Development of a qPCR assay for tracking the ecological niches of genetic sub-populations within Pseudo-nitzschia pungens (Bacillariophyceae)

Abstract

Three genetic sub-populations (Glade I, II and III) of Pseudo-nitzschia pungens, the potential toxic marine diatom, are known to have distinguishable growth characteristics under different culture conditions and distinct distributed patterns in the world. However, to date their exact eco-physiological traits are unrevealed in fields due to lack of the method to detect and/or measure abundances of each sub populations, hence, the qPCR (quantitative real-time polymerase chain reaction) assay was developed to detect and quantify the P. pungens cells of each Glade. Designed two specific primer sets, Pclal2F/R (for Glade I and II) and Pcla3F/R (for Glade III) only could amplify each target genomic DNA. The, significant linear relationships (R-2>0.998) was established between C-t (threshold cycle) value and the log of cell abundance for each Glade. Through the melting curve analysis, comparisons for gene copy numbers among the three clades and spike test for field study, our qPCR assay was reliable to quantify the cell numbers of each Glade. There was strong linear correlation (R-2>0.990) between cell abundances as estimated by qPCR assay and direct counting via light microscope in spike test, and 0.24 (clade I), 0.25 (Glade II) and 033 (Glade III) P. pungens cells per mL were detected markedly upon the use of specific two primer set. Finally, developed qPCR assay was applied on field samples successfully. Our study implicate that our qPCR assay is an accurate and sensitive technique to estimate the cell abundances of each Glade of P. pungens in field works. (C) 2017 Elsevier B.V. All rights reserved.