a Nikon epifluorescence microscope. Photographs were taken on Fuji Superia 400 colour print film, digitized and printed from Adobe Photoshop after contrast optimization using only functions affecting the whole image.
DNA was extracted from young leaf material of different gymnosperm species using the Qiagen DNAeasy Plant mini kit or the 2 x CTAB method. Isolated DNA was used directly for PCR amplification or digestion with the restriction enzymes HindIII and HaeIII. Digested DNA (5 µg) was separated on 1·2 % agarose gels, blotted, and probed with the following clones that were all labelled by PCR: (1) pDdgy1, a 417 bp fragment of reverse transcriptase of gypsy-like element from Dacrycarpus dacrydioides (A. Rich.) De Laub.; (2) pPongy2 and pPonty3, a 419 bp fragment of reverse transcriptase of a gypsy-like element from P. nivalis; (3) pPot3li, which contains a 257 bp fragment of reverse transcriptase of a copia-like retroelement (accession number AJ2(0731) from P. nivalis, labelled by PCR; and (4) pDdli1, a fragment of a LINElement isolated from D. dacrydioides, using the non-radioactive chemiluminiscence method Alk-Phos Direct (Amersham, Little Chalfont, UK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Four of the five Podocarpus species, those from New Zealand, showed a single pair of hybridization sites with the 45S rDNA probe. In P. totara (Fig. 1A), P. hallii and P. acutifolius the site was terminal on the largest acrocentric pair whereas in P. nivalis it was terminal on the second largest acrocentric pair (Fig. 1G). In the Australian species P. lawrencei, there was a major interstitial site on one of the small chromosome pairs and minor sites in terminal positions on two other pairs of chromosomes (Fig. 1D). In prophase cells, these sites had a somewhat reticulate appearance with brightly fluorescing regions separated by non-fluorescing ones, most notable in P. lawrencei (Fig. 1E). In the F1 hybrid ‘Country Park Fire’ (P. lawrencei x P. nivalis), there were clear differences in the degree of reticulation of the two sites; one was similar to those in P. lawrencei, whereas the other was less diffuse and consisted of three clear foci.
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The 5S rDNA probes hybridized to similar sites to the 45S sites in all the taxa studied, a result that was shown by both single-target hybridization to chromosomes with characteristic morphology and double-target hybridization. In Fig. 1A and C, the longest acrocentric chromosomes of P. totara were labelled with the two rDNA probes. Collocalization was also seen in the hybrid ‘Country Park Fire’ (Fig. 1F), although the chromosomes with the rDNA hybridization sites were of different sizes and the exact site location also differed between the two labelled chromosomes. At prophase, the hybridization pattern with the 5S rDNA probe appeared very similar to that with 45S rDNA.
The telomere probe showed clear hybridization sites at the ends of all chromosomes of all taxa although the size of the sites did appear to vary both between and within chromosomes (Fig. 1A and B). The telocentric chromosomes were clearly labelled at both ends. There were a number of interstitial sites that were distributed along the chromosome arms of most of the chromosomes (Fig. 1A and B). Two simple sequence repeats, (AAC)5 and (GATA)4, showed a diffuse pattern of hybridization sites along the length of all chromosomes (GATA; Fig. 1C). No hybridization was seen with the (ACG)7 probe or with the two gypsy-like retroelements obtained from P. totara and P. nivalis.
The in situ hybridization protocol revealed up to four pairs of DAPI bands in some metaphases of P. lawrencei and its hybrids (Fig. 1D). None of the other species showed DAPI banding of their chromosomes (Fig. 1A, C and G).
Five retroelement clones, pDdgy1 from Dacrycarpus dacrydioides, pPongy2 from Podocarpus nivalis, pPotli3 from P. totara, pPonty3 from P. nivalis and pDdLi1 from D. dacrydioides, were used for Southern hybridization to DNA digests from Podocarpus, Dacrycarpus, Halocarpus and other representative gymnosperm genera (Table 2; Fig. 2). Each clone showed unique hybridization patterns and revealed differences in organization of different retroelements between genera, although most sequences tested were present in several genera. Probe pDdgy1 (Fig. 2A) was abundant in all Podocarpaceae species and also hybridized to DNA from Ginkgo biloba, but no hybridization was detected to Cycas circinalis. Probe pPongy2 (Fig. 2B) showed no hybridization to P. totara DNA or to the HindIII digests of P. nivalis and D. dacrydioides but showed distinct, though different, bands with the other taxa. Probe pPot3li (Fig. 2C) showed weak hybridization signal to all Podocarpaceae species, but strong signal with Picea abies and Ginkgo biloba. Similar results were obtained with Southern hybridization with the copia and LINE retroelement clones pPonty3 (Fig. 2D) and pDdli1 (Fig. 2E). pPonty3 showed low abundance in Podocarpaceae species, but strong hybridization to Picea abies and Ginkgo biloba DNA. pDdli1 showed only one fragment with HindIII digested DNA from Dacrycarpus (from which it was isolated) but strong hybridization to digested DNA from Podocarpus nivalis, Ginkgo biloba and Picea abies.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Unlike most other genera in the conifers, Podocarpus shows extensive karyological variation: species have chromosome numbers that include n = 9, 10, 11, 17 and 19, and karyotypes that comprise various combinations of metacentric, acrocentric and telocentric chromosomes (Hair and Beuzenberg, 1958; Davies et al., 1997). At the molecular level, our in situ hybridization results highlight both variable and constant features of the karyotypes. There is variation in the location and number of rDNA sites in the genus. All the species examined have only one major 45S rDNA site, but this is located on morphologically different chromosomes: in three of the New Zealand species it is terminal on the longest acrocentric chromosome, but in the fourth it is terminal on the small acrocentric chromosome. In the Australian species P. lawrencei, the major 45S site is interstitial and it has small additional terminal sites on two other pairs of chromosomes.
The 5S rDNA sites were tightly linked to the 45S sites in all Podocarpus species examined, with 5S and 45S signal collocalizing on the chromosomes. This collocalization has not been found in other gymnosperms although the 45S and 5S sites are adjacent in species such as Pinus sylvestris, Picea abies and Picea sitchensis (Lubaretz et al., 1996; Brown and Carlson, 1997). In two bryophytes, one liverwort (Marchantia polymorpha L.) and one moss (Funaria hygrometrica Hedw.), the 5S rDNA has been found to be inserted into the 45S rDNA repeat (Sone et al., 1999). Sone et al. (1999) suggest that this arrangement of rDNAs evolved after the separation of the bryophytes from other land plants. The molecular organization of the two rDNA repeats is unknown in Podocarpus, but understanding the organization of these major gene clusters has consequences for interpretations of the phylogeny of the group.
With respect to the 45S rDNA, the organization of the clusters on chromosomes in Podocarpus differs from that found in Pinus, Picea and Larix. In Pinus and Picea, the number of major 45S rDNA sites is in the range of six to ten pairs (Hizume et al., 1992; Brown et al., 1993; Doudrick et al., 1995; Jacobs et al., 2000) and there are three major pairs in Larix (Lubaretz et al., 1996). Several studies have reported the presence of additional minor sites, some of which are found at or near the centromere (Doudrick et al., 1995; Friesen et al., 2001).
In situ hybridization with the telomeric probe gave similar results in all the Podocarpus species. In addition to terminal hybridization sites, there were large numbers of interstitial sites distributed along the length of all chromosomes. Other works have shown that Pinus sylvestris chromosomes have terminal and interstitial telomeric sequence sites, some of which were centromeric (Fuchs et al., 1995), while Schmidt et al. (2000) found multiple, major intercalary sites in Pinus elliottii. In Picea abies and Larix decidua, hybridization signals were confined to chromosome ends (Lubaretz et al., 1996). Variability in the interstitial sites and copy number of the telomeric sequences seems to be characteristic of gymnosperms and their distribution is mirrored by that of the other simple sequence repeats that we have studied. No distribution pattern was evident on the chromosomes.
The presence of distinct DAPI bands in the Australian species P. lawrencei suggests differences in genomic organization from the New Zealand species. Previous studies reported that DAPI bands were absent from the karyotypes of the New Zealand species of Podocarpus (Davies et al., 1997), as confirmed here. One of the few northern hemisphere species, P. macrophyllus (Thunb.) Lamb., also shows conspicuous DAPI bands on approx. two-thirds of its large telocentric chromosomes (Hizume et al., 1988). These differences in banding patterns between species from different areas may be taxonomically significant as the status of the New Zealand and Australian species of the genus is under review (Molloy pers. comm.).
The retroelements that we isolated from Podocarpus and Dacrydium species were found to hybridize to DNA from a wide variety of other gymnosperms (Fig. 2); similar widespread hybridization has been reported in several other groups of plants (Kamm et al., 1996; Brandes et al., 1997). The patterns of membrane hybridization of PCR amplified retroelement probes reflected differences in copy number and organization between different species. Some of these results are surprising. For example, a copia probe from P. nivalis showed no hybridization to the DNA of its close New Zealand relative P. totara, but showed two distinct bands with P. lawrencei from Australia and strong hybridization to Picea abies and Ginkgo biloba. Similar results were seen with the LINE and gypsy probes: pPot3li from P. totara showed little hybridization to P. nivalis and greatest hybridization to Picea abies and Ginkgo biloba, and pPongy2 from P. nivalis did not hybridize to P. totara DNA (Fig. 2B). The absence of clear signals following in situ hybridization with the LINE and gypsy probes on the chromosomes of several of the Podocarpus species and hybrids is not unexpected given the low level of hybridization of the same or similar probes on the gels. These results suggest that most families of retrotransposons were present in ancestral gymnosperms before the diversification of the recognized families, and that during the evolution of the different taxonomic lines different families of retrotransposons were amplified and multiplied (Friesen et al., 2001).
As a major and often dominant vegetation type in both northern and southern hemispheres, the differentiation and evolution of genomes and chromosomes in the gymnosperms are important elements in understanding the phylogeny, diversity and evolution of the species themselves. In Pinaceae, the level of polymorphism of genetic markers in the group is generally considered to be low (Nelson et al., 1993) and their karyotypes exhibit considerable uniformity in number and chromosome morphology. Study of the conifers as a whole shows that the repetitive DNA sequences differ widely within the group, as in other groups (Heslop-Harrison, 2000). These sequences provide useful diversity, and hence the study of their molecular cytogenetics at both the sequence and karyotypic level provides valuable information for determining the relationships, evolution and diversity of the group.
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ACKNOWLEDGEMENTS |
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B.G.M. thanks the University of Auckland for sabbatical leave. We thank Graham Hutchins, Country Park Nursery, Hornchurch, Essex for the supply of plants and Dr Brian Molloy, Landcare Research, Lincoln, N.Z. for information on Podocarpus taxonomy. We thank EU Framework IV grant PL 962125 for support of this project to J.S.H-H.
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