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DNA fingerprinting differentiation between beta-carotene hyperproducer strains of Dunaliella from around the world.

Olmos J, Ochoa L, Paniagua-Michel J, Contreras R - Saline Syst. (2009)

Bottom Line: In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and beta-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico.In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method.The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Microbiology Laboratory, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Department of Marine Biotechnology, Ensenada, B.C, México.

ABSTRACT

Background: Dunaliella salina is the most important species of the genus for beta-carotene production. Several investigations have demonstrated that D. salina produces more than 10% dry weight of pigment and that the species grows in salt saturated lagoons. High plasticity in the green stage and the almost indistinguishable differences in the red phase make identification and differentiation of species and ecotypes very difficult and time consuming.

Results: In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and beta-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico. All hyperproducer strains reached beta-carotene levels of about 10 pg/cell. Optical microscopy did not allow to differentiate between these Dunaliella strains; however, 18S rDNA fingerprinting methodology allowed us to differentiate D. salina from D. salina/bardawil.

Conclusion: In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method. The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.

No MeSH data available.


Amplification with conserved and specific oligonucleotides. Lane 1, molecular weight marker. Lanes 2 and 3 correspond to amplification with MA1–MA2 conserved primers, using D. bardawil (LB2538) and β-carotene hyperproducer species isolated from red hypersaline environments in Baja Mexico. Lanes 4 and 5, corresponds to amplification with DBs-MA2 specific-conserved oligonucleotides, from both β-carotene hyperproducers species. Lane 6 corresponds to amplification with DSs-MA2 specific-conserved oligonucleotides, from the LB2538 β-carotene hyperproducer species. Lane 7 corresponds to amplification with MA1–MA2 conserved primers, using 19/30 DNA sample.
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Figure 3: Amplification with conserved and specific oligonucleotides. Lane 1, molecular weight marker. Lanes 2 and 3 correspond to amplification with MA1–MA2 conserved primers, using D. bardawil (LB2538) and β-carotene hyperproducer species isolated from red hypersaline environments in Baja Mexico. Lanes 4 and 5, corresponds to amplification with DBs-MA2 specific-conserved oligonucleotides, from both β-carotene hyperproducers species. Lane 6 corresponds to amplification with DSs-MA2 specific-conserved oligonucleotides, from the LB2538 β-carotene hyperproducer species. Lane 7 corresponds to amplification with MA1–MA2 conserved primers, using 19/30 DNA sample.

Mentions: On the other hand, both species classified as D. salina/bardawil purchased from UTEX (LB2538) ten years ago and Dunaliella strain isolated from Baja Mexico (this work), turned red and reached 10% dry weight of β-carotene. In addition, both strains amplified a PCR product of ~2500 bp using MA1–MA2 primers (Fig. 3). Specific 18S rDNA fingerprints showed a PCR product of ~1000 bp using DBs-MA2 primers in both strains and no bands were amplified with DSs-MA2 (Fig. 3). With these results we established the fingerprinting profile of D. salina/bardawil as MA1–MA2 = ~2500 bp/DBs-MA2 = ~1000 bp. D. salina/bardawil species has not been reported from other places than Bardawil lagoon. In this sense, a controversy still exists about the authenticity of D. salina/bardawil as new species [2]. In addition, microscopic differentiation between D. salina and D. salina/bardawil species is difficult and time consuming. However, molecularly, D. salina/bardawil has an exclusive fingerprinting profile of MA1–MA2 = ~2500 bp/DBs-MA2 = ~1000 bp, different from the one presented by "D. salina var Teod" (MA1–MA2 = ~2100/DSs-MA2 = ~700). Certainly, more work must be done by taxonomic experts to definitively classify D. salina/bardawil as "D. bardawil" or as "D. salina var Bardal" or with another name. However, our methodology works well to differentiate between the two most important carotenogenic species of Dunaliella and to make a rapid and precise identification of them. Most importantly, this methodology is helpful in the beginning of the isolation process even in the green stage. This information is important both for commercial and scientific applications.


DNA fingerprinting differentiation between beta-carotene hyperproducer strains of Dunaliella from around the world.

Olmos J, Ochoa L, Paniagua-Michel J, Contreras R - Saline Syst. (2009)

Amplification with conserved and specific oligonucleotides. Lane 1, molecular weight marker. Lanes 2 and 3 correspond to amplification with MA1–MA2 conserved primers, using D. bardawil (LB2538) and β-carotene hyperproducer species isolated from red hypersaline environments in Baja Mexico. Lanes 4 and 5, corresponds to amplification with DBs-MA2 specific-conserved oligonucleotides, from both β-carotene hyperproducers species. Lane 6 corresponds to amplification with DSs-MA2 specific-conserved oligonucleotides, from the LB2538 β-carotene hyperproducer species. Lane 7 corresponds to amplification with MA1–MA2 conserved primers, using 19/30 DNA sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2710335&req=5

Figure 3: Amplification with conserved and specific oligonucleotides. Lane 1, molecular weight marker. Lanes 2 and 3 correspond to amplification with MA1–MA2 conserved primers, using D. bardawil (LB2538) and β-carotene hyperproducer species isolated from red hypersaline environments in Baja Mexico. Lanes 4 and 5, corresponds to amplification with DBs-MA2 specific-conserved oligonucleotides, from both β-carotene hyperproducers species. Lane 6 corresponds to amplification with DSs-MA2 specific-conserved oligonucleotides, from the LB2538 β-carotene hyperproducer species. Lane 7 corresponds to amplification with MA1–MA2 conserved primers, using 19/30 DNA sample.
Mentions: On the other hand, both species classified as D. salina/bardawil purchased from UTEX (LB2538) ten years ago and Dunaliella strain isolated from Baja Mexico (this work), turned red and reached 10% dry weight of β-carotene. In addition, both strains amplified a PCR product of ~2500 bp using MA1–MA2 primers (Fig. 3). Specific 18S rDNA fingerprints showed a PCR product of ~1000 bp using DBs-MA2 primers in both strains and no bands were amplified with DSs-MA2 (Fig. 3). With these results we established the fingerprinting profile of D. salina/bardawil as MA1–MA2 = ~2500 bp/DBs-MA2 = ~1000 bp. D. salina/bardawil species has not been reported from other places than Bardawil lagoon. In this sense, a controversy still exists about the authenticity of D. salina/bardawil as new species [2]. In addition, microscopic differentiation between D. salina and D. salina/bardawil species is difficult and time consuming. However, molecularly, D. salina/bardawil has an exclusive fingerprinting profile of MA1–MA2 = ~2500 bp/DBs-MA2 = ~1000 bp, different from the one presented by "D. salina var Teod" (MA1–MA2 = ~2100/DSs-MA2 = ~700). Certainly, more work must be done by taxonomic experts to definitively classify D. salina/bardawil as "D. bardawil" or as "D. salina var Bardal" or with another name. However, our methodology works well to differentiate between the two most important carotenogenic species of Dunaliella and to make a rapid and precise identification of them. Most importantly, this methodology is helpful in the beginning of the isolation process even in the green stage. This information is important both for commercial and scientific applications.

Bottom Line: In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and beta-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico.In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method.The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Microbiology Laboratory, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Department of Marine Biotechnology, Ensenada, B.C, México.

ABSTRACT

Background: Dunaliella salina is the most important species of the genus for beta-carotene production. Several investigations have demonstrated that D. salina produces more than 10% dry weight of pigment and that the species grows in salt saturated lagoons. High plasticity in the green stage and the almost indistinguishable differences in the red phase make identification and differentiation of species and ecotypes very difficult and time consuming.

Results: In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and beta-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico. All hyperproducer strains reached beta-carotene levels of about 10 pg/cell. Optical microscopy did not allow to differentiate between these Dunaliella strains; however, 18S rDNA fingerprinting methodology allowed us to differentiate D. salina from D. salina/bardawil.

Conclusion: In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method. The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.

No MeSH data available.