Environmental and Experimental Botany 55 (2006) 258–265
Genome size in Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae) populations: separation of “true” differences from environmental effects on DNA determination David J. Walker∗ , Inmaculada Mo˜nino, Enrique Correal Departamento de Recursos Naturales, Instituto Murciano de Investigaci´on y Desarrollo Agrario y Alimentario (IMIDA), Estaci´on Seric´ıcola, Calle Mayor, s/n, 30150 La Alberca (Murcia), Spain Accepted 16 November 2004
Abstract Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae) is a perennial legume of interest due to its use as a feed for livestock and its contents of furanocoumarins. Nuclear DNA contents of six populations of B. bituminosa, originating from mainland Spain (2), the Canary Islands (3) or Sardinia (1), were determined. Flow cytometry was used to estimate the 2C nuclear DNA contents of the selected populations, using leaf material from plants grown under field conditions in Southern Spain and analysed in December 2002, April 2003 and August 2003. For December and April, apparent mean values ranged from 0.998 to 1.121 pg, with Canarian populations exhibiting values lower than those from mainland Spain or Sardinia. Thus, there was true intraspecific variation in nuclear DNA content, related to the geographic origin of the populations and irrespective of environmental factors, for B. bituminosa. The mean nuclear DNA content was correlated significantly (P < 0.05) with the longitude (◦ E) and the mean temperature (◦ C) of the warmest month at the sites from which the populations originated. Except for the population from Mijas (mainland Spain), the estimates of nuclear DNA made using leaves collected in August (summer; average daily maximum temperature 35–36 ◦ C) were significantly lower (by between 3.8 and 7.4%) than those obtained in December (winter; average daily maximum temperature 18–21 ◦ C). This environmentally induced (artefactual) variation within populations, with apparent decreases in summer, may have been due to interference from leaf furanocoumarins, which are known to accumulate to a greater extent at higher temperatures. The results highlight the care that must be taken in the critical estimation of nuclear DNA contents by flow cytometry, particularly for species such as B. bituminosa which are capable, according to the environmental conditions, of accumulating significant amounts of secondary compounds. © 2004 Elsevier B.V. All rights reserved. Keywords: Bituminaria bituminosa (L.) C.H. Stirton (syn. Psoralea bituminosa L.); Flow cytometry; Fluorochrome interference; Intraspecific variation; Nuclear DNA; Secondary metabolites
∗
Corresponding author. Tel.: +34 968 366767; fax: +34 968 366792. E-mail address:
[email protected] (D.J. Walker).
0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.11.005
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1. Introduction When plant species having geographically-isolated populations have been investigated with respect to nuclear DNA content, only negligible differences have been found in certain cases (Lys´ak et al., 2000; Ellul et al., 2002), but significant differences have been reported also (Nandini et al., 1997; Keskitalo et al., 1998; Turpeinen et al., 1999; Emshwiller, 2002). Such intraspecific variation in DNA content has been attributed to both genetic instability (Price et al., 1983) and to environmental factors, such as nutrient supply, light and temperature (Johnston et al., 1996; Turpeinen et al., 1999). However, concerns, related to taxonomy and methodology, have been expressed and the true extent of intraspecific variation in genome size is unclear (Greilhuber, 1998; Price et al., 2000; Emshwiller, 2002; Noirot et al., 2003). One problem with the determination of nuclear DNA by flow cytometry is the possibility of interference by compounds in the plant tissue, which are released during sample preparation (Noirot et al., 2000, 2003; Price et al., 2000). The species used for the current study, Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae, Psoraleeae) (Stirton, 1981a,b) (Psoralea bituminosa L.), is a perennial species, widely distributed in the Mediterranean Basin and Macaronesia and used as a fodder crop (M´endez et al., 1991; Mu˜noz and Correal, 1998). The somatic chromosome number in the genus Bituminaria is 2n = 2x = 20 (Stirton, 1981b). M´endez et al. (1991) discriminated morphologically three varieties of B. bituminosa found in the Canary Islands: var. albomarginata (syn. var. palestina sensu Webb & Berth., pro parte), var. crassiuscula and var. bituminosa. The cultivar (cv.) “Tenerife” (var. bituminosa) is grown widely on the island of Tenerife to provide livestock feed. Determination of the nuclear DNA content of B. bituminosa may, potentially, be complicated by its contents of the furanocoumarins psoralen and angelicin (M´endez et al., 2001), which act as repellents and feeding deterrents (Calcagno et al., 2002) and are of commercial interest due to their use in cosmetics and photochemotherapy (Innocenti et al., 1991). UV-activated furanocoumarins can cause both crosslinking of DNA strands and (via oxidation of phenolics) breakage of strands (Ashwood-Smith et al., 1977; Appel, 1993; Sastry et al., 1997; Calcagno et al., 2002).
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For this study, field-grown plants of three Canary Island (Spain) populations, two populations from mainland Spain and a population from Sardinia (Italy) were chosen, to determine the extent of any intraspecific variation in nuclear DNA content among them, using flow cytometry. This represents one aspect of a project aimed at identifying and utilising the genetic, physiological and agronomic variability within B. bituminosa. To separate “true” intraspecific variation from possible furanocoumarin interference with estimation of nuclear DNA, we sampled material at three different times of year, since tissue levels of furanocoumarins are affected by environmental factors such as temperature and UV light (Bourgaud et al., 1992, 2001).
2. Materials and methods 2.1. Plant material Except for cv. Tenerife, the seeds of which were supplied by a local farmer, seeds of B. bituminosa were collected from native stands (Table 1). In 1998, seedlings raised from these seeds were planted in a field plot at La Alberca, Murcia (Spain) (37◦ 56 N, 1◦ 08 W). Plants were irrigated as necessary. It was decided to use fieldgrown plants, rather than growth chamber plants, to utilise the natural variation in climate at La Alberca and the fact that a meteorological data station is located only 40 m from the plants. Also, the summer extremes of temperature and irradiance in the field exceeded those which could be achieved in the growth chamber. 2.2. Flow cytometry The 2C nuclear DNA content was determined by flow cytometry. For the plants of B. bituminosa, cultivated in the field at La Alberca, this was performed in the first week of December 2002, the first week of April 2003 and the second week of August 2003. For each of the six populations, three growing leaves from each of four plants were analysed, one plant on each day of analysis. For each month, analyses were performed on four days, within a single week. Samples of growing leaf tissue of B. bituminosa and Glycine max (L.) Merr. cv. Polanka, cultivated in a growth chamber with a day/night temperature of 25/20 ◦ C, (approximately 20 mg fresh weight each),
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Table 1 Original locations, altitude and climate information of the populations of Bituminaria bituminosa Population Boca de Tauce cv. Tenerife Famara Sardinia Llano del Beal Mijas
Variety
Co-ordinates
Country of origin
Altitude (m)
Mean temperature in coldest/hottest month (◦ C)
crassiuscula bituminosa albomarginata bituminosa bituminosa bituminosa
28◦ 14 N,
Spain Spain Spain Italy Spain Spain
2200 450 600 7 300 209
4.0/17.4 11.7/21.4 11.1/22.8 10.0/23.1 10.7/26.1 11.5/25.9
16◦ 34 W
28◦ 10 N, 16◦ 29 W 29◦ 7 N, 13◦ 31 W 40◦ 34 N, 8◦ 19 E 37◦ 36 N, 0◦ 47 W 36◦ 36 N, 4◦ 38 W
were prepared together. A greater amount of G. max tissue was not used, in order to prevent merging of the G. max G0 /G1 peak with the G2 peak of B. bituminosa. Leaf material was chopped for 40–60 s with a razor blade, within a plastic Petri dish containing 0.2 ml of extraction buffer (Partec CyStain PI Absolute P Nuclei Extraction Buffer; Partec GMBH, M¨unster, Germany), containing the following anti-oxidants: polyvinylpyrrolidone-10 (5%, w/v), ascorbic acid (12 mM) and dithiothreitol (10 mM). In July 2002 (mid-summer), this combination of anti-oxidants was found to be optimal for minimisation of the % coefficient of variation (C.V.) = (100 × standard deviation)/mean of the nuclear DNA peaks. The resulting extract was diluted with 0.2 ml of extraction buffer (–anti-oxidants) and then passed through a 30-m filter into a 3.5-ml plastic tube. To this filtrate was added 1.6 ml of Partec CyStain PI Absolute P Staining Buffer, containing propidium iodide (PI) and RNase, to give final concentrations of 50 and 17.5 g ml−1 , respectively. All stages of the extraction and staining were performed at 2–5 ◦ C, in near-darkness to minimise possible interference from UV-activated furanocoumarins (Calcagno et al., 2002). Samples were kept at 4 ◦ C for 10–60 min (mean of 30–35 min for each population) before analysis by flow cytometry. The time spent at 4 ◦ C before analysis did not affect the estimation of DNA content. For example, estimates of the mean 2C nuclear DNA content of population Boca de Tauce, using leaves of plants grown in a growth chamber with a day/night temperature of 22/18 ◦ C and co-prepared with G. max, were 0.980 and 0.985 pg, respectively (n = 4), after 10 and 60 min incubation at 4 ◦ C. These two estimates did not differ significantly (P = 0.516) according to a student’s t-test for related values (t = −0.734). Samples were analysed with a Partec PA II flow cytometer, employing a 20 mW argon ion laser light
source (488 nm wavelength) (model PS9600, LG-Laser Technologies GmbH, Kleinostheim, Germany) with an RG 590 longpass filter. For each sample, 10,000 nuclei were analysed. B. bituminosa nuclear DNA was estimated by the internal standard method, using the ratio of the B. bituminosa/G. max G0 /G1 peak positions (Doleˇzel, 1997). The precision and linearity of the flow cytometer were checked on a daily basis using 3 m calibration beads (Partec). In addition, on each day of analysis, three leaf samples of G. max were co-prepared (20 mg fresh weight each) with leaf tissue of tomato (Lycopersicon esculentum Mill.) cv. Stupnick´e poln´ı (Doleˇzel et al., 1992; Suda et al., 2003), grown in the same growth chamber (day/night temperature 25/20 ◦ C). Any differences in tomato 2C nuclear DNA content, estimated using G. max as internal standard, would indicate that differences in B. bituminosa nuclear DNA observed among the three chosen sampling times were due to random variations in the cytometry equipment or method (Doleˇzel et al., 1998). 2.3. Statistical analysis For the log-transformed values of the nuclear DNA contents of the B. bituminaria populations and tomato (for which the standard deviations did not differ significantly, according to Cochran’s C-test and Hartley’s test), ANOVA (General Linear Model) was performed, using SPSS software version 11.0, to separate effects of population or time (month) of sampling. Differences between mean values were determined using the Student–Newman–Keuls test. Using SPSS software, Spearman rank correlation coefficients (r) were calculated between nuclear DNA content and the mean temperatures (◦ C) of the warmest and coldest months, the latitude (◦ N) and the longitude (◦ E) of the original locations of the populations.
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3. Results For estimation of the nuclear DNA amount in B. bituminaria, G. max was an appropriate internal standard, since its own 2C nuclear DNA, 2.50 pg (Doleˇzel et al., 1994; Suda et al., 2003), was similar to that of B. bituminaria (Fig. 1). For B. bituminosa, C.V. values for the G0 /G1 peak were 3.5–5.0%. Significant differences between populations (F = 105.64, P < 0.001) were found with respect to their mean 2C nuclear DNA content (Table 2). The Canarian populations (Famara, Boca de Tauce and cv. Tenerife) had lower values than the populations from Sardinia or mainland Spain (Llano del Beal and Mijas). Fig. 2 demonstrates clearly the greater nuclear DNA content of population Mijas compared to that of Boca de Tauce, for leaf material obtained from plants grown in a growth chamber with a day/night temperature of 22/18 ◦ C and co-prepared for flow cytometry with G. max. Famara had a greater DNA content than Boca de Tauce. The month of sampling had a significant effect on nuclear DNA estimation (F = 117.78, P < 0.001). Except for Mijas, all populations, when compared to
Fig. 1. Histogram of relative nuclear DNA content of Bituminaria bituminosa population Boca de Tauce, determined by flow cytometry analysis of propidium iodide-stained nuclei with Glycine max (2C nuclear DNA content 2.50 pg) as internal standard. Nuclei of B. bituminosa and G. max were isolated, stained and analysed simultaneously.
their December values, showed no change in April but a significant decline in August (between 3.6 and 7.4%). There was no significant correlation (P ≥ 0.05) between mean nuclear DNA content and mean temperature (◦ C) of the coldest month at the original lo-
Table 2 Flow cytometry estimates of 2C nuclear DNA content (mean ± S.D., n = 12) for six populations of B. bituminosa, using Glycine max as internal standard Population
Month of determination
2C Nuclear DNA (pg)a
Percentage of maximum value
Boca de Tauce
December April August
1.007 ± 0.023 c 1.000 ± 0.024 c 0.946 ± 0.013 a
100 99.3 93.9
cv. Tenerife
December April August
0.998 ± 0.018 c 1.000 ± 0.029 c 0.962 ± 0.020 ab
99.8 100 96.2
Famara
December April August
1.027 ± 0.018 cd 1.040 ± 0.023 d 0.971 ± 0.022 b
98.8 100 93.4
Sardinia
December April August
1.094 ± 0.043 f 1.091 ± 0.033 f 1.013 ± 0.026 c
100 99.7 92.6
Llano del Beal
December April August
1.066 ± 0.028 ef 1.080 ± 0.018 ef 1.019 ± 0.015 cd
98.7 100 94.3
Mijas
December April August
1.070 ± 0.032 ef 1.121 ± 0.025 g 1.062 ± 0.017 e
95.5 100 94.7
Assays were performed on four different days in each month. a Means followed by different letters (a–g) are significantly different (P < 0.05) according to the Student–Newman–Keuls test.
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D.J. Walker et al. / Environmental and Experimental Botany 55 (2006) 258–265 Table 3 Flow cytometry estimates of 2C nuclear DNA content (mean ± S.D., n = 12) in tomato (cv. Stupick´e poln´ı), using G. max as internal standard Month
2C Nuclear DNA (pg)a
December April August
2.044 ± 0.053 2.050 ± 0.028 2.039 ± 0.042
Assays were performed on the same days as for B. bituminosa (Table 3). a Means did not differ significantly (F = 2.31, P = 0.115) according to the ANOVA.
estimated values were highest for both tomato and B. bituminosa population Sardinia. Fig. 2. Histogram of relative nuclear DNA contents of B. bituminosa populations Boca de Tauce and Mijas, determined by flow cytometry analysis of propidium iodide-stained nuclei with G. max (2C nuclear DNA content 2.50 pg) as internal standard. Nuclei of B. bituminosa and G. max were isolated, stained and analysed simultaneously.
cations of the populations (Spearman rank correlation coefficients, r) (data not shown). The mean DNA contents in each of the three months when analyses were performed were correlated significantly with longitude (◦ E) (r = 0.771–0.886, P ≤ 0.036, n = 6), altitude (m) (r = −0.771 to −0.841, P ≤ 0.036, n = 6) and with the mean temperature of the warmest month at the original locations (◦ C) (r = 0.765–0.928, P ≤ 0.038). For latitude (◦ N), there were significant correlations with the mean nuclear DNA values of December and April (r = 0.812-0.943, P ≤ 0.025). To determine whether differences in nuclear DNA content of B. bituminaria among months were due to plant factors (e.g. due to climatic variations) or random variations in the procedure/equipment used, we determined tomato 2C nuclear DNA values with G. max as internal standard, using leaf material of both species grown under the same, controlled conditions. In Table 3, it can be seen that the mean estimate of tomato nuclear DNA did not differ significantly among the three months (F = 2.31, P = 0.115). This suggests that differences among sampling times in the 2C nuclear DNA values for the B. bituminosa populations were due to plant factors. However, in December, there were significant differences among days with respect to estimates of tomato nuclear DNA, which presumably arose as a result of uncontrolled experimental variations; for example, on the fourth day of determinations,
4. Discussion The main aim of this work was to determine, using flow cytometry, whether differences in nuclear DNA content exist among selected populations of B. bituminosa originating from different geographical locations. Flow cytometry is used widely for determination of plant genome size, due to its speed and simplicity (Doleˇzel, 1997; Bennett et al., 2000). However, secondary compounds, released during sample preparation, can interfere with the estimation of nuclear DNA content by flow cytometry, due to effects on dye-binding and other processes (Noirot et al., 2000, 2003; Price et al., 2000). This can produce apparent variations in nuclear DNA content, which can be attributed erroneously to a direct environmental effect (Johnston et al., 1996). To highlight any environmental effects on the plants which could interfere with the assays, the flow cytometric analyses were performed in December 2002, April 2003 and August 2003, with leaves of plants exposed to differing conditions of temperature and irradiance (mean maximum/minimum temperatures in the month prior to the analysis were 20/10, 21/10 and 36/22 ◦ C, for December 2002, April 2003 and August 2003, respectively). The apparent differences in B. bituminosa 2C nuclear DNA content observed among these months do not seem to be due to random variations in the procedure/equipment (Doleˇzel et al., 1998) since the estimates of tomato DNA content, determined with G. max as internal standard, did not differ among the three sampling times. So, variation among months
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with respect to estimation of B. bituminosa nuclear DNA was probably related to plant factors. B. bituminosa synthesises the furanocoumarins angelicin and psoralen (Innocenti et al., 1991; M´endez et al., 2001). It has been reported that leaf levels of psoralen increase under conditions of high temperature (32 ◦ C versus 21 ◦ C) in Psoralea cinerea (Bourgaud et al., 1992) and, more generally, that furanocoumarin levels increase in response to higher temperatures or UV supply (Bourgaud et al., 2001). This suggests that, in the current work, furanocoumarin accumulation, at summer temperatures in excess of 30 ◦ C, could have been responsible for the apparently lower nuclear DNA contents in August (Table 2). Furanocoumarins acting in situ in intact leaves would mean that performing DNA extraction in near-darkness and in the presence of anti-oxidants would have had little or no effect. Angelicin and psoralen could affect estimation of nuclear DNA in two ways. Firstly, upon activation by UV light, they can form interstrand cross-links and monoadducts, respectively, with DNA, resulting in chromosomal aberrations (Ashwood-Smith et al., 1977; Sastry et al., 1997). Secondly, furanocoumarins can oxidise phenolic compounds, leading to the formation of oxygen radicals which can break strands of DNA (Appel, 1993). However, there were intraspecific differences in nuclear DNA content among the studied populations of B. bituminosa, which were constant across the three months of analysis (Table 2). This apparent “true” intraspecific variation mirrors previous observations for other species (Nandini et al., 1997; Keskitalo et al., 1998; Turpeinen et al., 1999; Lys´ak et al., 2000; Emshwiller, 2002) for comparable work. Such differences may represent adaptations of populations to differing edapho-climatic conditions (Price et al., 1986; Turpeinen et al., 1999). There were significant correlations between nuclear DNA content and altitude (m) (negative correlation), longitude (◦ E) and the average temperature (◦ C) in the warmest month at the original location. For altitude and temperature, this may reflect effects on the growing season, since a smaller genome size may permit more rapid cell division and thus greater growth at sites with shorter growing seasons (Price et al., 1986; Turpeinen et al., 1999). The Canarian populations (Boca de Tauce, Famara and cv. Tenerife) had lower values than the populations from Sardinia or mainland Spain (Llano del Beal and
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Mijas). For populations Famara (var. albomarginata), due to low rainfall (108 mm per year), and Boca de Tauce (var. crassiuscula), due to lower temperatures, this may be due to shorter effective growing seasons, relative to the populations from mainland Spain and Sardinia. The smaller genome size of cv. Tenerife, less restricted by climatic conditions, may have resulted from a commercial selection of faster-growing plants. Suda et al. (2003), in a study of 104 Macaronesian angiosperms, found that the majority of taxa had 1C nuclear DNA amounts below 1.6 pg and that the values were generally lower than for their non-Macaronesian counterparts, in agreement with the current findings for B. bituminosa. Other members of the Fabaceae have been found, like B. bituminosa, to have a relatively small genome size (Bennett et al., 2000). Var. bituminosa occurs on all the Canary Islands, but var. crassiuscula (population Boca de Tauce) is restricted to altitudes of 1700–2200 m on Tenerife and var. albomarginata is endemic to Lanzarote (M´endez et al., 1991). Since cv. Tenerife and the studied populations from mainland Spain and Sardinia are all var. bituminosa, it is possible that the varieties albomarginata and crassiuscula represent subsequent colonisations and adaptations to differing local conditions, following the isolation on Lanzarote of a population of var. bituminosa (Suda et al., 2003). A principal component analysis using morphological traits, both quantitative (e.g. internode length) and qualitative (e.g. flower colour), also separated the Canarian populations from those of mainland Spain (Mu˜noz et al., 2000).
5. Conclusions This work highlights further some of the difficulties encountered in flow cytometric determination of plant nuclear DNA, particularly for species such as B. bituminosa which are capable, according to the environmental conditions, of accumulating significant amounts of secondary compounds. However, the results also show that “true” intraspecific variation in genome size seems to exist for B. bituminosa; 2C nuclear contents ranging from 1.000–1.040 for Canarian populations to 1.070–1.121 for populations from Sardinia or mainland Spain.
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Acknowledgements Dr. Pilar M´endez (Instituto Canario de Investigaciones Agrarias, La Laguna, Tenerife, Spain), for providing seeds of the Canarian populations of B. bituminosa, and Dr. J. Doleˇzel (Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic), for providing seeds of Glycine max cv. Polanka and tomato cv. Stupnick´e poln´ı. This work was funded by the Instituto Nacional de Investigaci´on Tecnolog´ıa Agraria y Alimentar´ıa (Spain), Project no. RTA01026-C3-2 “Caracterizaci´on, selecci´on y mejora de Bituminaria bituminosa para aprovechamiento ganadero y revegetaci´on de zonas degradadas”.
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