Genetic Markers for Metabarcoding of Freshwater Microalgae: Review

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 63 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.

A comparison of two gene regions for assessing community composition of eukaryotic marine microalgae from coastal ecosystems

“…Again, this could be related to the influence of dinoflagellate cell size on gene copy number, where larger cells tend to have more rDNA gene copies 48 , or intra-cellular variation of the rDNA gene regions detected as multiple ASVs. Previous studies have shown that community composition based on LM generally vary greatly in comparison to next-generation sequencing or metabarcoding 41 , with both more and less diversity reported 50 – 52 . Identification of EMC taxa by morphology is complicated, requiring time, effort, and often experts specialised in specific taxonomic groups to confidently assign specimens to genus or species level.…”

“…Interestingly, this greater depth of taxonomic resolution was not aligned with captured diversity overall and indicated a clear bias to dinoflagellates over other taxonomic groups. Higher taxonomic resolution achieved using the V4 marker for dinoflagellates may be a consequence of high sequence variation of dinoflagellates within the V4 region 41 , or the greater length of this region increasing the chances of species level nucleotide variation being captured. Dinoflagellates have been shown to have very high rDNA copy number with a lot of variation even within a strain 47 , 48 .…”

“…Selection of gene regions, primers and understanding of the bias they introduce are important considerations when using molecular techniques 39 , with taxonomic resolution and the availability of reference sequences also influencing results 16 , 40 . Upward of 12 gene regions are commonly used to characterise microalgal communities, dependent on the taxonomic group of interest 41 , with a combination of gene regions commonly used or suggested to overcome bias of any single gene region/primer pair 21 , 42 , 43 . Some of the most used regions include the 18S ribosomal DNA (rDNA) V4 and 18S rDNA V9 41 .…”

“…Upward of 12 gene regions are commonly used to characterise microalgal communities, dependent on the taxonomic group of interest 41 , with a combination of gene regions commonly used or suggested to overcome bias of any single gene region/primer pair 21 , 42 , 43 . Some of the most used regions include the 18S ribosomal DNA (rDNA) V4 and 18S rDNA V9 41 .…”

Treasures of Italian Microbial Culture Collections: An Overview of Preserved Biological Resources, Offered Services and Know-How, and Management

“…For viruses and microalgae, molecular identification is mainly carried out on the genes of the coat protein and rbcL (the large subunit of the CO 2 -fixing enzyme RuBisCO) or 18S and 28S ribosomal genes, respectively [50,51].…”