Single-Strand Conformation Polymorphism Fingerprint Method for Dictyostelids.

Phongthana Pasookhush,Pravech Ajawatanawong,Sureemas Buates,Prasit Palittapongarnpim,Suradej Siripattanapipong,Kowit Suwannahong,Kamolchanok Rukseree,Kanchiyaphat Ariyachaokun,Asmatullah Usmani

doi:10.3389/fmicb.2021.708685

Abstract

Dictyostelid social amoebae are a highly diverse group of eukaryotic soil microbes that are valuable resources for biological research. Genetic diversity study of these organisms solely relies on molecular phylogenetics of the SSU rDNA gene, which is not ideal for large-scale genetic diversity study. Here, we designed a set of PCR–single-strand conformation polymorphism (SSCP) primers and optimized the SSCP fingerprint method for the screening of dictyostelids. The optimized SSCP condition required gel purification of the SSCP amplicons followed by electrophoresis using a 9% polyacrylamide gel under 4°C. We also tested the optimized SSCP procedure with 73 Thai isolates of dictyostelid that had the SSU rDNA gene sequences published. The SSCP fingerprint patterns were related to the genus-level taxonomy of dictyostelids, but the fingerprint dendrogram did not reflect the deep phylogeny. This method is rapid, cost-effective, and suitable for large-scale sample screening as compared with the phylogenetic analysis of the SSU rDNA gene sequences.

Highlights

Dictyostelid social amoebae are a group of non-pathogenic, unicellular eukaryotic microbes commonly found in diverse ecosystems throughout the world (Baldauf and Strassmann, 2017)
To optimize the single-strand conformation polymorphism (SSCP) fingerprint method with reproducibility, ease, and information, we focused on two critical parameters – the concentration of the polyacrylamide gel and the temperature for SSCP run – that had a direct effect on the quality of the SSCP fingerprint
The results showed that cold temperatures, 4◦C, could maximize the discrimination power of the SSCP fingerprint method better than higher temperatures (Figure 3B)

Summary

Introduction

Dictyostelid social amoebae are a group of non-pathogenic, unicellular eukaryotic microbes commonly found in diverse ecosystems throughout the world (Baldauf and Strassmann, 2017). They are important eukaryotic microbes in soils that control the bacterial population and play a role in nutrient recycling (Cavender and Raper, 1965). The amoeba can aggregate and together differentiate into a multicellular slug-like organism that eventually transforms into a multicellular fruiting body.

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Conclusion