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Phycology |
Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Experimentelle Phykologie und Sammlung von Algenkulturen (SAG), Universität Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany; Culture Collection of Algae and Protozoa (CCAP), Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Dunbeg, Argyll, PA37 1QA, UK; DAMAR, Drum Road, Cupar Muir, Fife, KY15 5RJ, Scotland, UK; SequentiX–Digital DNA Processing, Dorfstr. 20, 18249 Klein Raden, Germany
Received for publication March 1, 2006. Accepted for publication March 13, 2007.
ABSTRACT
Cryopreservation is the long-term, indefinite storage of living biological resources at ultralow temperatures. It is almost universally assumed that cryogenic storage supports genetic and phenotypic stability of organisms. However, certain components of the cryopreservation process, particularly some cryoprotective additives (CPAs) and free radical mediated cryoinjury, may potentially cause genetic alterations. Genetic integrity in cryopreserved microalgae was assessed using a very sensitive molecular fingerprinting technique, AFLP, on 28 terrestrial microalgal strains. In about half of all investigated strains the AFLP fingerprints revealed, with high levels of reproducibility, clearly detectable genomic differences after cryopreservation employing a widely used standard two-step cooling protocol. Differences ranged from a single fragment position to multiple fragment changes and were compared to differences found between wild-type and UV-light- or radioisotope-induced mutants of Parachlorella kessleri. The basis of the changes are discussed in terms of their reversibility, as may be the case if they are attributed to DNA methylation and/or whether they are true mutations that may potentially manifest in the phenotype. The possibility that cryopreservation selects for genotypically different subpopulations of microalgae is also considered.
Key Words: AFLP • algae • cryopreservation • culture collections • DNA methylation • genetic stability
Microscopic algae are possibly the most diverse group of photosynthetically active organisms. They are one of the most important and abundant groups of global primary producers, thriving in almost all habitats on earth. Approximately 40–50% of the oxygen in the atmosphere can be attributed to them, and they constitute the original source of fossil carbon found in crude oil and natural gas (Andersen, 2005
). Furthermore, microalgae, including cyanobacteria, are employed in a broad range of biotechnological applications as health food, feed organisms, and sources of antioxidants or antimicrobial agents (Cohen, 1999
; Richmond, 2004
). Biotechnology, modern genomics, and increased concern about the loss/extinction of plant genetic resources all act as stimuli to increase the need to conserve ex situ, valuable, and representative plants including microalgal strains. In addition, there is an absolute need for genetically stable cultures of algae (and/or plant/animal germplasm) for use in biotechnology.
In microalgae the most common method for maintenance is serial subculturing of actively growing cultures under suboptimal conditions (Lorenz et al., 2005
). This method has the disadvantage of being labor intensive, and furthermore, the genetic and phenotypic stability of the strains cannot be guaranteed over years of routine maintenance (Day and Brand, 2005
). Loss of phenotypic characteristics during serial subculturing has been evidenced in several microalgal cultures (Day et al., 2005
); therefore, cryopreservation has been explored as an alternative option to serial culture. Cryopreservation is already used in addition to subculturing in several service algal culture collections worldwide (e.g., Culture Collection of Algae and Protozoa, CCAP, UK; Culture Collection of Algae at the University of Texas, UTEX, Texas, USA; National Institute for Environmental Studies, NIES, Japan). Cryopreservation obviates the need for labor-intensive serial subculture and, therefore, the number of specimens that may be held in a collection can be increased. Storage in liquid nitrogen or its vapor phase (ca. –139°C to –196°C) is assumed to prevent genetic changes, and certainly at extremely low subzero temperatures, metabolic activity is considered to cease (Grout, 1995
). Cryogenic storage has therefore increasingly been used for plants, animals, and medical applications (Fuller et al., 2004
). However, the concept of genetic stability after cryopreservation is largely untested. The cryopreservation process itself may induce various stresses on the organism via intracellular ice formation, cryoprotectant toxicity, and/or osmotic shock (Fleck, 1998
; Day et al., 2000
). During cryoinjury, cryoprotectants and/or oxidative stress may cause the formation of free radicals, which may potentially lead to genetic alterations (Fleck et al., 2000
; Benson and Bremner, 2004
). In the only report that evaluated genetic stability in microalgae after long-term cryostorage, no genomic differences could be detected between duplicate strains of the same isolate of Chlorella vulgaris Beijerinck from the CCAP culture collection after continuous subculture over several decades or storage for >20 yr at ultralow temperatures (Müller et al., 2005
). Because there has been no systematic study on the genetic stability of other algae after cryopreservation, genetic stability assessments of microalgae after cryopreservation are both timely and essential for assisting cryopreservation protocol evaluation and their validation.
Among genetic marker techniques with high resolution (i.e., distinguishing below the level of species), randomly amplified polymorphic DNA (RAPD; De Verno et al., 1999
), simple sequence repeat analysis (SSR; Harding and Benson, 2001
), or amplified fragment length polymorphism (AFLP; Wilkinson et al., 2003
) have provided evidence for stability in higher plant material after cryopreservation (De Verno et al., 1999
; for review see Harding, 2004
; Harding et al., 2005
). In the present study, we generated AFLP fingerprint patterns before and after cryopreservation to assess genetic stability in selected strains of microalgae. The AFLP technique described by Vos et al. (1995)
is a fingerprint technique that is widely used to study biodiversity of higher plants at the molecular level. It combines the advantages of RAPDs, known to have reproducibility problems, but the entire genome can be randomly sampled, and of SSRs, that are highly reproducible, but noncoding regions are selectively sampled over the genome.
AFLP permits the simultaneous analysis of many loci widely spread over the entire genome, without prior sequence knowledge of the organisms under study. It has been found to be a very sensitive, reliable fingerprinting technique to resolve differences between isolates of the same species in a broad range of taxa including bacteria, animals, plants, and microalgae (e.g., Mueller and LaReesa, 1999
; Savelkoul et al., 1999
; Müller et al., 2005
). Resolution of AFLP patterns established genetic distances between geographical isolates of algae (Donaldson et al., 1998
; Mannschreck et al., 2002
; Schaeffer et al., 2002
; Murphy and Schaffelke, 2003
; De Bruin et al., 2004
; Erting et al., 2004
; John et al., 2004
) as well as genetic differences among different isolates of the same species, Chlorella vulgaris (Müller et al., 2005
).
In the present study, 28 terrestrial, eukaryotic microalgae including one brackish isolate were examined. The test strains represent a variety of different cell morphologies such as unicells with different types of cell walls, colonies, motile and nonmotile cells, and filaments. A widely used un-optimized two-step cooling cryopreservation protocol (Taylor and Fletcher, 1999
) with dimethylsulfoxide (DMSO) or methanol as the cryoprotectant was used for all strains. In addition, the possible effects of stress caused by three successive cycles of freezing/thawing were investigated for five strains. Reproducibility of the AFLP technique was tested for all strains, allowing one to distinguish between nonreproducible fragments (e.g., due to PCR anomalies) and true postcryopreservation differences. To test the resolution of the AFLP technique and to provide a marker to detect genomic differences, we compared UV light- and radioisotope-induced mutants and wild-type strain of Parachlorella kessleri.
MATERIALS AND METHODS
Algal strains, culture conditions
Algal test strains (Table 1) were mainly from Chlorophyta (25 species, with representatives from the classes Chlorophyceae, Trebouxiophyceae, and Ulvophyceae), and one member was from Streptophyta (Cosmarium cucumis), one from Euglenophyta (Euglena gracilis), and one from the Bacillariophyta (Phaeodactylum tricornutum). The algal strains investigated had not been cryopreserved previously (except strain CCAP 216/1), so that there was no possibility that the strains were preconditioned by any treatments. However, the algal strains chosen were known to differentially survive cryopreservation; with high levels of viability, with low viability levels (e.g., Chlamydomonas reinhardtii [Crutchfield et al., 1999
; Brand and Diller, 2004
]; or Euglena gracilis [Morris and Canning, 1978
; Day et al., 2000
]), as well as cryorecalcitrant organisms (e.g., Pseudendocloniopsis, Chlamydocapsa, J. G. Day, unpublished data). Algal cultures were obtained from the Culture Collection of Algae at Göttingen University (SAG, Göttingen, Germany, http://www.epsag.uni-goettingen.de). In addition, Coccomyxa arvernensis CCAP 216/1 was obtained from the Culture Collection of Algae and Protozoa (CCAP, Dunbeg, UK, http://www.ccap.ac.uk). This is a duplicate strain of strain SAG 216-1, but CCAP 216/1 has been kept in a cryopreserved state for 27 yr at the CCAP culture collection, while strain SAG 216-1 was maintained by serial transfer and was not cryopreserved before this study. Mutants of Parachlorella kessleri SAG 211-11h previously induced by radioisotopes (SAG 211-11h/9) or UV light (SAG 11.80) were used for comparison. Both mutants differ phenotypically from their green wild type in pigment composition, i.e., by the yellow or orange-brown appearance of the algal colonies on agar. All strains were cultured in the appropriate liquid or solid medium as recommended in the SAG and CCAP culture catalogues/web sites and maintained under identical culture conditions (14 : 10-h light : dark regime at 20°C and light intensity of 50 µmol photons·m–2·s–1). Prior to cryopreservation and following a post-thaw re-growth phase, the axenic state of the cultures was confirmed by checking both macro- and microscopically for contamination after duplicate cultures were incubated on Trebouxia Organic Medium (Ahmadjian, 1967
) for 3 d with one set under standard algal culture conditions and the other set in darkness at 37°C.
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). Genomic DNA was extracted using the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) following the instructions given by the manufacturer. Genomic DNA was extracted twice from each strain, on two different days in order to detect variations in AFLP banding patterns due to differences in the extraction processes. For the AFLP fingerprints, DNA needs to be of optimal quality to allow complete digestion, and this step is crucial for the production of high quality AFLP fingerprints. Only samples with high molecular DNA, i.e., those migrating at the same level with the 20-kb fragment of a lambda DNA standard in agarose gel electrophoresis, were processed further. Restriction digestion using enzymes EcoRI and MseI, ligation of adapters, and preselective (without selective nucleotides) and selective amplifications were performed as described by Müller et al. (2005)
with the following enzyme–primer combinations during selective amplification: EcoRI+A/MseI+C, EcoRI+G/MseI+C and EcoRI+C/MseI+C. However, for some strains other combinations (randomly chosen from primers in the literature on AFLP studies with higher plants) were required in order to decrease the number of amplified fragments: EcoRI+A/MseI+CG, EcoRI+C/MseI+CG and EcoRI+G/MseI+CG were used for Macrochloris radiosa, Uronema belkae, Trebouxia sp. and Chloromonas rosae; EcoRI+AAG/MseI+Cand EcoRI+ACT/MseI+C were used for Cosmarium cucumis, Euglena gracilis and Phaeodactylum tricornutum. In the case of Chlamydomonas reinhardtii, EcoRI+A/MseI+TG, EcoRI+C/MseI+TG and EcoRI+G/MseI+TG were used according to Werner et al. (2001)
. The amplified fluorescently labelled DNA fragments were separated by capillary electrophoresis using an ABI Prism 3100 (Applied Biosystems, Foster City, California, USA) automated sequencer and analyzed in the range of 100 to 500 bp above 50 relative fluorescent units with GeneScan software (Applied Biosystems). The reproducibility of the resulting AFLP patterns before cryopreservation was confirmed by three replicate reactions for each strain on two different DNA extractions: two reactions from the first and one of the second DNA extractions as described by Müller et al. (2005)
. Post-cryopreservation (post-cryo) AFLP evaluations included DNA to be extracted twice: (1) from the algal culture directly after thawing, recovery in fresh medium, and 12 h growth in darkness (t = 0), and (2), after a regrowth phase of 2 to 3 wk (t = 2–3), resulting in a total of five AFLP reactions from four different DNA extractions per sample. After the initial cryopreservation, five morphologically different strains were selected: three green algae, a diatom, and E. gracilis; these taxa were subjected to two further successive cryopreservation cycles of freezing, thawing, and regrowth (Table 2). The AFLP analyses were performed after the regrowth phase of each cycle, but not directly after thawing as in the initial cryocycle. Altogether, seven AFLP patterns were obtained and compared for each of these five selected strains. All pre- and post-cryo AFLP patterns were compared by eye using GeneScan software. A fragment position was counted as nonreproducible if it appeared in only one or two of the three replicated pre-cryopreservation (pre-cryo) AFLP patterns. The nonreproducible fragment positions were calculated as a percentage of the total number of fragment positions. Post-cryo patterns were manually compared with their corresponding patterns from the three pre-cryo AFLP reactions, and additional and/or missing fragments were noted. Fragment positions that appeared to be nonreproducible in the pre-cryo AFLPs were excluded from post-cryo comparison.
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; Taylor and Fletcher, 1999
). In the case of Chlamydomonas reinhardtii and E. gracilis, methanol (5 and 10%, v/v, respectively) was used following the methods described by Crutchfield et al. (1999)
and Brand and Diller (2004)
. Algae were pretreated with the cryoprotectant in cryovials (1.5 mL, Corning, Europe) for 10 min on ice prior to transfer to a controlled-rate cooler (Planer, UK), precooled to 4°C. Cultures were cooled at a cooling rate of –1°C·min–1 to an intermediate holding temperature of –35°C and held at that temperature for 40 min following Withers and King (1980)
. An additional cooling step of –1°C·min–1 to –45°C was used to guarantee that the samples were held at a temperature below –35°C (the temperature of homogeneous ice nucleation) during transfer from the controlled rate cooler to the cryostore vessel where the samples were directly placed into the vapor phase of liquid nitrogen (ca. –176°C). Vapor phase transfer was considered important (Fountain et al., 1997
) for this particular study to obviate the potential leakage of liquid nitrogen into cryovials because this has the potential to confound AFLP analyses through contamination. Samples were stored for at least 24 h in the cryostore vessel and thawed by direct immersion of the cryovials in a water bath of 45°C until the ice had melted (2–3 min). After thawing, either 200 µL of the undiluted samples were directly plated on agar slopes and/or samples were diluted 20-fold with fresh liquid medium (to reduce the concentration of the cryoprotectant, i.e., to 0.25% v/v DMSO or 0.25–0.5% v/v methanol) in the post-cryo culture, then diluted samples were also transferred onto agarized medium. All samples were kept in darkness for 12 h and then incubated under standard culture regimes as outlined. Post-thaw viabilities (PTV) were determined by visual inspection 2–3 weeks following thawing. This was assigned to four categories: no, poor, good, or very good regrowth (Tables 1, 2). On agar these corresponded to 
1 x 108, 4 x 108 and 6 x 108 cells·mL–1 for Chlorella and other algae with small cells (<20 µm), or 1 x 105, 3 x 105 and 5 x 105 cells·mL–1 for Cosmarium and other algae with larger cells (<200 µm). In liquid cultures, these categories corresponded to approximately 1 x 106, 4 x 106 or 7 x 106 cells·mL–1 for Chlamydomonas and other algae with small cells (<60 µm), or 2 x 105, 6 x 105 and 9 x 105 cells·mL–1 for Euglena and other algae with larger cells (<400 µm). RESULTS
Prior to cryopreservation, all 28 algal strains were analyzed using an identical protocol for DNA extraction and AFLP reactions, with the exception that the choice of primers in selective amplification was adapted for seven specific strains. Resulting AFLP patterns had between 113 and 371 DNA fragments for all samples (Table 1). Patterns for each strain are from a total of three independent AFLP reactions, which were performed on two independent DNA extractions. Reproducibility within these three replications was on average 99.1%, and the percentage of nonreproducible fragments ranged from 0 to 3.6% (Table 1). Amphikrikos cf. nanus samples had an exceptionally high level of nonreproducible fragments (8.5%) and were excluded from calculating the pre-cryo mean value of nonreproducible fragments as well as from post-cryo AFLP analyses (Table 1).
Examples for nonreproducible fragments are shown in Fig. 1A–C for Macrochloris radiosa and in Fig. 2A–C for Chloromonas rosae. The greatest number of nonreproducible AFLP fragments was observed on comparing the two independent pre-cryo DNA extractions, with fewer nonreproducible fragments were obtained on comparison of the two replicated AFLP reactions from the same DNA extraction. Furthermore, in most cases the shape and height of AFLP fragments obtained from the same DNA extraction were more similar compared to those, from patterns of the two independent DNA extractions (Fig. 1A–C). However, this was not the situation for all samples. Patterns obtained for Chloromonas rosae had the same amount of dissimilarities and similarities between all three pre-cryo replicates (Fig. 2A–C). It was noted that, approximately one-third of the nonreproducible fragments had relatively low peak intensities, close to background levels (e.g., both nonreproducible fragments in Fig. 1B, C).
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Post-thaw viability assessments between liquid and agar cultures were comparable; however, regrowth rates of diluted samples placed on agar were slightly lower compared to those of the undiluted samples. The PTV levels in Table 1 correspond to regrowth on agar of undiluted post-cryo samples. The only exception was Chlamydomonas moewusii SAG 11-61b for which a higher PTV was obtained for the diluted culture (Table 1). In general, the post-thaw viability levels did not correlate with the variation in post-cryo AFLP patterns.
AFLP patterns of both strains of Coccomyxa arvernensis, CCAP 216/1 taken from cryogenic storage and SAG 216-1, not previously cryopreserved prior to our study, were identical despite the CCAP strain being maintained under different regimes for more than 20 yr. However, reproducibility of the AFLP fragment pattern before cryopreservation was poorer in CCAP 216/1 compared to the corresponding strain from the SAG culture collection (Table 1). After cryopreservation, the CCAP strain differed in six fragment positions after the regrowth phase, while just one position differed in the corresponding SAG strain. Interestingly, the CCAP strain shared the same additional fragment position with the SAG strain after thawing and regrowth, which was absent in both strains before cryopreservation (results not shown).
A group of strains that had no differences in the AFLP patterns directly after thawing and after the regrowth phase, including three strains of green algae with different morphologies, a diatom P. tricornutum and Euglena gracilis, were selected to be refrozen in two additional successive cycles of cryopreservation. In only two of these selected strains, Chlorella vulgaris and E. gracilis, differences were observed after the third cycle (Table 2). These differences, apparently induced by further cryo-cycles, were small, 0.8 and 0.3%, respectively, corresponding to just a single fragment position, with viability levels remaining almost unchanged after three successive cycles. However, in E. gracilis, recovery after thawing was slow, with very poor or no growth observed after a 2-wk incubation under standard conditions after the second and third cycle, although they regrew for both cycles after 5 wk.
DISCUSSION
AFLP analysis was employed to assess the genetic stability of 28 cryopreserved microalgae. Identification of nonreproducible fragments before cryopreservation (e.g., due to PCR anomalies) allowed the detection of truly differing fragments after cryopreservation. No differences were found between AFLP patterns before and after cryopreservation in 10 strains, with 14 strains clearly differing in their respective patterns (Table 1). The remaining four strains either failed to regrow, were contaminated after thawing, or yielded poor quality DNA (Table 1). In the 14 strains with differences, half of them had AFLP pattern differences in the same range (>1.8%) as those found for two phenotypically distinct mutants of Parachlorella kessleri (Table 1). Essential prerequisites for assessing genetic stability with AFLP are that the method provide sufficient sensitivity to detect minor genomic alterations and that the observed differences be highly reproducible. These aspects were further investigated in the present study prior to pre- and post-cryo pattern comparisons.
Sensitivity and reproducibility of AFLPs
A variety of recent studies in microalgae have demonstrated that the AFLP technique discriminates between isolates from different geographical origins or mating types of the same species (Werner et al., 2001
; De Bruin et al., 2004
; John et al., 2004
; Müller et al., 2005
). In the present study, AFLPs were capable of clearly distinguishing between two different mutants of Parachlorella kessleri and their corresponding wild-type strain (Table 1, Fig. 3). The differences in pigmentation of strains SAG 11.80 (orange/brown colonies) and SAG 211-11h/9 (yellow colonies) correlated with the differences in the AFLP patterns (Fig. 3). The UV-light-induced mutant SAG 11.80 was more similar to the wild type than to the radioisotope-induced mutant SAG 211-11h/9, indicating that the radiation-induced mutation results in more detectable genomic alterations.
As described in Müller et al. (2005)
, to identify nonreproducible fragments in the AFLP patterns, we performed three AFLP reactions with two independent DNA extractions. The percentages of nonreproducible fragments for the 27 strains (one strain, Amphikrikos, was excluded due to poor DNA quality, discussed later) prior to cryopreservation were between 0.3 and 3.6% with a mean of 0.9% (Table 1, e.g., Fig. 2). This verifies the successful application of the AFLP methodology to a variety of microalgae using an identical, standardized protocol. This level of reproducibility is similar to that reported by other workers using AFLP to examine microalgae, plants, or animals, where values below 5% were obtained (Hansen et al., 1999
; Bonin et al., 2004
; John et al., 2004
). Nonreproducible fragments can result from restriction/amplification anomalies due to poor DNA quality, or errors during AFLP reactions. Other sources of nonreproducible fragments include microbial contamination of the cultures that introduce "foreign" DNA into algal DNA extractions and the presence of distinct algal subpopulations in the culture. In the last case, where a culture consists of several genetically non-identical subpopulations, the ratio of the DNAs from the various subpopulations could vary in different DNA extractions and thus lead to nonreproducible fragments. A certain error rate can be anticipated for any restriction or PCR-based technique, owing to nonselective restriction or amplification (e.g., Koonjul et al., 1999
), but this is difficult to experimentally determine. In the present study, about one-third of the nonreproducible fragment positions were fragments of low intensity, close to the background (e.g., Fig. 1), which are likely to have been generated by nonselective amplification (Bonin et al., 2004
). The purity and integrity of high molecular weight DNA has been found to be the most critical factor for the quality of AFLP fingerprints (Donaldson et al., 2000
; McLenachan et al., 2000
; Mannschreck et al., 2002
; Bonin et al., 2004
), and this was confirmed here. Most of the nonreproducible fragments were found in the comparison of the two different DNA extractions, but not with the same DNA extraction, reflecting small variations in the purity/integrity of the DNA. The effect of poor DNA quality on AFLP patterns was most obvious in Amphikrikos cf. nanus where the amount of nonreproducible fragments was 8.5%, almost 10 times more than the average. We assume that this was due to poor DNA quality. For this strain, it was already known that obtaining DNA of sufficient quality can be particularly difficult. Extractions can even sometimes yield DNA that cannot be amplified by PCR (Hepperle et al., 2000
), which would also lead to unreliable AFLP analyses.
The influence of contaminant organisms on AFLP patterns was demonstrated here for Chloromonas rosae after cryopreservation, by the presence of additional and missing fragments compared to an axenic culture (Fig. 2). Similarly, the effect of otherwise undetected contaminants on AFLP patterns has been demonstrated in fungi, in which a bacterial contaminant almost "caused" the reporting of a unique genotype (Dyer and Leonard, 2000
). Missing fragments can be explained by a suppression of algae-specific fragments, where preferential amplification of contaminant DNA occurs, similar to reports by RAPD analyses (Dyer and Leonard, 2000
). Therefore, all samples should be tested for purity prior to AFLP studies to detect any contaminants.
Differences between AFLP patterns can also result from human error in scoring AFLP fragments and has accounted for up to 2.1% differences between two independent manual assessments (Bonin et al., 2004
). In the present study, this source of error was minimized by having a single person evaluate the patterns within a short time. An evaluation with automated scanning software could not be used here because it can lead to an overestimation of differences between virtually identical patterns (Müller et al., 2005
).
Genomic alterations observed after cryopreservation
Nonreproducible fragment positions were excluded prior to assessing AFLP differences after cryopreservation. Hence, residual differences reflect genomic alterations and not artefacts. In the studied terrestrial microalgal strains, AFLPs clearly revealed some genomic alterations after cryopreservation (Table 1). The differences in AFLPs could indicate that the two-step cooling protocol with DMSO or methanol as a cryoprotectant may be suboptimal for some strains. AFLPs may therefore be useful as a means of evaluating the effectiveness of cryopreservation protocols. Suboptimal cryopreservation protocols increase, for example, the occurrence of gas-vacuolated-deficient mutants of the cyanobacterium Microcystis aeruginosa (Day et al., 2005
). Although we did not detect any obvious phenotypic differences (e.g., changes in the color of algal colonies on agar) in our cultures after thawing and regrowth, the differences in AFLPs observed for several cryopreserved strains were in the same size range as the differences found for pigment mutants of Parachlorella kessleri. However, one has to consider that genetic alterations may not necessarily be correlated with phenotypic changes. A single AFLP fragment position corresponds to about 0.5% of the counted fragments (Table 1) and may or may not lead to phenotypic changes. In cases were post-cryo differences in AFLPs were detected, an extended array of physiological and biochemical characters (e.g., photosynthetic activity, production of metabolites, or vital staining) should be investigated to assess AFLP patterns with respect to phenotypic correlations.
The post-cryo differences in the present study (0.5 to 6.3%, mean 1.3% of 113–371 AFLP fragments) were higher than differences obtained in AFLP investigations after cryopreservation in higher plants. In Prunus 0.3% of 565 AFLP fragments were variable between those of noncryopreserved and cryopreserved plantlets (Helliot et al., 2002
). Studies on strawberry, employing 16 primer combinations, resulted in a single post-cryo missing fragment (Hao et al., 2002a
). Furthermore, no post-cryo differences were found in a variety of other higher plants with a variable number of fragment positions, i.e., Asteraceae (433 fragments), Haemodoraceae (95 fragments), and 195 fragments in apple shoots (Turner et al., 2001
; Wilkinson et al., 2003
; Liu et al., 2004
). All the studies on higher plants discussed used samples of the same species or genus, and one could reasonably speculate that the higher level of post-cryo AFLP differences observed in this study may be attributable to greater morphological and taxonomical diversity among the algal taxa studied.
Cell "reactions" to cryopreservation stresses, i.e., the amount of detectable post-cryopreservation differences, may vary during different freezing events with the same organism and the same protocol. Strains CCAP 216/1 and SAG 216-1 of C. arvernensis represented the same isolate (i.e., revealed identical AFLP patterns before cryopreservation in this study), but varied in the number of fragment positions that were different in the post-cryo patterns after cryopreservation (Table 1). Strain CCAP 216/1 has been maintained for about 27 yr in a cryopreserved state, and when thawed for this study, there was no difference in the AFLPs, suggesting that the first freezing event had no effect, in contrast to the second freezing/thawing. This may be due to the slightly different cooling regimes that were used. Similarly, in the five strains that were subjected to a total of three successive cycles of freezing/thawing, no differences were detectable after the first freeze/thaw, but after the second or third cycle, effects were observed for the two strains (Table 2).
The two strains used for C. avernensis in this study represent an interesting example in which the same algal isolate has been kept separated in different states in different culture collections for many years. Despite literally thousands of generations separating the two strains, both had identical AFLP patterns before cryopreservation in this study. This comparative study is in accordance with observations in other simple coccoid members of Trebouxiophyceae, Chlorella vulgaris (Müller et al., 2005
). These findings do not support the hypothesis of "evolution in culture," i.e., that cultures maintained by serial transfer are under selective pressure and that mutations occur in a period of a few years. However, this aspect certainly needs to be tested further with a broader sample of morphologically and taxonomically diverse algae.
It has been suggested that high post-thaw viability levels above 60% are optimal to guarantee representative, successfully cryopreserved, and genetically stable cultures (Morris, 1981
). In the present study, all strains without post-cryo AFLP differences had good or very good post-thaw viability, which seems to support this view (Table 1). However, overall we found no correlation between the variation in post-thaw viability levels with the post-cryo AFLP differences. Some strains, such as Uronema belkae and Scenedesmus obliquus, with very good post-thaw viability, had differences in their post-cryo AFLPs, whereas strains such as Chlorogonium elongatum and Chlamydocapsa maxima that did not regrow after thawing had either no differences or only one differing fragment (Table 1).
Possible causes of genomic alterations after cryopreservation
There is a variety of reasons why genomic alterations may occur as a result of cryopreservation, for example, due to the formation of free radicals, which can damage enzyme functions, and the effects of the cryoprotectants. In addition, individual components of the process, e.g., cryoprotectant treatment, cooling, freezing, thawing, or post-thaw manipulations may individually or synergistically have induced the changes in AFLP patterns obtained; this is particularly important for suboptimal protocols. In this study the molecular analysis was undertaken after the cryopreservation process and as such was used to screen for potential changes in the entire preservation protocol, rather than in the individual components of the protocol. In future studies, the contribution of the different components of a cryopreservation protocol to the genetic stability profiles should be examined.
Understanding the impacts of cryoinjury may be particularly important because protective mechanisms against oxidative stress may become saturated under environmental stress so that DNA is "attacked" by free radicals, resulting in genomic alterations (Benson, 1990
; Benson and Bremner, 2004
). Large temperature variations may influence enzyme functions leading to inefficient DNA repair systems (Calcott and Gargett, 1981
). Penetrating cryoprotectants, such as DMSO, which otherwise protect DNA as they "scavenge" free radicals (Benson, 1990
), may also be mutagenic. In our experiment, the algae were exposed to CPAs at a relatively high concentration before cryopreservation and during a nongrowth phase for 10 min on ice, but only to residual levels of CPAs after cryopreservation during cell culture after thawing. AFLP fingerprints were performed on thawed samples after an exposure of 12 h (t = 0) or several weeks (t = 2–3) to CPA that was diluted down to 0.25–0.5%. In cases where the thawed samples were plated on agar media, the CPA may have become even more diluted as it diffused into an even larger volume of agar. While the effects of exposure to DMSO at 5% (v/v) and higher on growing Euglena cells were significant, exposure to 2.5% (v/v) DMSO produced no effect during a 48-h treatment (Vannini and Poli, 1983
). In a separate study (data not shown), we compared growth rates of algal cells on culture media with 2.5% methanol and those on culture media without methanol and found no differences. It is therefore unlikely that the low concentrations of CPAs to which our samples were exposed during growth after thawing caused genetic differences. However, the possible residual effects of dilute cryoprotectants on cell growth and on the genome cannot be eliminated because their actions are unknown and thus require further investigation.
It is also possible that the differences in AFLPs after cryopreservation may not necessarily represent mutations induced by the cryopreservation process. Changes in the DNA methylation status have been argued as the most likely explanation for differences after cryopreservation that are detectable in restriction enzyme-generated fingerprints such as AFLP patterns (Helliot et al., 2002
; Harding, 2004
; Harding et al., 2005
). Helliot et al. (2002)
attributed about 0.3% of the fragment differences to methylated restriction sites that cannot be cleaved by methylation-sensitive restriction enzymes such as EcoRI. Changes in DNA methylation status are a reversible effect and often regarded as an adaptive response to high osmotic stress to which cells are exposed at various stages of the cryopreservation process (Turner et al., 2001
). Osmotic stress associated with colligative damage incurred during controlled rate cooling may also occur to a greater extent for organisms that are suboptimally cryoprotected by penetrating additives. An increased level of DNA methylation was detected in cryopreserved strawberry and apple plants using a modified AFLP technique (MSAP, methylation-sensitive amplified polymorphism, Hao et al., 2001
, 2002b
). It is known that the DNA methylation status can change over a short time and in response to environmental stimuli (Jarvis et al., 1992
). But once cryopreservation changes in the DNA methylation status are detectable, the duration of these changes in a microalgal culture is as yet unknown. It is possible that the 2 to 3 weeks after thawing, when AFLP patterns were detected from our selected strains, may not have been sufficient time to fully recover from the methylation induction phase to reverse the DNA methylation status to that before cryopreservation. The DNA methylation status of Chlorella vulgaris SAG 211-11b, examined with high performance liquid chromatography (HPLC), 2 mo after cryopreservation was 13% (K. Harding, unpublished data, see Harding et al., 2005
; Johnston et al., 2005
), an unexpectedly high level compared to the study of Jarvis et al. (1992)
on microalgae in which an average of 2% was found with HPLC. Future analyses of cryopreserved microalgal strains using AFLP should therefore also include an assessment of the DNA methylation status.
The observation that post-cryo differences were more frequent and greater after a regrowth phase of 2–3 wk than directly after thawing (Table 1) could also be explained by the possibility that the procedure is "selective" for specific subpopulations. Potentially, a freeze-tolerant subpopulation may survive cryopreservation better than the pre-cryo population (Pearson et al., 1990
). For example, it is feasible that Euglena gracilis may have required some degree of cryo-tolerance after three successive cycles of freezing and thawing, until a subpopulation became dominant during the 5-wk regrowth phase, when one differing fragment was detectable (Table 2). It is now common practice for an algal strain to be derived from the isolation of a single cell (Andersen and Kawachi, 2005
), and all the microalgal strains in this study were derived from an original clonal cell. Even for organisms, where sexual reproduction is not known, the rate of asexual recombination could be higher than is commonly supposed (Drake et al., 1998
). Spontaneous mutations in microalgae, for which sexual reproduction is not known (the majority of strains in the present study) or for cultures containing a single mating type (e.g., Chlamydomonas reinhardtii or Chlorogonium elongatum), may still be sufficiently frequent to form subpopulations that are adapted to extreme environments (Drake et al., 1998
; Flores-Moya et al., 2005
). The findings reported in the present study can be interpreted such that cryopreservation (particularly where a suboptimal protocol is applied) may lead to the selection of subpopulations, which differ in their genotypic properties as compared to their nonfrozen counterparts, but in the absence of phenotypic changes, may not represent a selective advantage. Within the same culture, several pre-existing subpopulations differing in their sensitivity toward cryopreservation may be present, including those adapted cells resulting from de novo mutations.
Fundamental to any conservation technique is the risk of genomic alteration that cannot be excluded, but it is necessary to quantitatively determine its magnitude. This study has demonstrated that using the AFLP method, the cryopreservation of microalgae can result in genomic alterations that are clearly detectable. Whether they are reversible, as would be the case due to changes in DNA methylation or they represent true mutations (with possible phenotypic correlates) induced by cryopreservation or a combination of both, is still unclear at this stage. No obvious correlations can be made between those algae with detectable AFLP changes and those without changes. Clearly, AFLP changes are not solely attributed to the physical forces of freezing and thawing, but more likely express adaptive responses to stress. The work presented here may be seen as a starting point, and it is anticipated that it will stimulate other workers to develop this area further.
FOOTNOTES
1 The authors thank H. Timmermann for her excellent assistance with cryopreservation; E. E. Benson for training J.M. in cryopreservation techniques, stimulating discussion, and valuable comments on this manuscript; and J. Johnston for HPLC analyses. Financial support was provided by the European Commission, project COBRA (The conservation of a vital European scientific and biotechnological resource: microalgae and cyanobacteria), contract no. QLRI-CT-2001-01645. The authors appreciate the support extended to J.M. by Dr. G. Berthold, which was made possible through his generous donation to the SAG Culture Collection at University of Göttingen. 
6 Author for correspondence (tfriedl{at}uni-goettingen.de
)
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