FBIP: Survey of invertebrate and microbial diversity of the Prince Edward Islands system

Study Extent

Survey and metabarcoding of microorganism in Prince Edward and Marion Islands

Sampling

- Soil/root samples were collected from various plants occuring on Marion Island, these samples were stored at -20oC and sent to the laboratory for analysis. Despite being frozen isolations were performed. - Surface seawater (taken from a depth of 5 m) and for the illumina samples (depth was approx 5m) was filtered through 100 µm mesh, followed by polyethersulfone membrane filters (0.22 µm) to collect bacteria. Filtered seawater and the filters were stored separately at -20ºC.

Quality Control

No

Method steps

1. Standard mycological culturing methods, used standard sequencing methods: - 16S rRNA genes using bacterial targeting and archaea targeting primers and 18S rRNA genes using universal eukaryote primers were amplified via polymerase chain reactions using genomic DNA extracted from biomass which was collected on the polyethersulfone membrane filters. Polymerase chain reaction products were sized on an agarose gel and extracted. The cleaned up extractions were then sequenced via pyrosequencing and Illumina sequencing. Sequence reads were curated using Mothur. - Extraction of genomic DNA from soil samples, followed by polymerase chain reaction (PCR) amplification of 16S rRNA and 18S rRNA gene for selected samples. PCR products were cut out from agarose gels to ensure the correct PCR product was obtain, each correct product was extracted from the gel and then purified, followed by sequencing on the Illumina platform. Raw sequence reads were curated using Mothur and classified according to the Silva v132 database. - Extraction of genomic DNA from root samples, followed by polymerase chain reaction (PCR) amplification of the ITS1 region. PCR products were cut out from agarose gels to ensure the correct PCR product was obtain, each correct product was extracted from the gel and then purified, followed by sequencing on the Illumina platform.
   - DNA was extracted from the isolated fungal endophytes, the ITS region was PCR amplified and amplicons were sent for sanger sequencing. When sequences were determined individual isolates were identified using BLAST on the NCBI website

microbial diversity

1. Bacteria
   rank: kingdom
2. Archaea
   rank: kingdom
3. Eukaryota
   rank: kingdom
4. Fungi
   rank: kingdom

Prince Edward and Marion Islands