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1984; Diem and Dommergues 1990) and C. equisetifolia and C. glauca are among ... S. Brown. Cytometric determination of genome size and base composition.
Plant Cell Reports (1998) 18: 346–349

© Springer-Verlag 1998

J. Schwencke · J.-M. Bureau · M.-T. Crosnier S. Brown

Cytometric determination of genome size and base composition of tree species of three genera of Casuarinaceae

Received: 20 December 1997 / Revision received: 13 March 1998 / Accepted: 30 March 1998

Abstract The genome size and base composition of diploid plant species from three genera of the Casuarinaceae family were determined by flow cytometry. Casuarina glauca Sieb. ex Spring. and Gymnostoma deplancheana (Miq.) L. Johnson showed a small genome with 2C = 0.70 pg, 58.6% AT, 40.5% GC for the first species and 2C = 0.75 pg, 58.7% AT, 40.5% GC for the second. Allocasuarina verticillata (Lam.) L. Johnson had a larger genome: 2C = 1.90 pg, 59.3% AT, 41.1% GC. One haploid genome of C. glauca is therefore about 340×106 base pairs. In leaves, roots or bark of these three species, polysomaty was virtually absent: a maximum frequency of 4C nuclei of only 0.08 was found in bark of C. glauca. The genome sizes of C. glauca and G. deplancheana are among the smallest described for higher plants. Small genome size, diploidy and the absence of polysomaty are advantageous traits for facilitating molecular approaches to improvement of these actinorhizal plants and developing the study of their symbiotic interactions with Frankia. Key words Casuarina glauca · Gymnostoma deplancheana · Allocasuarina verticillata · Actinorhizal plants · Frankia symbiosis

Introduction

Plants from the family Casuarinaceae are included among the large group of actinorhizal plants which can form endosymbiotic associations with species of Frankia, an actinomycete able to fix atmospheric nitrogen both in vitro and in planta (Baker and Schwintzer 1990; Akkermanns et al. 1992). Actinorhizal plants have economic potential as timber and fuel wood, as well as in forestry, agroforestry, biomass production, land reclamation or rehabilitation, and amenity planting (Midgley et al. 1981; Dawson 1986; Dommergues 1996). Trees from the Casuarinaceae have been largely utilised in a variety of climates from humid tropical to arid conditions (National Research Council 1984; Diem and Dommergues 1990) and C. equisetifolia and C. glauca are among the rare useful trees for desert afforestation and agroforestry (El-Lakany 1983, 1994). Differences in tree performance and susceptibility to insects (Hassan 1990) have triggered studies on clonal selection (Sougoufara et al. 1992) and improvement by molecular genetics for these species (Franche et al. 1994; Diouf et al. 1995). This encouraged us to analyse the genome size of species from three genera of Casuarinaceae. All three species had relatively small genome sizes and virtually no polysomaty. These are advantageous properties for species improvement by molecular genetics, for research in plant biology and for studying the Frankia-plant symbiotic interactions.

Communicated by A. M. Boudet J. Schwencke (½) 1 Biotechnologie des Symbioses Forestières Tropicales (ORSTOM-CIRAD-Forêt), 45 bis Av. de la Belle Gabrielle, F-94376 Nogent-sur-Marne, France J.-M. Bureau · M.-T. Crosnier · S. Brown Institut des Sciences Végétales, CNRS, UPR40, F-91198 Gif-sur-Yvette, France Present address: 1 LIGP-DIEP, Bât. 152, C.E.A.-Saclay, F-91191 Gif-sur-Yvette, France e-mail: [email protected]

Materials and methods Plant material Diploid species from the genera Allocasuarina, Casuarina and Gymnostoma of the Casuarinaceae were studied. Their chromosome number has been reviewed by Turnbull (1990). Our specific samples were not cytogenetically verified but originated from the seed collection of the BSFT: C. glauca Sieb. ex Spring. (2 n = 18) is lot 277 obtained from H. El-Lakany (DDC, Cairo), Allocasuarina verticillata (Lam.) L. Johnson (2 n = 20–28, generally 26) is lot 250 from Australia, and

347 Gymnostoma deplancheana (Miq.) L. Johnson (2 n = 16) is lot 278 from New Caledonia. Three field samples of C. glauca from Rabat, Morocco, and of G. deplancheana from New Caledonia were also analysed. For a botanical description of these species see Wilson and Johnson (1989). Our routine internal standard for flow cytometry was diploid Petunia hybrida P×PC6 (2 n = 2 x = 14) obtained from H. Dulieu (INRA, Dijon) and cytogenetically checked by ourselves. It has 2C = 2.85 pg with 41.0% GC (Marie and Brown 1993). Glasshouse conditions Seeds of Casuarina species were sterilised by immersing them for 2 min in concentrated H2SO4, then washed with sterile distilled water until they reached neutral pH. Sterilised seeds were germinated in a sterile bed of 2 : 1 vermiculite : sand mixture. Two-month-old seedlings were transferred into pots containing sterilised sand : vermiculite mixture (two plants for every 12-cm-diameter pot), irrigated twice a week with 1/4 Hoagland with NH+4 (Hoagland and Arnon 1950). Tissue samples for cytometry were from 6- to 12-month-old plants. To obtain hydroponic plants, 2-month-old seedlings growing in sterilised sand : vermiculite were directly transferred to 1/4 liquid Hoagland in appropriate containers. Evaporated water was replaced once a week and fresh Hoagland replaced every month. Nuclei preparation and genome size determinations Nuclei were prepared from the leaf tissue and stained for flow cytometry according to Galbraith et al. (1983) and Marie and Brown (1993). Fragments of the given species and from P. hybrida were chopped with a razor blade in 500 µl modified Galbraith buffer (0.2% Triton X-100 and 5 µg/ml 2-mercaptoethanol). After filtration through a 30-µm nylon filtrette (Brown et al. 1991), 5 units/ml DNase-free RNase A (Boehringer) and 50 µg/ml propidium iodide (Sigma) were added. After an incubation for 20 min at room temperature, the fluorescence intensity of the 2C nuclei relative to those of P. hybrida was assessed by flow cytometry as described by Marie and Brown (1993). Analyses were repeated three times for at least three glasshouse-grown plants, and for natural stands, with the exception of A. verticillata for which no natural stands could be obtained.

Results and discussion

The cytometric procedure was robust for this material inasmuch as the histograms were symmetrical and reproducible: the standard errors were low (Table 1). The average coefficients of variation of the 2C nuclei population were about 6% with propidium iodide, 8% with Hoechst 33342 and 9% with mithramycin. Unfortunately, leaves of A. verticillata were not amenable to analysis with mithramycin, even in alternative buffers. This did not prevent determination of the AT base composition. Nevertheless, the GC composition had to be estimated for A. verticillata using bark or roots. Genome size All three species had small genomes (Table 1). The 2C (pg) values found for these glasshouse-grown C. glauca (0.70) and G. deplancheana (0.75) are among the lowest known for higher plants (Bennett and Leitch 1995). The haploid (1C) genome of C. glauca is therefore 0.35 pg which converts to 340×106 base pairs. For comparison, Arabidopsis thaliana has 2C = 0.33 (Marie and Brown 1993), diploid Prunus spp. 2C = 0.55–0.77 pg (Arumuganathan and Earle 1991) and diploid Medicago truncatula (host plant of nitrogen-fixing Rhizobium meliloti) has 2C = 1.0 pg (Blondon et al. 1994). Values for A. verticillata (1.90) were threefold higher. These genome values are the first reported for trees from Casuarinaceae. Although our results concern only a small sample, there were no evident exceptions within these populations. For instance, mean values (n = 2–6) for three samples of G. deplancheana from natural stands growing in New Caledonia were 0.76, 0.78 and 0.78 pg and for three

Base composition The base composition of the genome was determined by independently measuring the 2C nuclear fluorescence relative to P. hybrida with either 5 µg/ml of bisbenzimide Hoechst 33342 (Aldrich), 50 µg/ml propidium iodide (Sigma) or 50 µg/ml mithramycin (Serva) with 50 mM MgCl2 and then applying the following equations (Godelle et al. 1993): %ATspecimen = %ATpetunia · (RHo/RPi)1/5

(1)

and %GCspecimen = %GCpetunia · (RMi/RPi)1/3

(2)

where RPi = intensityspecimen/intensitypetunia with the intercalating dye propidium iodide, RHo with Hoechst (AT dependent), and RMi with mithramycin (GC dependent).

Table 1 Genome size and base composition assessed by flow cytometry. All genome size values are from leaves of glasshouse-grown plants with the intercalating dye propidium iodide. The AT% was obtained using Hoechst 33342 and the GC% independently using mithramycin by the procedure of Godelle et al. (1993). Accordingly, the sum (AT + GC) may not by exactly 100%. Confidence intervals are not shown since this is a non-linear statistic. However, precision should be similar to that calculated for 2C. The internal reference was Petunia hybrida cv. P×PC6. These estimates all come from leaves except the GC value for A. verticillata obtained from bark, an identical value being found when using roots. Values in parentheses are the number of replicates Species

2n

Flow cytometry The flow cytometer (EPICS V, Coultronics France, Margency) with a 100-µm nozzle was configured as follows: for Hoechst 33342, 80 mW at 351 + 364 nm, filters 408LP and 530SP; for mithramycin, 50 mW at 458 nm, filter 570LP; for propidium iodide, 300 mW at 514 nm with filter 610LP The integral fluorescence intensity was taken after both linear and logarithmic amplification (with logical elimination of any debris and doublets through light scatter and peakarea analysis). Each histogram corresponded to about 5000 nuclei.

Genome size (2C ±SE) (pg)

Base composition (%) AT

GC

AT + GC

59.3 (7)

41.1 (8) 100.4

Allocasuarina verticillata

20–28 1.90 ±0.01 (8)

Casuarina glauca

18

0.70 ±0.02 (15) 58.6 (21) 40.5 (9)

99.1

Gymnostoma deplancheana

16

0.75 ±0.03 (7)

99.2

58.7 (12) 40.5 (2)

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young glasshouse-grown plants 0.74, 0.76 and 0.77 pg per 2C genome. The mean AT composition estimated with these field samples (12 months after the glasshouse samples) was 59.2% (n = 22) and for GC 41.3% (n = 20), which we consider simply confirms the data of Table 1. Chromosome number increases from G. deplancheana (2 n = 16) to C. glauca (2 n = 18) and finally A. verticillata (2 n = 26). Allocasuarina species reportedly also have larger chromosomes, concordant with our size assessment here. The haploid number (x = 9) is thought to be the basic one for the family Casuarinaceae (Barlow 1959 a, b; Turnbull 1990). Genome analysis repeated after 1 year for all glasshouse-grown plants gave essentially the same results, i.e. for G. deplancheana mean estimates with propidium iodide were within 1.7%. Our mean estimates (n = 4) on nuclei from bark and roots were within 1.5% for A. verticillata and 3.8% for C. glauca, differences which we attribute to methodological factors. Plants with small genome size may show strong polysomaty, as does, e.g. A. thaliana (Galbraith et al. 1991). However, differentiated leaf tissue from these species of the Casuarinaceae had nuclei which were essentially 2C (Fig. 1) as common in tree species: for example, as found for poplar (Jehan et al. 1994), Acacia spp. (Coulaud et al. 1995) and seven oak species (Zoldos et al. 1998). This was the case whether analysing leaves, roots (from hydroponic C. glauca plants and soil-grown A. verticillata) or bark of young (glasshouse grown) and old plants (naturally growing): the maximum frequency of 4C nuclei found was 0.08 in C. glauca bark, the rest being 2C only. Molecular phylogeny has shown that in the Casuarinaceae, the genus Gymnostoma derived before the genera Casuarina and Allocasuarina, these more recent genera being more selective towards Frankia strains (Maggia and Bousquet 1994). With a haploid chromosome number of 13, Allocasuarina probably derived by evolutionary hybridisation followed by doubling to a tetraploid genome. Recent interest for the improvement of trees from the Casuarinaceae by molecular techniques (Franche et al. 1994; Diouf et al. 1995; Bogusz et al. 1996) has been triggered by the known variation in their performance in the field, as well as their sensitivity to some insects (Hassan 1990; Sougoufara et al. 1992). In this context, the quite small genomic size found here for species from the Casuarina and Gymnostoma genera implies that these plants could be good biological material for genetic engineering. Since plants from the Gymnostoma genus, including G. deplancheana, are usually difficult to culture in vitro and in greenhouse conditions, C. glauca appears to be the better choice. Allocasuarina species are known to have larger chromosomes, to present polyploid states and apomixis (Turnbull 1990). Infective and effective Frankia strains have been isolated from nodules of all these trees and, moreover, improved exponential growth conditions for these Frankia strains have been described (Girgis and Schwencke 1993; Selim and Schwencke 1994; Selim et al. 1996). Furthermore, reliable nodulation conditions have been set up for Casuarina (Selim and Schwencke 1995). Therefore, these actinorhizal species appear to be interest-

Fig. 1 Typical histograms of fluorescence intensity (linear scale) of nuclei obtained from roots or leaves of plants from the Casuarinaceae, using the 2C nuclei of Petunia hybrida as internal standard, after staining with Hoechst 33342. Results represent measurements of about 5000 individual nuclei

ing models for the study of the specific regulatory mechanisms which control the expression of the symbiotic genes. In reality, most of the available information on this important subject is restricted today to the legume-rhizobia symbiosis. Acknowledgements We gratefully acknowledge Prof. E. Duhoux and Dr. Y. D. Dommergues for critical review, Danièle De Nay for flow cytometry, Dr. Y. Kouchkovsky for help with graphics and the co-operation of R. Pelletier with greenhouse conditions. We are deeply indebted to Dr. M. Arahou (University Hassan IV, Morocco) and Dr. T. Jaffré (ORSTOM, New Caledonia) for providing us with field samples of C. glauca and G. deplancheana. This work was partially supported by a grant from the EEC.

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