Optimization of PCR Primer Systems to qualify and quantify Nitrogen Cycling Microbes in Coral Reef Octocorals. Nitrogen cycling microbiome in octocoral holobionts

Nitrogen cycling microbes are ubiquitously associated with hexacorals (usually reef-building) and play a critical role in functioning the coral holobiont. Observed phase shifts toward to octocoral-dominant state in global coral reefs shows the potentially ecological significance of octocorals in future marine ecosystem. However, molecular evidence for nitrogen cycling microbes in octocorals remain poorly proposed. Thus here, we evaluated the gene specific primers previously used in hexacorals for our octocorals, and subsequently optimized the standard and real-time PCR systems to qualify and quantify nitrogen cycling genes in two aquarium strains, the octocoral Xenia umbellata and Pinnigorgia flava. In total 11 forward and 12 reverse primers with different binding combinations were screened in the standard PCR. Only one primer pair worked successfully for nitrogen fixation nifH amplification (IGK3 and YAA) and one for denitrifying nirS amplification (1F and qR). After gene absolute quantifications by real-time PCR, we found 9.9 to 98.8 nifH gene copies ng-1 of DNA, and 3.1 to 30.9 nirS gene copies ng-1 of DNA in two octocoral specimens. Yet, bacterial and archaeal amoA genes in both octocoral holobionts were below the limits of detection of real-time PCR, i.e., 0.8 and 6.2 gene copies ng-1 of DNA separately. In summary, our work has provided a comprehensive and comparative overview of primers used to detect nitrogen cycling microbes in the octocoral holobiont. Our results suggested that nitrogen cycling microbes might be also commonly associated with coral reef octocorals.