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GNPS - 2022WoodFrogFeeding_UnderstandingHerptileMicrobiomes_FecalPelletExtractions

ABSTRACT: Methanol extractions of fecal pellets collected from laboratory-reared wood frogs that were selectively inoculated with the herptile gut fungus, Basidiobolus.

INSTRUMENT(S): 6545 Q-TOF LC/MS

ORGANISM(S): Lithobates Sylvaticus

SUBMITTER: Kerry McPhail

PROVIDER: MSV000095549 | GNPS | Wed Aug 07 17:11:00 BST 2024

REPOSITORIES: GNPS

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Project description:Using whole genome microarrays based on the annotated genomes of Postia placenta, we monitored the changes in its transcriptomes relevant to cell wall degradation during growth on three chemically distinct Populus trichocarpa (poplar) wood substrates. The research goal is to identtify genes essential for cellulose depolymerization. From a data set of 12438 unique gene models, each NimbleGen (Madison, WI) array targeted 9959 genes and featured 10 unique 60mers per gene, all in triplicate. RNA and protein were obtained from P. placenta strain MAD-698-R (USDA Forest Mycology Center, Forest Products Laboratory, Madison WI) grown in malt extract agar for 10 days prior to inoculation with wood wafers. Three poplar wood substrates with distinct cell wall chemical properties were selected from several hundred 4-year old poplar (Populus trichocarpa) trees grown in a common garden field trial at the University of British Columbia (Canada). We selected three poplar genotypes based on cell wall chemical traits. Substrate A corresponds to a genotype with a higher than average lignin content and lower that average glucose content; Substrate B, a lower than average lignin content and higher that average glucose content; Substrate C lignin and glucose contents are near the population average. Poplar wood stems were cut into 0.5 mm wafers on a microtome, sterilized for 20 min at 121°C, dried at 50°C overnight, and cooled to room temperature. The specimens were then inoculated in Petri dishes with actively growing mycelia. Approximately 5 g of wood wafers were placed in each Petri dish (exact weights were recorded), sealed and incubated at 22°C and 70 ± 5% relative humidity for 10, 20 or 30 days. For RNA, the degraded wafers were removed from the Petri dishes, immediately snap-frozen in liquid nitrogen and stored at -80°C for later use. Total RNA was converted to Cy3 labelled cDNA, hybridized to microarrays and scanned as previously described by Vanden Wymelenberg et al 2010 (Appl Enviro Microbiol 76:3599-3610).The 24 arrays per fungal species were scanned and data extracted using NimbleScan v.2.4. The raw data was loaded into GeneSpring, where the intensities were converted to log2 and quantile normalized, and all probes per gene averaged. This data was then exported and further analyzed in R. For substrates “A” and “B”, three replicates were used for each wood substrate/fungus and incubation period combination. For substrate “C” only 2 biological replicates were employed.

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GNPS - 2022WoodFrogFeeding_UnderstandingHerptileMicrobiomes_FecalPelletExtractions

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