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A novel cost effective and high-throughput isolation and identification method for marine microalgae.

Jahn MT, Schmidt K, Mock T - Plant Methods (2014)

Bottom Line: For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean.We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks.Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK ; Current address: Department of Botany II, Julius-Maximilians University Würzburg, Julius-von-Sachs-Platz 3, 97082 Würzburg, Germany.

ABSTRACT

Background: Marine microalgae are of major ecologic and emerging economic importance. Biotechnological screening schemes of microalgae for specific traits and laboratory experiments to advance our knowledge on algal biology and evolution strongly benefit from culture collections reflecting a maximum of the natural inter- and intraspecific diversity. However, standard procedures for strain isolation and identification, namely DNA extraction, purification, amplification, sequencing and taxonomic identification still include considerable constraints increasing the time required to establish new cultures.

Results: In this study, we report a cost effective and high-throughput isolation and identification method for marine microalgae. The throughput was increased by applying strain isolation on plates and taxonomic identification by direct PCR (dPCR) of phylogenetic marker genes in combination with a novel sequencing electropherogram based screening method to assess the taxonomic diversity and identity of the isolated cultures. For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean. These cultures include the isolate of a novel marine Chlorophyceae strain among several different diatoms.

Conclusions: We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks. Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures.

No MeSH data available.


Related in: MedlinePlus

Representative sequencing electropherogram sections. Compared are the base calling signal noise of (a) unialgal Thalassiosira pseudonana laboratory culture (b) non-unialgal culture 1-80-15-M with 2 morphospecies (c) unialgal classified culture of Skeletonema marinoi (2-80-8-M). The color code refers to the per base Quality Values (QV) as the –10 log10(Pe), with Pe as the base call error probability.
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Figure 2: Representative sequencing electropherogram sections. Compared are the base calling signal noise of (a) unialgal Thalassiosira pseudonana laboratory culture (b) non-unialgal culture 1-80-15-M with 2 morphospecies (c) unialgal classified culture of Skeletonema marinoi (2-80-8-M). The color code refers to the per base Quality Values (QV) as the –10 log10(Pe), with Pe as the base call error probability.

Mentions: Using the high-throughput isolation technique of streaking enriched natural microalgal communities across agar plates, on 59.3% (35 of 59) of the incubated plates algal growth was detected. From about three quarters (77.1%; 27 of 35) of these plates, it was possible to pick single colonies. Moreover, all (158 of 158) of the picked colonies transferred to 12-well plates showed visible growth under the microscope after 1.5 weeks of cultivation. In a preliminary study, primers amplifying the whole 18S rDNA (~1750 bp) region were used for unialgal assessment and taxonomic identification. However, dPCR amplicon sequencing from the 5′ end of the whole 18S rDNA region lacked sufficient variability compared to the V4 sub-region on the 18S rRNA gene. By combining the dPCRs of the whole 18S rDNA and of the V4 region of 18S rDNA, the dPCR approach succeeded in 70.25% (85 of 121) of the reactions. Furthermore, the amplicons obtained by dPCR, as shown in Figure 1, had identical size compared to the control PCR conducted with extracted DNA. Also, no additional bands were visible for dPCRs.About 65% of the screened cultures (24 of 37) were identified as unialgal based on our new electropherogram-based assessment. Figure 2 illustrates the discrimination principle between sequences from unialgal cultures and mixed populations.


A novel cost effective and high-throughput isolation and identification method for marine microalgae.

Jahn MT, Schmidt K, Mock T - Plant Methods (2014)

Representative sequencing electropherogram sections. Compared are the base calling signal noise of (a) unialgal Thalassiosira pseudonana laboratory culture (b) non-unialgal culture 1-80-15-M with 2 morphospecies (c) unialgal classified culture of Skeletonema marinoi (2-80-8-M). The color code refers to the per base Quality Values (QV) as the –10 log10(Pe), with Pe as the base call error probability.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128616&req=5

Figure 2: Representative sequencing electropherogram sections. Compared are the base calling signal noise of (a) unialgal Thalassiosira pseudonana laboratory culture (b) non-unialgal culture 1-80-15-M with 2 morphospecies (c) unialgal classified culture of Skeletonema marinoi (2-80-8-M). The color code refers to the per base Quality Values (QV) as the –10 log10(Pe), with Pe as the base call error probability.
Mentions: Using the high-throughput isolation technique of streaking enriched natural microalgal communities across agar plates, on 59.3% (35 of 59) of the incubated plates algal growth was detected. From about three quarters (77.1%; 27 of 35) of these plates, it was possible to pick single colonies. Moreover, all (158 of 158) of the picked colonies transferred to 12-well plates showed visible growth under the microscope after 1.5 weeks of cultivation. In a preliminary study, primers amplifying the whole 18S rDNA (~1750 bp) region were used for unialgal assessment and taxonomic identification. However, dPCR amplicon sequencing from the 5′ end of the whole 18S rDNA region lacked sufficient variability compared to the V4 sub-region on the 18S rRNA gene. By combining the dPCRs of the whole 18S rDNA and of the V4 region of 18S rDNA, the dPCR approach succeeded in 70.25% (85 of 121) of the reactions. Furthermore, the amplicons obtained by dPCR, as shown in Figure 1, had identical size compared to the control PCR conducted with extracted DNA. Also, no additional bands were visible for dPCRs.About 65% of the screened cultures (24 of 37) were identified as unialgal based on our new electropherogram-based assessment. Figure 2 illustrates the discrimination principle between sequences from unialgal cultures and mixed populations.

Bottom Line: For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean.We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks.Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK ; Current address: Department of Botany II, Julius-Maximilians University Würzburg, Julius-von-Sachs-Platz 3, 97082 Würzburg, Germany.

ABSTRACT

Background: Marine microalgae are of major ecologic and emerging economic importance. Biotechnological screening schemes of microalgae for specific traits and laboratory experiments to advance our knowledge on algal biology and evolution strongly benefit from culture collections reflecting a maximum of the natural inter- and intraspecific diversity. However, standard procedures for strain isolation and identification, namely DNA extraction, purification, amplification, sequencing and taxonomic identification still include considerable constraints increasing the time required to establish new cultures.

Results: In this study, we report a cost effective and high-throughput isolation and identification method for marine microalgae. The throughput was increased by applying strain isolation on plates and taxonomic identification by direct PCR (dPCR) of phylogenetic marker genes in combination with a novel sequencing electropherogram based screening method to assess the taxonomic diversity and identity of the isolated cultures. For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean. These cultures include the isolate of a novel marine Chlorophyceae strain among several different diatoms.

Conclusions: We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks. Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures.

No MeSH data available.


Related in: MedlinePlus