Quantitative PCR of functional genes from microbial communities throughout Mitchell Peninsula, Browning Peninsula and Robinson Ridge in the Windmill Islands

Full description

In 2005, 9 soil samples were obtained from 3 separate sites in the Windmill Islands; Mitchell Peninsula (66º31´S, 110º59´E), Browning Peninsula (66°27’S, 110°32’E) and Robinson Ridge (66°22′S, 110°35′E). At each site, samples were collected from 3 distance points (0m, 100m and 200m) along 3 parallel, longitudinal transects. Soils were obtained from the top 10cm. Soils were stored at -80°C until use.

Community gDNA was extracted from 0.25-0.3g of each soil sample using the FastDNA SPIN kit for soil (MP Biomedicals, NSW, Australia). QPCR was performed upon each gDNA extract to quantify the copy numbers of rbcL1E, hhyL and 16S rRNA per gram of soil. Previously published qPCR primers targeting rbcL1E (rbcL1Ef/rbcL1Er; Ji et al., 2017), hhyL (NiFe-244f/NiFe-568r; Constant et al., 2010, 2011), and the 16S rRNA gene (Eub1048f/Eub1194r; Maeda et al., 2003; Table 1) were used. The RuBisCO type IE qPCR primer set (rbcL1Ef/rbcL1Er) was validated for use in our specific soils by amplicon sequencing DNA lysates from the three different sites.

Positive controls for QPCR were synthetically designed gene fragments (gBlocks; Integrated DNA Technologies, VIC, Australia) composed of representative rbcL1E (JX458468.1), hhyL (AB894417.1), and 16S rRNA (MF689012.1) gene sequences. Standard curves were generated over 5–7 orders of magnitude.
QPCR reaction mixtures for rbcL1E and the hhyL target genes were prepared using 10 μl QuantiFast SYBR Green PCR Master Mix (Qiagen, VIC, Australia), 0.5 μl of each 40 μM primer (Integrated DNA Technologies), 8 μl UltraPure DNase/ RNase-free distilled water (Invitrogen), and 1 μl diluted (1:10) gDNA. The reaction mix for the 16S rRNA gene was identical, except that 7 μl molecular water and 1 μl of 5 ng/μl T4gene32 Protein (Sigma-Aldrich, NSW, Australia) were added to reduce non-specific amplification (Baugh et al., 2001; Villalva et al., 2001).

Thermocycling reactions was completed using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, NSW, Australia) under standard two-step conditions; 94°C for 5 min, 45 cycles of 94°C for 20 s, and 54°C for 50 s, followed by a melt-curve step from 50 to 95°C. The quantitative fluorescence data were spectrophotometrically collected during the 54°C step.

CFX manager software (Bio-Rad Laboratories) was used for data analysis. Melt peak analysis confirmed amplification specificity. Amplification within the most dilute standard was detected at least 5 Ct before the negative template controls. The average Ct values across replicates were determined, and then standard curve efficiencies and copy numbers were converted into copies/g of soil. The R2 value for each qPCR was equal to or greater than 0.99. The genetic copy
numbers of rbcL1E quantified were corrected against the proportion of rbcL1E target reads observed during site-specific primer validation.

Sample Collection:
During the 2005-2008 Windmill Island expeditions

Data Collection:
QPCR was conducted in 2017 and 2018.

Sample collection:
Soils were collected from the top 10cm of soil during an expedition occurring in 2005. Samples were collected from 3 distance points along 3 parallel longitudinal transects, resulting in 9 samples obtained from each site. These sampling sites were Mitchell Peninsula (66º31´S, 110º59´E), Browning Peninsula (66°27’S, 110°32’E) and Robinson Ridge (66°22′S, 110°35′E). The transect information for each soil studied is included in the dataframe:
- Barcode- Barcode of sample assigned by AAD upon sampling
- Location- Indicates the site that the soil was sampled from
- Transect_Information- Indicates the transect number and distance along the transect from which the soil was obtained.
- Gene Quantified- indicates which gene was targeted during the QPCR assay
- Copies/g soil- indicates the final calculated copy number detected within the soil, based upon the QPCR results obtained.

Further information about the methods used to produce this data can be obtained in the following publication:
Ray, A. E., Zhang, E., Terauds, A., Ji, M., Kong, W., & Ferrari, B. C. (2020). Soil Microbiomes With the Genetic Capacity for Atmospheric Chemosynthesis Are Widespread Across the Poles and Are Associated With Moisture, Carbon, and Nitrogen Limitation. 11(1936). doi:10.3389/fmicb.2020.01936

Please cite the above article when using this data.

Lineage

Progress Code: completed

Statement: There were no issues with data collection or analysis.