Genetic variation among seven strains of Dunaliella salina (Chlorophyta) with industrial potential,...

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Aquaculture 233 (2004) 149–162

Genetic variation among seven strains of Dunaliella

salina (Chlorophyta) with industrial potential,

based on RAPD banding patterns and on

nuclear ITS rDNA sequences

Patricia I. Gomez*, Mariela A. Gonzalez

Departamento de Botanica, Facultad de Ciencias Naturales y Oceanograficas, Universidad de Concepcion,

Casilla 160-C, Concepcion, Chile

Received 27 October 2003; received in revised form 27 October 2003; accepted 12 November 2003

Abstract

Dunaliella salina (Chlorophyta) is a halophilic microalga cultivated as a natural source of h-carotene in several countries, including Chile. Previous studies of some Chilean strains of this

microalga have shown a great variability in their physiological and genetic attributes. Random

amplified polymorphic DNA (RAPD) band patterns and nuclear ribosomal DNA internal

transcribed spacer (ITS-1 and ITS-2) sequences were used to genotypically characterize three

Chilean and four foreign (from Mexico, China, Australia and Israel) strains of D. salina with

industrial potential. Unweighted pair group mean average (UPGMA) cluster analysis of RAPD

data distinguished, at 70% similarity, two clusters. One cluster included all of the foreign strains,

except the one from Australia, and the other grouped two of the Chilean strains (CONC-001 and

CONC-007). The other Chilean strain (CONC-006) and the Australian strain appeared as single

entities. Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML)

analyses of the ITS sequence data yielded four clades, most of them well supported: there were

two clades containing exclusively one strain, CONC-006 from Chile (86–94% support) and the

one from Australia (98–100% support); the third clade grouped the strains from Mexico, China

and Israel (99–100% support), and, the fourth clade, the strains CONC-001 and CONC-007

from Chile (100% support). Both RAPD banding pattern and ITS sequence data were consistent

in resolving the same genetic relatedness among the strains analyzed. For both approaches, the

strain CONC-006 from Chile and the strain from Australia were the most divergent entities

within the group. Furthermore, the strain CONC-006 appeared genetically more related to the

foreign strains than to the other Chilean ones. The physiological attributes exhibited by these

0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2003.11.005

* Corresponding author. Tel.: +56-41-204156; fax: +56-41-246005.

E-mail address: [email protected] (P.I. Gomez).

P.I. Gomez, M.A. Gonzalez / Aquaculture 233 (2004) 149–162150

strains of D. salina are mostly in agreement with the genetic relationships deduced from this

study.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Dunaliella salina strains; RAPD; Ribosomal ITS sequences; Genotypic characterization

1. Introduction

Dunaliella salina is a halotolerant green microalga widely used in mass culture for

commercial purposes. Under appropriate culture conditions, some strains of D. salina can

accumulate up to 10% carotenoids, mostly h-carotene, becoming the richest eukaryotic

source of this pigment known so far (Ben-Amotz et al., 1982; Ben-Amotz and Avron,

1983; Borowitzka and Borowitzka, 1988).

Physiological variability has been reported at the intraspecific level in D. salina

(Araneda et al., 1992a,b; Cifuentes et al., 1992, 1996a,b; Markovits et al., 1993; Gomez

et al., 1999). A negative consequence of this variability is that it could lead to erroneous

assumptions about the industrial potential of this microalga, when information coming

from one strain is extrapolated to the whole species. On the other hand, this intraspecific

diversity is advantageous from a biotechnological point of view, since it permits the

application of strain selection procedures, easily improving fine chemicals production

(Lopez Alonso et al., 1992a,b; 1994).

The global demand of natural beta-carotene is around 1430 tons per annum (tpa), while

the availability is just 35 tpa produced by the companies Henkel-Cognis (Australia),

Nature Beta Technologies (a subsidiary of Nikken Sohonsha, Israel) and Dutch State

Mines (DSM; Food Specialties, Netherlands). The balance demand is met through

synthetic carotene manufactured by Roche and BASF (online business magazine do-

main-b.com, June 2002, http://www.domain-b.com).

Henkel-Cognis (previously Betatene) is the largest producer of D. salina in the world;

the company has two sites, one at Hutt Lagoon, Western Australia and the other at

Whyalla, South Australia, which are the world’s largest algal farms with a total capacity of

800 ha. Similarly, but at a lower scale, Nature Beta Technologies (NBT) which was

founded to commercialize the algal research at the Weizmann Institute of Science in

Rehovot, Israel, also cultivates D. salina as a natural carotene source. Unlike the other

companies, DSM Food Specialties produces the pigment by a fermentation process of the

fungi Blakestea trispora.

D. salina is probably the most successful microalga for mass cultivation described so

far, especially due to its high salinity requirement that minimizes the number of

competitors and predators. Furthermore, due to its lack of cell wall, dried Dunaliella

is easily and fully digestible by animals and humans (Borowitzka and Borowitzka, 1988;

Ben-Amotz and Avron, 1989). In spite of these advantages, the mass culture of D. salina

as a natural carotene source still needs to be developed to satisfy all the required

demand.

The development of mass culture conditions has required growth optimization of

growth as well as the amount of the carotene produced per unit time and culture volume.

http:\\www.domain-b.com

P.I. Gomez, M.A. Gonzalez / Aquaculture 233 (2004) 149–162 151

This objective is complicated due to the fact that the conditions for maximum cell

growth are opposite of those for maximum carotene accumulation per cell. There is no

doubt that the selection of strains with improved growth and carotenogenic attributes

would help to make the mass culture of D. salina economically competitive with its

synthetic counterpart.

The genetic characterization of strains with industrial potential has a great relevance in

applied science because it permits partitioning of phenotypic variation into environmental

(phenotypic plasticity) and genetic components. Furthermore, genetic diversity studies

permit exotic genotypes detection, which could be the sources of genes with biotechno-

logical potential.

Many different molecular techniques can be used to reveal genetic polymorphisms. One

of them is random amplified polymorphic DNA (RAPD), a DNA-fingerprinting method

that allows the detection of multi-locus genetic variation (Welsh and Mc Clelland, 1990;

Williams et al., 1990). The RAPD technique has been successfully employed to identify

microalgae up to the strain level (Neilan, 1995; Nishihara et al., 1997; Bolch et al.,

1999a,b). By using RAPD, a recent genetic analysis of eight Chilean strains of D. salina

showed that the strain CONC-006 from an alpine salt lake (‘‘Salar de Atacama’’) was

genetically more similar to CONC-001 (42% similarity) from a coastal lagoon (‘‘La

Rinconada’’) than to almost all of the other strains collected in the ‘‘Salar de Atacama’’,

including CONC-007 (39% similarity) (Gomez and Gonzalez, 2001).

On the other hand, some non-coding rapidly evolving sequences have been used to

resolve intra- and interspecific relationships among a variety of organisms. This is the case

of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA

(Schlotterer, 2001). The ITS-1 and ITS-2 sequences have proved very useful in assessing

genetic relatedness in green algae, both unicellular and colonial (e.g. Coleman et al., 1994;

Mai and Coleman, 1997; Angeler et al., 1999).

In Dunaliella, Gonzalez et al. (1999), using ITS-RFLP analysis, did not find a

correlation between polymorphism in the ITS region (ITS1 + 5.8S rDNA+ ITS2) and

the morpho-physiological attributes used for the taxonomy within the genus. However,

after a reappraisal of the physiological attributes of nine strains of Dunaliella (excluded D.

salina) (Cifuentes et al., 2001), Gonzalez et al. (2001) recommended renaming several

strains of Dunaliella based on the phylogenetic relationships deduced from the ITS

sequences which, this time, were congruent with the morpho-physiological attributes of

the strains. Moreover, they found a much higher sequence variation among strains of D.

salina than among strains belonging to other species of the genus, stressing the need of

further work to elucidate the species status of this complex taxon.

Comparison of the physiological response within D. salina (different strains) is

rather difficult since the data come from different physiological studies, most of them

done under different experimental conditions. The only previous data that account for

physiological variability within different strains of Dunaliella is the paper published by

Cifuentes et al. (1992), which includes eight Chilean strains. Recently, an exhaustive

study on D. salina evaluated growth and carotenogenesis in three Chilean (CONC-001,

CONC-006, CONC-007) and four foreign (from Mexico, China, Australia and Israel)

strains cultivated in combined conditions of temperature and irradiance (Gomez and

Gonzalez, unpublished data). All the foreign strains, in spite of coming from


geographically very distant locations, exhibited similar physiological responses to the

Chilean ones. In this study, our aim was to find out whether physiologically similar

strains of D. salina are also genetically related. To do this, RAPD patterns and ITS

rDNA sequence data were analyzed to quantify genetic polymorphism and to establish

phylogenetic relationships among these strains.

2. Materials and methods

2.1. Strain culture and DNA extraction

In this study, unialgal cultures of seven strains of D. salina were included. All of them

are maintained in the Culture Collection of Microalgae at the Universidad de Concepcion,

Concepcion, Chile. Table 1 summarizes the geographic origin and the source of each

strain. All the strains were cultivated in 250-ml Erlenmeyer flasks with 100 ml of modified

Johnson’s medium (Borowitzka, 1988) with 15% NaCl under a continuous photon flux

density of 100 Amol m� 2 s� 1 and 25F 2 jC. Axenic culture preparation and total DNA

extraction were carried out as in Gomez and Gonzalez (2001).

2.2. RAPD reaction conditions

RAPD amplifications were done in a 25 Al volume. A total of 50 primers from the D, P,

OPA and OPD (Operon) series were used. PCR-reaction composition, amplification

program and electrophoresis conditions were performed according to previously described

methodology (Gomez and Gonzalez, 2001). Amplifications were repeated and checked for

reproducibility between reactions.

Table 1

Strains of D. salina included in this study

Strain Source and Geographic GenBank accession numberstrain number origin

ITS-1 ITS-2

D. salina

CONC-001

CONC-001 La Rinconada Lagoon,

Antofagasta, Chile

AF546091 AF546092

D. salina

CONC-006

CONC-006 Sector Burro Muerto,

Salar de Atacama, Chile

AF313424 AF313425

D. salina

CONC-007

CONC-007 Sector Puilar, Salar de

Atacama, Chile

AF313426 AF313427

D. salina

Mexican

Provided by

Ernesto Retamales,

U. Antofagasta, Chile

Yucatan, Mexico AF546093 AF546094

D. salina

Chinese

Provided by

Dr. Ralph Lewin,

Scripps Institution, USA

Tanggu, China AF546095 AF546096

D. salina

Australian

CCAP 19/18 Hutt Lagoon, Australia AF546097 AF546098

D. bardawil ATCC 30861 Bardawil Lake, Israel/Egypt AF313430 AF313431

Geographic origins and the GenBank accession number of ITS sequences are also shown for each strain.

2.3. ITS spacers sequencing

The primers used to amplify the ITS region (ITS1 + 5.8S rDNA+ ITS2) were AB28

and TW81 described in Goff et al. (1994). The amplification reaction mixtures and the

PCR program were performed as in Gonzalez et al. (1999). PCR products were analyzed

by electrophoresis of 2 Al of the reaction mixture through 2% agarose gel and visualized

by ethidium bromide staining. ITS fragments were gel purified using the kit Wizard

PCR Purification System (Promega, Madison, WI) and used directly as templates for

sequencing (without prior cloning). Sequencing was performed with an ABI Prism 377

DNA sequencer (Centro de Sıntesis y Analisis de Biomoleculas, Universidad de Chile,

Chile).

2.4. Data analysis

For RAPD-PCR data, just strong and reproducible bands were scored. Co-migrating

bands were scored as either present (score 1) or absent (score 0) to produce a binary matrix

for all the strains examined. The presence/absence matrix was used to calculate pairwise

RAPD distances between strains using the mean-distance metric of PAUP (4.0b8 version,

Swofford, 1999) and to construct a clustering dendrogram using the unweighted pair group

mean average (UPGMA) algorithm. For ITS analysis, combined ITS-1 and ITS-2 data sets

were analyzed. ITS sequences from all of the D. salina strains were aligned manually to

sequences of two reference outgroups: Dunaliella viridis (strain CONC-002; Genbank No.

AF313418, AF313419) and Dunaliella tertiolecta (strain UTEX 999; Genbank No.

AF313434, AF313435) according to Gonzalez et al. (2001). The resulting alignment

was subjected to a cladistic analysis. Both distance-based and character state-based tree-

building methods were used. Three different optimality criteria were employed: neighbor-

joining (NJ), maximum parsimony (MP) and maximum likelihood (ML). NJ analysis was

performed with the Kimura two-parameter distance model (Kimura, 1980) using the

MEGA 1.02 (Kumar et al., 1993) program. Bootstrapping the data with 500 replications

tested the robustness of each branch. In addition, sequences were analyzed with the MP

and ML algorithms of PAUP; both analyses were performed by branch and bound search

and subjected to 1000 and 500 bootstrap replicates, respectively. All sites were treated as

independent, unordered and equally weighted characters. ML analysis was based on the

Hasegawa–Kishino–Yano model (Hasegawa et al., 1985).


3. Results

3.1. RAPD-PCR analysis

From the total 50 primers initially tested, 12 gave clear and reproducible amplification

patterns, and consequently, were used for the comparative analysis (Table 2). The number

of bands generated with these primers was between 5 (with primers P-100, OPA-11 and

OPA-13) and 11 (with primers OPD-11 and OPD-18), with fragment size ranging between

300 and 2000 bp.

Table 2

Sequences of RAPD primers used to compare the seven strains of D. salina studied, and the total number of bands

scored with each one

Primer Sequence 5V! 3V Total number

of bands scored

P-2 >ACA.ACT.GCT.C< 8

P-3 >TGA.CTG.ACG.C< 7

P-10 >GCG.ATC.CCC.A< 8

P-100 >ATC.GGG.TCC.G< 5

OPA-01 >CAG.GCC.CTT.C< 10

OPA-04 >AAT.CGG.GCT.G< 8

OPA-09 >GGG.TAA.CGC.C< 8

OPA-11 >CAA.TCG.CCG.T < 5

OPA-13 >CAG.CAC.CCA.C< 5

OPA-18 >AGG.TGA.CCG.T < 8

OPD-11 >AGC.GCC.ATT.G < 11

OPD-18 >GAG.AGC.CAA.C< 7

Total 94


The primers generated a total of 94 genetic markers (Table 2), all of them polymorphic

since none was present in all of the seven strains. RAPD patterns obtained with some of

the primers employed are shown in Fig. 1.

RAPD-PCR reactions were highly reproducible; identical banding patterns were

produced when DNA from independent cultures of the same strain were analyzed (data

not shown).

The similarity values obtained by the RAPD analysis ranged between 53.2% for the

strains pair CONC-007/China and 81.9% for the Mexico/China pair (Table 3).

Fig. 1. RAPD-PCR banding patterns of the seven strains of D. salina obtained with the primers P-100 (lanes 2 to

8) and OPA-11 (lanes 9 to 15). Lanes 2 and 9: D. salina CONC-001, 3 and 10: D. salina CONC-006; 4 and 11: D.

salina CONC-007; 5 and 12: D. salina Mexican; 6 and 13: D. salina Chinese; 7 and 14: D. salina Australian; 8

and 15: D. bardawil. Lane 1: fX174/HaeIII and pBR322/PstI.

Table 3

Similarity matrix, derived from RAPD-PCR data, among the seven strains of D. salina included in this study

CONC-001 CONC-006 CONC-007 Mexican Chinese Australian D. bardawil

CONC-001 – 0.61702 0.74468 0.62766 0.55319 0.61702 0.57447

CONC-006 36 – 0.59574 0.62766 0.57447 0.65957 0.61702

CONC-007 24 38 – 0.58511 0.53191 0.59574 0.59574

Mexican 35 35 39 – 0.81915 0.58511 0.73404

Chinese 42 40 44 17 – 0.55319 0.76596

Australian 36 32 38 39 42 – 0.61702

D. bardawil 40 36 38 25 22 36 –

Above the diagonal: similarity among strains.

Under the diagonal: number of different characters among strains (of a total of 94 characters).


UPGMA cluster analysis of RAPD data showed two clusters and two single strains, at a

threshold level of 70% similarity (Fig. 2). Cluster I includes all of the foreign strains,

except the one from Australia, and cluster II groups together the strains CONC-001 and

CONC-007 from Chile. At this similarity level, the strain CONC-006 from Chile and the

strain from Australia appeared as individual entities (III and IV in Fig. 2).

3.2. ITS sequence analysis

A single band of the ITS region (ITS1 + 5.8S rDNA+ ITS2) was produced by PCR

amplification: its size was very similar (ca. 640 pb) among all the strains. Table 1 shows

the GenBank accession number for the sequences of ITS-1 and ITS-2 spacers in the strains

studied.

Fig. 2. UPGMA cluster analysis for seven strains of D. salina derived from RAPD data.


In all the D. salina strains, the ITS-2 exhibited a slightly higher variability (sub-

stitutions, insertions and deletions 49/235 sites = 20.8%) than ITS-1 (32/215 sites = 14.9%)

(Fig. 3). Among substitutions, transitions were more common than transversions as

indicated by a transition/transversion ratio of 2.2 estimated via ML, over all nucleotide

positions.

Divergence values, estimated via the Kimura two-parameter model, ranged between

11.4% (CONC-006/CONC-007) and 0.2% (China–Mexico) (data not shown).

Fig. 3. Aligned sequences of ITS-1 and ITS-2 in the seven strains of D. salina studied. D. viridis (D. vir) and

D. tertiolecta (D. tert) were included as outgroups. A dash (–) indicates introduced gaps. Letters in bold and

underlined indicate mutations that characterize the Chilean strains.


There were DNA motifs shared by all the strains (including outgroups) and others

unique to certain D. salina strains. The three Chilean strains shared six substitutions along

the ITS spacers when compared with the foreign strains; these are located at positions 55,

56 and 120 in ITS-1 and 18, 112 and 222 in ITS-2. In addition, two short insertions in the

ITS-2 (between nucleotides 21–26 and 31–35) are found only in strains CONC-001 and

CONC-007 (Fig. 3).

The strain CONC-007 was the most divergent within the group (Fig. 4). Its farthest

conspecific was CONC-006, a strain isolated from the same biogeographic region (‘‘Salar

de Atacama’’) with which it differs by 11.4%, a value much higher than the ones observed

among the foreign strains (0.2% for the Mexico/China pair and 3.0% for the Australia/

Dunaliella bardawil pair) (data not shown).

In the parsimony analysis, 52 out of 456 characters (11.4%) were parsimony

informative and only one most parsimony tree was found at 142 steps. The data showed

low homoplasy (H.I. = 0.2289) and high consistency index (C.I. = 0.7711).

All the phylogenetic trees (distance, MP and ML) were consistent in showing five

clades: clade D includes the strains from Israel, Mexico and China; clade C consists of the

Australian strain; clade B consists of the strain CONC-006 and clade A includes the strains

CONC-001 and CONC-007. All these clades were solved with high bootstrap values (86–

100%) in the three types of phylogenetic trees. While the strain CONC-006 from Chile

Fig. 4. Phylogram based on maximum parsimony analysis of the entire ITS-1 and ITS-2 sequences of D. salina

strains. Tree shown is the only most parsimonious one at 142 steps recovered in a branch and bound search. D.

viridis and D. tertiolecta were included as outgroups. Bootstrap values are shown above (1000 replicates for

maximum parsimony-MP) and below (500 replicates for maximum likelihood-ML) branches. Consistency index

excluding uninformative characters (CI) = 0.7711; retention index (RI) = 0.7979 and rescaled consistency index

(RI) = 0.6911. Scale bar for one nucleotide change shown at the bottom.


(clade B) and the one from Australia (clade C) each stand by itself, the strains from Israel,

Mexico and China and the two from Chile (CONC-001 and CONC-007) form two clades

(A and D), each one very well supported (bootstrap values: 99–100%), indicating a high

genetic relatedness within each group (Fig. 4).

4. Discussion

DNA-based methods have provided new possibilities for obtaining information about

genetic diversity of organisms, even at the individual level. The information provided by

this type of studies is fundamental in many fields of biology, both basic and applied (Karp

and Edwards, 1998; Weising et al., 1995).

The advantages provided by RAPD-PCR analysis have been recognized in several

papers (for reviews see Weising et al., 1995; Caetano-Anolles and Gresshoff, 1997).

However, one traditional drawback of using RAPD markers is that they represent

dominant markers, precluding discrimination between homozygous and heterozygous

diploid organisms (Van Oppen et al., 1996; Weising et al., 1995). The advantages of using

RAPD markers in the characterization of eukaryotic haploid organisms have been already

demonstrated in studies of the toxic dinoflagellate Gymnodinium catenatum (Bolch et al.,

1999b) and on strains of D. salina (Gomez and Gonzalez, 2001).

Van Oppen et al. (1994), Valatka et al. (2000), and others have stressed that RAPD

should be used only for comparisons among very closely related taxa, i.e. at the

intraspecific and/or interspecific level. In microalgae, RAPD markers have been employed

in the genetic characterization of strains of Cyanophyceae (Neilan, 1995; Nishihara et al.,

1997; Bolch et al., 1999a), Dinophyceae (Bolch et al., 1999b) and Chlorophyceae (Gomez

and Gonzalez, 2001). In the present work, the high resolution power of RAPD-markers is

demonstrated by the generation of polymorphic markers with all of the primers employed

(Fig. 1, Table 2), reinforcing previous conclusions (Gomez and Gonzalez, 2001) about the

usefulness of this technique to genetically characterize strains of D. salina with different

physiological attributes.

Gomez and Gonzalez (2001) had previously reported the high degree of genetic

variability among the Chilean strains CONC-001, CONC-006 and CONC-007 shown by

RAPD band pattern analysis. In this study a good agreement was obtained between the

phenetic and cladistic analyses of RAPD data and ITS sequences, respectively.

Different regions of the genome are exposed to different selective pressures, depending

on the genetic product for which they code (Parker et al., 1998). Even though ITS

sequences exhibit high variation rates in Volvocales, their secondary structure presents

highly conserved domains, probably because these regions affect the processing of the

rRNA primary transcript (Mai and Coleman, 1997; Coleman et al., 1998). RAPD markers,

on the other hand, are randomly generated from many loci and they can reveal genetic

differences where other markers reach their resolution limit (e.g., Van Oppen et al., 1994;

Leignel et al., 1997). Fabry et al. (1999) mentioned that RAPD data revealed substantial

differences (ca. 35%) in two African isolates of Gonium pectorale (Volvocales), which

appeared nearly identical according to intron (ypt4 and act-IX) and ITS sequences analysis

(Kohler and Fabry, unpublished results). Although in the present research both RAPD and


ITS analyses revealed genetic differences among the strains of D. salina studied, much

higher divergence values were obtained from RAPD data than from ITS sequences,

demonstrating the higher resolution power of RAPD.

When Gonzalez et al. (2001) sequenced the ITS spacers of the Chilean strains CONC-

006 and CONC-007, they found that the two strains did not group together as expected,

based on their geographic proximity; on the contrary, they were associated with strains of

distant geographic origins (CONC-006 with ATCC 30861 from Israel and CONC-007

with UTEX 1644 from Mexico). When more strains of D. salina are added to the analysis

(this study), the Chilean strains are again the most divergent ones, independent of the

optimality tree-building criteria used (NJ, MP and ML) (Fig. 4). On the other hand, the two

deletions in the ITS-2 reported by Gonzalez et al. (2001) as a unique character for the

strains ATCC 30861 (D. bardawil) and CONC-006, turned out to be a rule rather than an

exception in D. salina, since we detected these deletions in all of the strains analyzed,

except for CONC-001 and CONC-007 (Fig. 3, ITS-2 nucleotides 21–26 and 31–35).

When considering the physiological attributes of the analyzed strains, it should be noted

that the two strains that stand by themselves based on molecular data (this paper), namely

strains CONC-006 and Australia, exhibited contrasting physiological attributes. Thus,

CONC-006 accumulates a significantly higher amount of carotenes per cell than the

Australian strain (61.1 and 33.9 pg cell� 1, respectively); on the other hand, the Australian

strain accumulates a significantly higher amount of carotenes per culture volume than

CONC-006 (40.6 and 10 mg l� 1, respectively); this difference is due to the different

carrying capacities of both strains (Gomez and Gonzalez, unpublished data). Furthermore,

CONC-006 accumulates a-carotene (between 25% and 35%) under any culture condition

(15 or 26 jC), while the Australian strain accumulates V 10% and only at low temperature

(15 jC). The same correlation is true for all the other foreign strains (from Israel, China,

Mexico): all of them exhibited very similar physiological attributes (Gomez and Gonzalez,

unpublished data) and were grouped together based on molecular data. For comparison,

although CONC-001 and CONC-007 differed significantly in their physiological

responses (in terms of carotene content per culture volume and per cell, carotenoids/

chlorophyll ratio, and growth rate), they were grouped together based on molecular data.

The ITS data strongly support the phylogenetic closeness of CONC-001 and CONC-007,

although the RAPD data revealed a 25.5% difference between both strains. Again, RAPD

markers, as mentioned previously, can reveal genetic differences when ITS (in this case)

has reached its resolution limit.

Since intraspecific variability is a fundamental aspect in speciation processes, the

physiological diversity that exists among the strains of D. salina studied (Gomez and

Gonzalez, unpublished data) and the genetic basis that support it (this paper) might

indicate the presence of more than one species among them. Mating experiments would be

the most direct way of resolving this problem; nevertheless, due to self-mating (homo-

thalism), we have not been able to determinate interbreeding ability between the strains.

Through a series of papers, Coleman and co-workers have demonstrated that in Volvocales

(D. salina belongs to this order) there is good agreement between mating ability and ITS

sequence identity, especially in some highly conserved regions of the secondary structure

of ITS-2 (Coleman and Mai, 1997; Coleman, 1999, 2000, 2001). Future studies should be

focused on determining the secondary structure of this ribosomal spacer in the strains of D.


salina analyzed, and looking for a qualitative type of sequence change within its highly

conserved region (hairpin loop II and III), a useful alternative in this case where the

biological species status is difficult to test by means of sexual compatibility.

Summarizing, the correlation detected between the differences in the carotenogenic

capacity of the D. salina strains and the genetic diversity among them is a clear

demonstration of the genome participation in determining these attributes. Obviously,

for mass cultivation, this is a very important result since the existence of genetically

characterized strains will minimize incorrect assumptions about the biotechnological

important traits of different strains, which could be due to phenotypic plasticity.

Acknowledgements

We thank Dr. Annette W. Coleman (Brown University, USA) for reading the

manuscript and helping us with the English. This study was supported by a grant to P.

Gomez from FONDECYT (Grant #2990047).

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Genetic variation among seven strains of Dunaliella salina (Chlorophyta) with industrial potential,...

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