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Gene. 2016 Feb 1;576(2 Pt 1):708-16. doi: 10.1016/j.gene.2015.10.033. Epub 2015 Oct 23.

Application of cryopreservation to genetic analyses of a photosynthetic picoeukaryote community.

Author information

1
Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan; JST, CREST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: kawach9i@nies.go.jp.
2
Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan; JST, CREST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan.
3
Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan.
4
JST, CREST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan; Tohoku National Fisheries Research Institute, 3-27-5 Shinhama-cho, Shiogama, Miyagi 985-0001, Japan.
5
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.

Abstract

Cryopreservation is useful for long-term maintenance of living strains in microbial culture collections. We applied this technique to environmental specimens from two monitoring sites at Sendai Bay, Japan and compared the microbial diversity of photosynthetic picoeukaryotes in samples before and after cryopreservation. Flow cytometry (FCM) showed no considerable differences between specimens. We used 2500 cells sorted with FCM for next-generation sequencing of 18S rRNA gene amplicons and after removing low-quality sequences obtained 10,088-37,454 reads. Cluster analysis and comparative correlation analysis of observed high-level operational taxonomic units indicated similarity between specimens before and after cryopreservation. The effects of cryopreservation on cells were assessed with representative culture strains, including fragile cryptophyte cells. We confirmed the usefulness of cryopreservation for genetic studies on environmental specimens, and found that small changes in FCM cytograms after cryopreservation may affect biodiversity estimation.

KEYWORDS:

18S rRNA gene; 454 pyrosequencing; Cryopreservation; Photosynthetic picoeukaryotes

PMID:
26506442
DOI:
10.1016/j.gene.2015.10.033
[Indexed for MEDLINE]

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