Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

This study visually matched the spatial and temporal distribution of key methane-cycling microbial genes in South Texas coastal wetlands, it explores how environmental factors regulate methane dynamics. Our findings reveal that the abundance of methane-cycling genes in South Texas coastal wetlands is sensitive to environmental conditions, including salinity and season. In particular mmoX, a type of methane monooxygenase gene, being most abundant in cooler seasons.

This protocol addresses the research in detecting methane-cycling microbial genes by offering novel tools to visualize spatial and seasonal microbial dynamics influenced by environmental factors. By integrating optimized PCR methods with GIS visualization, this protocol provides enhanced detection and spatial mapping of methane-cycling genes while capturing dynamic, spatial, and seasonal microbial patterns influenced by environmental factors.

[Narrator] To begin, prepare a 25-microliter cPCR reaction mixture with the eDNA and other components. Place the tubes in the PCR machine and run the cPCR reaction with appropriate parameters. After cPCR, prepare the qPCR reaction mixture containing cyber green, excluding the template DNA. Dispense 57 microliters of the prepared reaction mixture into each PCR tube. Add 3 microliters of template DNA, standard or nuclease-free water to each tube and tap the tube gently to mix. Aliquot 20 microliters of the prepared reaction mixture from each tube into designated wells of a 96-well qPCR plate. Seal the PCR plate with an adhesive PCR sealing film using an applicator. And centrifuge it at 1,000 x g for one minute to ensure proper mixing, and eliminate any bubbles within the wells. Place the PCR plate in the thermal cycler. Turn on the qPCR machine and open the related software to set up the protocol. Set up the qPCR program and initiate the run. Open the geographic information system software, ArcGIS Pro, and save the project file with the name Study Area in the specified folder on the computer. Click on Map, then select Basemap, and choose terrain with labels as the base map. Click on Locate, then click Search. When the search bar opens, type the name of the study area to locate it on the map. To draw the specific area using geo-referencing, click on View, then Catalog Pane from the top layer. Double-click on the folder from the catalog and select the required file name. Right-click on the geo-database .gdb file. Click New and select Feature Class. Then the Create Feature Class window will appear. Type the Name and Alias box and click Finish at the bottom. Now, click on View, followed by Contents. The alias name will show up in the Contents pane. Click on Edit from the top layer, followed by Create. The Create Features pane will open. Double-click on the alias in the Create Features pane and the Configure Tool Feedback options will appear. Select lines, then sketch lines on the map to create the study area boundary. Double-click the map to finish. Minimize the GIS software and open a spreadsheet. Type the sample name in the first column, latitude in the second column, and longitude in the third column. Use the next four columns for qPCR data of pmoA1, pmoA2, mmoX, and mcrA. Save the file in CSV format in a specific folder on the computer. Next, open the GIS software and click Add Data, followed by XY Point Data. Select the saved CSV file from the folder in the Input Table box. Rename the file in the Output Feature Class box, and click Run to display the sampling points on the map. Now, click on the search bar at the top and search Kriging. Select the sampling station file and choose pmoA1. Then click the environment, select the sampling station in layer and mask, and click Run. To create a layout of the map, click on Insert, New Layout, and select ANSI-Landscape. Click on Map Frame, select the map with kriging, and place all the maps in the layout by drawing rectangles. Select North Arrow and place it in the layout to indicate the north direction. Then select Scale Bar to display the scale of the area on the map. Click on Legend, then place it in the layout to display map legends. Click on Grid, then select any black graticule option to create a grid with latitude and longitude. The grid will appear in the Contents pane with the label Black Horizontal Label Graticule. Double-click on Black Horizontal Label Graticule, select Components, and remove Ticks 1 and Grid by clicking on the cross sign to their right. Finally, click on Share from the top layer, then click Export Layout. Select the file type as PDF, save the file on the computer using the Name box, set the vertical resolution to 500 DPI, and click Export to create the PDF file of the map. The cPCR analysis revealed spatial variability in methane cycling-related gene detection, with mcrA genes detected in all LB and RV samples, but absent in BC samples, linking salinity to methanogen distribution. The cPCR showed brighter bands for pmoA1 with A189-mb661 primer pairs, confirming low but detectable abundance in RV samples, while it was absent in LB and BC. The qPCR analysis showed high seasonal variation, with mmoX genes most abundant during the cool season and pmoA1 most abundant during the warm season.