Single-cell genotyping of phytoplankton from ocean water by gel-based cell manipulation.

ABSTRACT

A comprehensive understanding of phytoplankton diversity is valuable for assessing an environment of interest as phytoplankton are primary producers in various aquatic food webs. Microscopic analyses are useful for diversity assessment based on characteristic cell morphologies. However, phylogenetic classification based solely on morphology requires an extremely high level of expertise. The genetic approach is another option for evaluating phytoplankton diversity; however, it cannot reveal morphological information. To integrate these two approaches, an original technology was developed, that is referred to as microcavity array (MCA)/gel-based cell manipulation (GCM). The model experiments using monocultures of various phytoplankton indicated that the efficiencies of cell recovery and isolation of single-cell plankton were dependent on cell size and shape. Cells with widths larger than the cavity width showed high level of recovery and isolation efficiency. Subsequent whole-genome amplification (WGA) of isolated single-cell plankton provided a sufficient amount (≈30 µg) of WGA products for genetic analyses. Furthermore, it is showed that MCA/GCM could directly analyze phytoplankton in ocean water obtained from Suruga Bay, Japan, without any cumbersome pretreatment. These results indicate that MCA/GCM technology is a powerful tool for elucidating the phytoplankton diversity in marine environment.