Genetic Markers for Metabarcoding of Freshwater Microalgae: Review

SIMPLE SUMMARY: The metabarcoding approach is widely used for studying the diversity and distribution of freshwater microalgae and for routine biomonitoring. Due to microalgae being a phylogenetically diverse group, the choice of a genetic marker directly affects the metabarcoding results. Specific markers are good for identifying only concrete groups, while universal markers may miss classes or lack the variability necessary for differentiating taxa at the species and sometimes genus levels. An analysis of publications on the subject showed that metabarcoding studies of eukaryotic freshwater microalgae used 12 markers (different nuclear regions 18S and ITS and plastid regions rbcL, 23S and 16S). Studies that compared outcomes from different markers show that the resulting lists of taxa do not match. The plastid marker rbcL is widely used for diatom metabarcoding, as it differentiates taxa at the species and intraspecies levels, and there is a specific set of primers designed for identifying Eustigmatophyceae. The V9 18S region is more variable than V4 18S and provides more diversity at higher taxonomic levels (supergroup and phylum). The ITS1 and ITS2 regions are used rarely and may be underestimated. These barcodes amplify well with the standard primers and are variable enough to identify sequences at the species level. Plastid markers (23S and 16S rDNA) focused on the plastid-containing eukaryotic algae and Cyanobacteria, conserved regions, identify taxa to the genus level and higher. Using specialized curated databases for data interpretation significantly improves the quality of the results. ABSTRACT: The metabarcoding methods for studying the diversity of freshwater microalgae and routine biomonitoring are actively used in modern research. A lot of experience has been accumulated already, and many methodological questions have been solved (such as the influence of the methods and time of sample conservation, DNA extraction and bioinformatical processing). The reproducibility of the method has been tested and confirmed. However, one of the main problems—choosing a genetic marker for the study—still lacks a clear answer. We analyzed 70 publications and found out that studies on eukaryotic freshwater microalgae use 12 markers (different nuclear regions 18S and ITS and plastids rbcL, 23S and 16S). Each marker has its peculiarities; they amplify differently and have various levels of efficiency (variability) in different groups of algae. The V4 and V9 18S and rbcL regions are used most often. We concentrated especially on the studies that compare the results of using different markers and microscopy. We summarize the data on the primers for each region and on how the choice of a marker affects the taxonomic composition of a community.