## 4 Discussion
Among the samples used in this study, _cnorB_ and _nirK_ (64.0%, 16/25), _napA_ (60.0%, 15/25), _narG_ (56.0%, 14/25), and _amoA_ (52.0%, 13/25) were detected at particularly high rates, whereas other genes were detected at rates of 24.0-48.0%. South Korea is located at 33-38\({}^{\circ}\) N, 126-132\({}^{\circ}\) E, and the sampling sites for this study were Gapyeong and Yangpyeong in Gyeonggi-do and Cheongju in Chungcheongbuk-do, which are mainly in the central region and have cold climates. These regions have four distinct seasons; the main characteristics of the climate are as follows. From early March to early May, warm, dry spring weather appears under the influence of mobile anticyclones and Yangtze air masses. From May to mid-August, hot, humid summer weather appears under the influence of the North Pacific air mass. From late August to late October, the autumn weather is clear and dry under the influence of high pressure from mainland China and the Yangtze air mass. From November, the temperature and humidity gradually decrease under the influence of the Siberian air mass. From December to February, cold, dry winter weather appears under the influence of the Siberian air mass [30]. In this study, N cycle-related genes were identified at higher rates in autumn, whereas these genes had similar levels in the summer and rainy season and lower levels in spring and winter. [PERSON] et al. [31] reported a seasonal variation in the abundance of several N cycle-related genes in the forest soils from Bukhansen, South Korea. Among the six genes, _nifS_, _nirK_, and _norB_ showed seasonal changes, whereas _nifH_, _narG_, and _nosZ_ changed little over time; _nirS_ and _nirK_ were at low abundance in January and September but at high abundance in April and October, and _norB_ was rare in November, January, and December but abundant in April, September, October, and July. In this study, N cycle-related gene amplification was rare in spring and summer in the forest soils of the regions sampled in this study but was common in autumn, and several genes were identified in the rainy season and winter. The _nirS_ gene did not appear in all seasons, whereas _nirK_ and _cnorB_ were identified in autumn and the rainy season, _nifH_ and _nosZ_ appeared in autumn, and _narG_ appeared in autumn and winter. These patterns may change depending on environmental factors, such as the season, as well as other factors, such as tree composition. PCR methods were unavailable for the genes _amoC_, _hao_, _hzo_, _nirB_, and _nxrB_ at the time of this study; therefore, our findings should be supplemented in future research. PCR amplification of the total DNA extracted from environmental samples, such as soil, can offer visual confirmation of unknown, non-specific amplification, as seen in this study (Supplementary Figure S15). However, because the target band can be purified by gel electrophoresis, this issue is not expected to have a significant effect on subsequent studies, such as gene diversity analysis. Quantitative PCR (qPCR) can be used to rapidly monitor specific genes in environmental samples, such as soil; however, because qPCR mainly amplifies nucleotide sequences of less than approximately 200 bp, there is a limit to gene diversity analyses using amplification products. Among the non-selective PCR methods reviewed in this study, _amoA_ method #2 (110 bp), _napA_ method #1 (152 bp), _narG_ method #4 (173 bp), _nirK_ method #5 (165 bp), _nirS_ methods #4 and #5 (244 and 257 bp), and _nosZ_ method #6 (279 bp) may be suitable for rapid monitoring. Approximately 95% of N in its natural state is found in soil organic matter, which is a determinant of soil quality and an important component of the carbon cycle. The N concentration in soil organic matter largely determines the degree of mineralization and immobilization, as well as N-cycle processes, including N\({}_{2}\) fixation, nitrification, and denitrification. Thus, soil is an important medium for research on all of these processes [32; 33; 34].