Identifying potential biomineralization genes in the coccolithophorid Gephyrocapsa oceanica

The Prymnesiophyceae class includes the order Isochrysidales, which consists of coccolith forming sister-species Gephyrocapsa oceanica, Emiliania huxleyi, and the noncoccolith forming species Isochrysis galbana. These coccolithophores form their coccoliths through the process of biomineralization. The molecular mechanisms and genes involved in the process remains unclear. The aim of this study was to identify potentialgenes involved in biomineralization in the species E. huxleyi and G. oceanica and compare them to the non-coccolith forming species I. galbana by transcript profiling using a highthroughput RNA-sequencing (RNA-seq) method. The transcriptome profiles were generated for cells grown in calcium replete conditions and calcium deplete conditions,with or without the addition of sodium bicarbonate. Physiological parameters including cell growth, rate of calcium uptake and photosynthesis further supported the evidence thatphotosynthesis and calcification are independent processes. The effects of calcium replete conditions and calcium deplete conditions on calcification and coccolith morphology weremonitored via SEM. The data further supported the finding that cells grown in calcium deplete conditions have fragments of coccoliths while cells in calcium replete conditionshave well-formed and intact coccoliths. The RNA-seq identified 295 differently expressed genes using negative binomial distribution to compare transcript levels for all three speciesgrown under calcifying 9 mM Ca2+ and 0 mM Ca2+ conditions. Real time RT-PCR was used to independently validate the differential expression of 21 potential calcifying genes.Real time RT-PCR validated a total of 17 out of 21 differentially expressed genes, for which 6 were considered significantly up-regulated and 11 were considered significantlydown- regulated. G. oceanica real time RT-PCR analyses independently validated 12 out of 21 genes (57%), while E. huxleyi analyses validated 13 out of 21 genes (61%) of thedifferentially expressed genes when compared to the RNA-seq data. Keywords: Gephyrocapsa oceanica, Emiliania huxleyi, Isochrysis galbana, Isochrysidales, biomineralization, coccolith, coccolithophorid, scanning electron microscopy, high-throughput RNA sequencing, transcriptome profiling, real-time RT-PCR.