Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae

Frontiers in Microbiology
Osama S SaadSenjie Lin

Abstract

Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8-99.3%; R2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicabilit...Continue Reading

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Datasets Mentioned

BETA
PRJNA599937

Methods Mentioned

BETA
environmental stress
electrophoresis
Assay
PCR
light microscopy
Illumina sequencing

Software Mentioned

cMix
gMix
GraphPad Prism
MEGA7
BLAST
R
QIIME
Blast search
UPARSE
ClustalW

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