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Biological Researchversin impresaISSN 0716-9760

Biol. Res. v.34 n.1 Santiago 2001

doi: 10.4067/S0716-97602001000100012

Genetic polymorphism in eight Chilean strains of thecarotenogenic microalga Dunaliella salinaTeodoresco(Chlorophyta)

PATRICIA I GOMEZ1, MARIELA A GONZALEZ2

1Departamento de Biologa Molecular, Facultad de Ciencias Biolgicas, 2Departamentode Botnica,Facultad de C iencias Naturales y Oceanogrficas, Universidad de C oncepcin. Chile

ABSTRACT

Eight Chilean strains of Dunaliella salina obtained within a restrictedgeographic range, but exhibiting a high variability in their morphology, rate of growth and carotenogenic capacitwere analyzed by Random Amplified Polymorphic DNA (RAPD-PCR). Twenty of the 50 random primers (D, P, Oand OPD series) that were tested amplified reproducible bands and were useful for comparative analysis of tstrains. Of 107 polymorphic genetic markers,49 were strain-specific. A great genetic variability was found amothe strains in spite of their geographic proximity. In addition, phenetic analysis of the data showed cloagreement between the morpho-physiological attributes and the genetic diversity of the strains.

Key terms: -carotene, Dunaliella salina, genetic polymorphism, RAPD

INTRODUCTION

Dunaliella salinaTeodoresco is a halophilic walless microalga that accumulates large amounts of -carotene whcultivated under high light intensity and growth-limiting conditions (Ben-Amotz et al. 1982; Ben-Amotz and Avr1983; Borowitzka and Borowitzka 1988). -carotene is accumulated within oily globules in the interthylakospaces of the chloroplast and is composed mainly of two isomers: 9-cis and all-trans (Ben-Amotz et al. 1981988), with 9-cis -carotene being the most important for commercial purposes (Borowitzka 1986; Ben-Amoand Avron 1990; Becker 1994).

Dunaliella salina is highly suitable for mass cultivation; its ability to grow in media with high salt concentratioenables it to be cultivated outdoors in a relatively pure culture and with few potential predators . In addition, ihigh carotene content (which protects it from intense solar irradiation) and halotolerance allow cultivation in aror desert areas with access to brackish water or seawater (Borowitzka 1986; Borowitzka and Borowitzka 198Ben-Amotz et al. 1991).

The presence of this microalga in Chile was reported in 1990 (Parra et al. 1990a, b). Eight strains have beisolated since then, all from the northern regions of the country. Previous studies carried out with these straihave shown great variability in morphology, growth rate and accumulation of total carotenes when cultivatunder different conditions (Araneda et al. 1992a, b; Cifuentes et al. 1992; 1996a, b). Moreover, quality analysof the -carotene produced by three of these strains (CONC-001, CONC-006 and CONC-007) revealed significavariability in the -carotene isomers ratio (Markovits et al. 1993; Gmez et al. 1999). Although the physiologicvariation detected in the Chilean strains of D. salinapresumably reflect genotypic differences, as yet there hbeen little or no study of genetic diversity in these populations.

DNA-based molecular methods have provided new possibilities for studying the intraspecific differencesorganisms. Random Amplified Polymorphic DNA (RAPD) (Welsh and McClelland 1990; Williams et al. 1990) allothe detection of multi-locus genetic variation using short primers of arbitrary sequence. In contrast to othstandard molecular techniques, such as restriction fragment length polymorphism (RFLP), DNA sequencing, a

allozymes, the RAPD technique is very easy to perform and requires no prior knowledge of the genomes undinvestigation (a general review in Weising et al. 1995).

At the intraspecific level, RAPD markers have been successfully employed in microalgae for identificationtoxigenic strains in Cyanophyceae (Neilan 1995; Nishihara et al. 1997; Bolch et al. 1999a) and in Dinophyce(Bolch et al. 1999b). However, studies using this technique for generating genetic markers among strainsmicroalgae with industrial potential have not been previously published.

8/13/2019 Biological Research - Genetic Polymorphism in Eight Chilean Strains of the Carotenogenic Microalga Dunaliella Sali

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23/07/12 Biological Research - Genetic polymorphism in eight Chilean strains of the carotenogenic microalga

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By using RAPD, this study searched for the presence of genetic polymorphism among 8 Chilean strains of D. salifrom very close geographical locations but with striking differences in morphology and physiological behavior.

METHODS

All the strains of D. salinacame from the north of Chile; one of them (CONC-001) from the La Rinconadapond,thalassohaline water body located 30 km north of the city of Antofagasta (2339'S; 7024'W) and the oth

seven (CONC-003 to CONC-009) from the Salar de Atacama, a 3000 km2 salt-flat at 2340 m above sea lev(2330'S; 6815'W). Unialgal cultures of all of these strains are maintained in the Microalgae Culture CollectioUniversity of Concepcin, Chile.

The strains were grown in Johnson medium (Borowitzka and Borowitzka 1988) with 15% NaCl under a continuophoton flux density of 100 molm-2s-1and 252 C. In order to obtain axenic cultures, exponentially-growing celwere washed three times with fresh medium by centrifuging at 2000 rpm for 5 min. The cultures were treated t

following day with an appropriate volume of an antibiotic mixture to obtain a final concentration of 200 mg l

penicillin, 100 mg l-1 streptomycin and 20 mg l-1 chloramphenicol (Hoshaw and Rosowski 1973). After 5 days, 1from each tube was transferred aseptically to flasks containing 50 ml of antibiotic-free culture medium.

Total DNA was extracted from 50 ml of 21-day cultures. Cells were collected by centrifugation at 5000 rpm formin; the pellet was resuspended in 500 l of TEN buffer (Tris pH 8 10 mM, EDTA 10 mM, NaCl 150 mM), vortexecentrifuged 10 s, and the supernatant discarded. The cells were resuspended in 150 l of sterile distilled waterwhich 300 l of buffer SDS-EB (SDS 2%, NaCl 400 mM, EDTA 40 mM, Tris pH 8 100 mM) was added. The proteiwere extracted once with 300 l of a phenol:chloroform/isoamylic alcohol (24/1) [1:1] mixture, and stirredvortexing. The aqueous phase was separated by c entrifuging and re-extracted with 300 l of chloroform:isoamylalcohol (24:1). The aqueous phase was separated again by centrifugation, the DNA was precipitated by addi

two volumes of absolute alcohol at -20C for 1 hr. Finally, it was centrifuged at 13000 rpm for 5 min, and tpellet was washed with 200 l of 70% ethanol, dried and re-dissolved in 150 l buffer TE (Tris pH 8 10 mM, EDpH 8 1 mM) until use.

RAPD amplifications employing the polymerase chain reaction (PCR) were carried out in a 25 l volume containi10 ng of genomic DNA, 1X reaction buffer, 1.5 mM MgCl2, 0.1 mM each of dNTP, 0.2 M primer and 1 U of T

polymerase. A total of 50 primers of arbitrary sequence and 10 bp in length from the D, P, OPA and OPD (Operoseries were used. The PCR reaction was carried out in a Perkin Elmer thermocycler with the following program:initial denaturation step of 94C for 6 min followed by 35 cycles of denaturation (94C, 1min), annealing (45C,min) and extension (72C, 2 min) with a terminal extension of 72C for 6 min. In order to ensure reproducibilitRAPD patterns from two independent axenic cultures were obtained for each strain, and PCR for each DNA sampwas replicated at least twice.

The amplified fragments were eletrophoretically separated in 2% agarose gels in TAE at 60V for 90 min, stained

ethidium bromide (0.5 g ml-1

) for 15 min, and photographed under a UV transiluminator with Polaroid film 66The phenetic analysis of the data was carried out with the unweighted pair group method with arithmetic me(UPGMA) using the PAUP version 4.0 beta program.

RESULTS

Only 20 primers (Table I) generated appropriate amplification patterns (clear bands and low backgroufluorescence) and were used in the comparative analysis among the strains. As observed in Figure 1, RAanalysis revealed a high degree of genetic polymorphism among some of the strains of D. salina analyzed. Tnumber of RAPD bands generated per primer varied between 1 (P-8 primer) and 11 (OPA-18 primer). A total107 reproducible bands were scored, ranging in size from 300 to 2000 bp.

TABLE I

Sequences of the RAPD primers employed on this study

Primer Sequence5' ---> 3'

Totalnumber of

bandsscored

D-02P-2P-3P-8P-10P-100OPA-01OPA-04

OPA-09OPA-10OPA-11OPA-13OPA-18OPD-02OPD-08

>GGA.CCC.AAC.CACA.ACT.GCT.CTGA.CTG.ACG.CCCG.CAG.CCA.AGCG.ATC.CCC.AATC.GGG.TCC.GCAG.GCC.CTT.CAAT.CGG.GCT.GGGG.TAA.CGC.CGTG.ATC.GCA.GCAA.TCG.CCG.TCAG.CAC.CCA.CAGG.TGA.CCG.TGGA.CCC.AAC.CGTG.TGC.CCC.AAGC.GCC.ATT.GGGG.GTG.ACG.AAGG.GCG.TAA.GGAG.AGC.CAA.CACC.CGG.TCA.C