Molecular Soil Biology 2026, Vol.17, No.1, 12-25 http://bioscipublisher.com/index.php/msb 16 Figure 2 Conceptual comparison between the static chamber method and the eddy covariance method for measuring greenhouse gas fluxes in paddy fields. The static chamber method measures gas accumulation at the plot scale, whereas the eddy covariance method captures continuous ecosystem-scale gas exchange (Adopted from Butterbach-Bahl et al., 2016) 4 Microbial Community Structure and Functional Gene Analysis 4.1 High-throughput sequencing and community diversity analysis When studying microbial communities in paddy fields, amplicon sequencing has become a common tool, such as 16S rRNA for bacteria and archaea, and ITS for fungi. Through this method, changes in community composition under different irrigation conditions can be observed at a high throughput (Knight et al., 2018). During the analysis, the α diversity indicators, such as Shannon or Chao1, are usually examined first, to roughly reflect the species richness and evenness; if one wants to compare the differences in communities between different treatments, β diversity and ordination methods, such as PCoA or NMDS based on Bray-Curtis distance, are often used. However, the research focus has been changing in recent years. Often, it is not only about "what microorganisms are present", but also about what they might be doing. Therefore, metagenomic shotgun sequencing has gradually been used to analyze functional potential and metabolic pathways, especially when discussing carbon cycling, nitrogen cycling, and methane-related genes. Some studies have compared three irrigation patterns - continuous flooding (FI), alternate wetting and drying (AI, also known as AWD), and another intermittent irrigation (RI) - together. The results showed that WGS not only could reveal the differences in taxonomic composition but also simultaneously present changes in functional pathways (Zhang et al., 2021). The resulting results often provide a clearer functional background for subsequent network analysis or the identification of key functional groups. 4.2 Determination of functional gene abundance and expression levels When studying the methane cycle in paddy fields, mcrA and pmoA are often regarded as two key functional gene indicators. The former corresponds to the key enzyme in the final step of methane production, while the latter is related to the components of methane-oxidizing enzymes. Therefore, many studies use the abundance or expression levels of these genes to roughly assess the potential activity of methane production and methane oxidation (Yang et al., 2019). Some reviews suggest that the ratio of mcrA/pmoA under certain conditions can indicate whether methane is more likely to be "produced" or "consumed". However, this explanation is not reliable in all cases. For example, the sampling location (whether in the surface soil or rhizosphere), the soil moisture condition, and the availability of substrates can all affect the results. Therefore, the more common
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