Genomic analysis of red-tide water bloomed with Heterosigma akashiwo in Geoje

Abstract

Microbial community structures of harmful algal bloom (HAB) caused by Heterosigma akashiwo in Geoje were analyzed using the MiSeq platform. To analyze phytoplankton communities without cross-reactivity with predominant bacteria, a new phytoplankton-specific 23S universal primer set was designed by modifying two previously used ones. The new universal primer set turned out to be a useful tool for the analysis of the phytoplankton community; it showed a high specificity for phytoplankton without cross-reactivity to bacterial sequences as well as the wide taxon coverage presenting from prokaryotic cyanobacteria to eukaryotic algae. Next Generation Sequencing (NGS) data generated by two universal primer sets (16S and 23S) provided useful information about the H. akashiwo bloom. According to the 23S universal primer set, proportions of H. akashiwo increased by more than 200-fold as the bloom occurred and its numbers were high enough to detect in control sites. Its operational taxonomic units (OTUs) were detected in the bloom sites at low proportions suggesting that the 16S universal primer set may not be as effective for monitoring harmful algal blooming (HAB) as the 23S universal primer set. In addition, several abundant OTUs in Chlorophyta were not presented by the 16S universal primer set in this study. However, the 16S primer set was useful for detecting decreases in Foraminifera as HAB occurred suggesting that genomic analyses using two universal primer sets would provide more reliable data for understanding microbial community changes by various environmental or ecological events, including HAB. Genomic analyses using two universal primer sets was also useful for determining a correlation between microbial components as HAB occurred. Heterosigma akashiwo was positively correlated with other bloom species, including Karenia mikimotoi, Teleaulax amphioxeia, and bacteria in Verrucomicrobia.

Diversity, community structure, and quantity of eukaryotic phytoplankton revealed using 18S rRNA and plastid 16S rRNA genes and pigment markers: a case study of the Pearl River Estuary

“…The qPCR assays were performed as previously described (Gong et al 2013 ), with some modifications. Since the primers for plastid 16S rRNA genes also target heterotrophic bacteria, another set of primers was selected that specifically targeted the plastid 23S rRNA gene of eukaryotic algae (Kang et al 2018 ). These two plastid genes are thought to have identical copy numbers in a chloroplastid genome.…”

“…The PCR reaction mixture (20 µL) contained 10 µL Master, 1 µL each primer, 2 µL DNA template and 6 µL double-distilled water. The primer sets 345F (5′-AAGGAAGGCAGCAGGCG-3′) and 499R (5′-CACCAGACTTGCCCTCYAAT-3′) (Zhu et al 2005 ), and P23MISQF1 (5′-GGACARWAAGACCCTATGMAG-3′) and P23MISQR1 (5′-AGATYAGCCTGTTATCCCT-3′) (Kang et al 2018 ) were used for amplifying 18S and 23S rRNA gene abundances of phytoplankton, respectively. The qPCR assay was based on the fluorescence intensity of the SYBR Green dye, and reactions for each sample were carried out in a Roche LightCycler 96 System (Roche Diagnostics, Mannheim, Germany).…”

Environmental DNA Metabarcoding Analysis of Fish Assemblages and Phytoplankton Communities in a Furrowed Seabed Area Caused by Aggregate Mining

“…Environmental DNA (eDNA) was extracted with genomic DNA using DNeasy R Plant Mini Kit (QIAGEN, Hilden, Germany), in accordance with a slightly modified version of the manufacturer's protocol (Yoon et al, 2016;Kang et al, 2018). The membrane was initially put into lysis buffer containing 1/4" ceramic spheres (MP Biomedicals, Santa Ana, United States) and homogenized by FastPrep-2 (MP Biomedicals).…”

“…The libraries for metabarcoding analysis of fish and phytoplankton taxa were constructed for the Illumina MiSeq system, including negative controls for eDNA extraction and PCR reaction mixture. MiFish and plastid 23S (p23S) rRNA universal primer sets were used for analyzing the fish assemblages and phytoplankton community structure, respectively (Miya et al, 2015;Kang et al, 2018). Primary PCR was conducted with each corresponding universal primer set overhanging the adapter sequences (Table 1).…”

“…Primary PCR was conducted with each corresponding universal primer set overhanging the adapter sequences (Table 1). PCR conditions for library preparation of MiFish and p23S followed those used in previous studies (Miya et al, 2015;Kang et al, 2018). The quantity and quality of the amplicon libraries were measured using a Quantus Fluorometer (Promega, Madison, United States) and BioAnalyzer 2100 (Agilent Technologies, Santa Clara, United States).…”