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

Abstract

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).

DNA Content Variation and Its Significance in the Evolution of the Genus Micrasterias (Desmidiales, Streptophyta)

“…Beside the problems with nuclei isolation due to the complexity of cell wall discussed previously [31] we noticed the peak pattern variation in histograms obtained from individual measurements.…”

“…This is also reflected in the amount of the Kew Plant DNA C-values database algae entries of only 253, in contrast to the presently over 7500 angiosperm C-value entries [62]. The results reported in this paper constitute one of few flow cytometric analysis in microalgae [31], [63]–[65] and first such analysis in desmids.…”

The Plant DNA C‐values database (release 7.1): an updated online repository of plant genome size data for comparative studies

“…those belonging to Heterokonta, such as diatoms, and the brown algae (Phaeophyta)). They are often neglected because of the methodological challenges they pose when estimating genome size (Voglmayr, ; Mazalová et al , ). Evolutionary relationships among many algal lineages are still controversial, but recent phylogenetic studies provide strong support for three of the streptophyte algal lineages (i.e.…”

Characterization of Two Zygnema Strains (Zygnema circumcarinatum SAG 698-1a and SAG 698-1b) and a Rapid Method to Estimate Nuclear Genome Size of Zygnematophycean Green Algae

“…In order to obtain enough high-quality nuclei for FC and DAPI analysis, we have also tried generating protoplasts following the methods described by Mazalová et al (2011) and Ohiwa (1977Ohiwa ( , 1981. Unfortunately, these methods failed as the cell walls of SAG 698-1b were particularly hard to be removed with cell wall digesting enzymes (cellulases, hemicellulases and pectinases) (Supplementary Figure 10).…”