Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Gall, Y. Le; Brown, Spencer C.; Marie, Dominique; Mejjad, M.; Kloareg, Bernard

doi:10.1007/bf01379001

Cited by 91 publications

(83 citation statements)

References 26 publications

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“…The intraspecific differences were also documented by ŠMarda et al (2008) on fescues which range from 42.53% to 46.41%. Similar variation with a range from 42.9% to 46.7% was detected for several algae species by le Gall et al (1993). The most marked discrepancy caused by the use of specifically binding dyes was shown by dOležel et al (1992).…”

Section: Methodssupporting

confidence: 63%

“…Although flow cytometry (FC) is routinely used in higher plants, it has rarely been used for microalgae (le Gall et al 1993;SiMOn et al 1994;VeldhuiS et al 1997). Moreover these studies estimated nuclear DNA content from whole cells, but recently it turned out that only estimates based on isolated nuclei are accurate enough for general comparisons (kaPraun 2007).…”

Section: Introductionmentioning

confidence: 99%

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Quantification of DNA content in freshwater microalgae using flow cytometry: a modified protocol for selected green microalgae.

Mazalová

Šarhanová²,

Ondřej³

et al. 2011

Fottea

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Abstract:The study of genome size variation in microalgae lags behind that of comparable research in higher plants and seaweeds. This situation is essentially caused by: (1) difficulties in obtaining sufficient biomass for experiments; (2) problems with protoplast isolation due to cell-wall heterogeneity and complexity; and (3) the absence of suitable standards for routine measurements. We propose a multi-step protocol that leads to the quantification of DNA content in desmids using flow cytometry. We present detailed culture conditions, the minimal biomass necessary for three repetitive measurements, a method to isolate protoplasts and selection of suitable standards. Our protocol, which is mainly based on studies with higher plants and commercially available enzyme mixtures, is useful in Streptophyta, especially members of the Zygnematophyceae, because of their close phylogenetic relationship to higher plants, in particular the similarity of their cell wall organization. Moreover, the suggested protocol also works for some Chlorophyta (Chloroidium ellipsoideum, Tetraselmis subcordiformis) and Heterokontophyta (Tribonema vulgare). We suggest and characterize a new standard for flow cytometry of microalgae (Micrasterias pinnatifida). Modification of the enzyme mixture is probably necessary for microalgae whose cell walls are surrounded by a mucilaginous envelope (Planktosphaeria), those that contain alganan (Chlorella), monads with a pellicle or chlamys (Euglena, Chlamydomonas). While we did not anticipate any success with diatoms (Pinnularia), because of their silica frustules, the enzyme mixture also failed for some other green microalgae (Xanthidium, Kentrosphaera, Stigeoclonium, Trentepohlia and Pseudendoclonium).

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Section: Methodssupporting

confidence: 63%

Section: Introductionmentioning

confidence: 99%

Quantification of DNA content in freshwater microalgae using flow cytometry: a modified protocol for selected green microalgae.

Mazalová

Šarhanová²,

Ondřej³

et al. 2011

Fottea

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show abstract

“…The entire life cycle can be completed in the laboratory within 2-3 months. The haploid genome of Porphyra is relatively small in size (2.7×10 8 5.3×10 8 bp) [4]. These features make it a good material for studying genetics and genomics.…”

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confidence: 99%

Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology

et al. 2011

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With high-throughput Solexa sequencing technology, we profiled Porphyra yezoensis transcriptomes from 8 different samples. More than 1200 megabases from 13333334 quality paired-end reads were generated, which were assembled into 31538 unigenes. Blast analysis showed that 56.7% unigenes were novel, which represented the specific genes of Porphyra and/or rhodophytes. Several hundreds of unigenes related to stress tolerance were discovered, including genes related to desiccation-(211) and high light-tolerance (31), flavonoid biosynthesis (10), reactive oxygen scavenging (48) and other stress-tolerance processes (208), which indicated there existed complex and diversity modes of stress tolerance in this species. A complete set of essential genes involved in the C3-(57) and C4-(44) carbon fixation pathway (except pyruvate phosphate dikinase) were discovered, which not only proved that they were actively transcribed but also clearly outlined the panoptic view of carbon fixation in Porphyra. Moreover, by statistically analyzing the types, proportions and frequencies of the interspersed repeats (TEs) and simple sequence repeats (SSRs), we discovered that the top three types of TEs were all retrotransposons and the trinucleotide was the absolute predominant type among SSRs, promoting our understanding of structural characteristics of the transcriptome. This study substantially improved the global view of the Porphyra genome and provided a valuable resource for future research.Porphyra yezoensis, red algae, transcriptome, Solexa sequencing technology, expressed sequence tags, functional analysis, transposable elements, microsatellites Citation:

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“…These species are considered model seaweeds for marine biotechnology (Fukuda et al 2008;Oohusa 1993) because of their biological and economical importance, as well as small genome size. The haploid genomes of Porphyra are estimated to be approximately 260-500 Mbp (Kapraun et al 1991;Le Gall et al 1991;Matsuyama-Serisawa et al 2007). However, heterologous gene expression to manipulate molecular pathways has been hindered by the difficulty of genetic transformation in Porphyra (Fukuda et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Development of an expression system using the heat shock protein 70 promoter in the red macroalga, Porphyra tenera

et al. 2011

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Porphyra is a commercially valuable source of food and drugs, and represents an important model organism for algal research. However, genetic research on P. tenera has been limited due to lack of a heterologous gene expression system. In the present study, we isolated a native promoter, the PtHSP70 promoter, for efficient expression of foreign genes in this organism. This promoter lies approximately 1 kb upstream of the coding sequence for Heat Shock Protein 70 (HSP70) and was isolated using adapter-mediated genomic PCR. Promoter activity was evaluated using the synthetic GUS gene (PyGUS) with optimized codons for Porphyra yezoensis. Interestingly, the PtHSP70 promoter allowed equivalent expression of PyGUS in both P. tenera and P. yezoensis, whereas the GAPDH promoter from P. yezoensis was not fully functional in P. tenera. These data suggest that the PtHSP70 promoter has a more conserved regulatory mechanism than the PyGAPDH promoter between these species.We also established an efficient transient transformation system for P. tenera by evaluating various transformation parameters such as quantity of gold particles, pressure of helium and vacuum, developmental stages of leafy gametophytes, and target distance. Under the optimal conditions of transient transformation, the frequency of GUS expression was determined by histochemical staining to be 30-50 cells per bombardment. Therefore, the new transient transformation system using the PtHSP70 promoter can be used for foreign gene expression in P. tenera, which may advance the development of P. tenera as a model organism.

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Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Cited by 91 publications

References 26 publications

Quantification of DNA content in freshwater microalgae using flow cytometry: a modified protocol for selected green microalgae.

Quantification of DNA content in freshwater microalgae using flow cytometry: a modified protocol for selected green microalgae.

Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology

Development of an expression system using the heat shock protein 70 promoter in the red macroalga, Porphyra tenera

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