## 4 Discussion  
In this study, we selected suitable PCR methods for the study of N cycle-related genes in soil microorganisms. Among 18 detailed genes related to the N cycle, we identified several PCR methods with excellent applicability for surface soil and gene diversity studies in all but five genes (_amoC_, _hao_, _hao_, _nirB_, and _nxxB_). To the extent possible, we selected those PCR methods that amplified a base sequence of 400-700 nt (\(\geq\)300 nt) to take full advantage of the abilities of a conventional PCR (e.g., follow-up research, such as microbiome gene diversity testing) [15; 24; 25]. However, we were only able to select the PCR methods that produced amplicons of \(<\)300 bp in length for four genes: _cnorB_ (177 bp), _hzs_ (200-300 bp), _nosZ_ (267 bp), and _nrfA_ (236 bp). The PCR methods selected in this study are expected to be useful for the study of N cycle-related microbial communities in various soil types. We also analyzed the microorganism genes related to the N cycle among surface soil land-use types and five seasons (four conventional seasons and the rainy season). However, we were unable to identify suitable PCR methods for five genes: _amoC_, _hao_, _hzo_, _nirB_, and _nxxB_. Among 65 reactions (13 detailed N cycle-related genes in five replicate samples per point), we analyzed 16.9% of the field samples (11/65), 23.1% of the forest samples (15/65), 26.2% of the bare land samples (17/65), and 80.8% of the grassland samples (average, 52.5/65). Among the 25 reactions attempted for each detailed gene, we observed positive reactions for _amoA_ (13), _amoB_ (8), _cnorB_ (16), _qnorB_ (8), _hzs_ (6), _nupA_ (15), _narpG_ (14), _nirR_ (10), _nirK_ (16), _nirS_ (9), _nosZ_ (11), _nrfA_ (12), and _nxxA_ (10). Among the 65 responses according to season, reactions were observed in 32.3% of the spring samples (21/65), 50.8% of the summer samples (33/65), 49.2% of the rainy season samples (32/65  
Among the field type samples, the _amoA_, _cnorB_, _hzs_, _napA_, _nifH_, _nirK_, _nosZ_, and _nxrA_ genes were amplified and mainly appeared during summer and the rainy season. Among the forest samples, ten genes (_amoA_, _cnorB_, _hzs_, _napA_, _narG_, _nifH_, _nirK_, _nosZ_, _nrfA_, and _nxrA_) were amplified: _cnorB_, _nirK_, and _nnxrA_ mainly in the rainy season and autumn samples, _amoA_, _hzs_, _nifH_, _nosZ_, and _nrfA_ only in the autumn samples, and _napA_ and _narG_ in the autumn and winter samples. The bare land samples showed similar results to the forest samples, except for the amplification of _qnorB_ and _nirS_ in the autumn samples, perhaps because the bare land soils were sampled at a short distance (1.6 km) from the forest sampling location. Among the four land use types examined in this study, a high percentage of N cycle-related genes were analyzed in the grassland samples, and 13 N cycle-related detailed genes were amplified (Table 3 and Supplementary Figure S15). [PERSON] et al. [26] reported that the abundance of N-cycle genes and the potential greenhouse gas fluxes depend mainly on the land use type and less on the soil aggregate size. In this study, the amplified genes were found to differ according to the soil land use type. Notably, the grassland microorganism genes related to the N cycle appeared in most samples regardless of the season, unlike those in the samples from the other three land use types. This phenomenon may be explained by the fact that grasslands have the highest level of biomass on Earth, and natural grasslands have the N balance of a managed grassland system, with carbon:N:organic and sulfur:organic phosphorus ratios maintained at nearly constant values [27; 28; 29]. Because grassland is an important land use type for the soil's N cycle, it may be necessary to examine samples from more diverse land use types and more seasonal periods in future research. The 13 detailed genes used in this study showed 6-16 reactions per 25 samples (average, 11.4).