# Comparison of Microbial Gene Diversity in Grassland Topsoil Depending on Soil Quality
## 4 Discussion  
Numerous studies on soil quality based on chemical measurements have been reported, but research on biological quality is limited; therefore, it is necessary to obtain basic data. In this study, we analyzed eukaryotic and prokaryotic genetic diversity related to the quality of grassland toposoil and suggested several biomarkers of soil quality. As the topsoil is the main network space where biological activities occur in the soil [1; 2], it was selected as the material for this study. There is a possibility that the quality of grassland toposoil is relatively diverse compared to agricultural toposoil, such as fields, where the chemical-based soil quality diversity is at a medium-high level, and bare land, where the soil quality is low. In this study, grassland was assumed to be a suitable land use type. However, as this study involved analyses of data from six sites, it might be necessary \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Soil Quality**} & \multirow{2}{*}{**Rank**} & \multirow{2}{*}{**Division**} & \multicolumn{2}{c}{**Identification (Genus**} & \multicolumn{2}{c}{**Rate (\%)**} \\ \cline{3-6}  & & & **or Species-Level)** & **High** & **Low** & **High-Low** \\ \hline \multirow{8}{*}{Low} & 20 & Eu & _Archaenorhabditis_ & - & 0.33 & \(-\)0.33 \\  & 19 & Eu & _Rhynchobodo_ & - & 0.35 & \(-\)0.35 \\  & 18 & Eu & _Triplonchida_ & - & 0.40 & \(-\)0.40 \\  & 17 & Pro & _Bryobacter_ & 0.85 & 1.28 & \(-\)0.43 \\  & 16 & Eu & _Spunella_ & 0.40 & 0.85 & \(-\)0.45 \\  & 15 & Pro & _Rhodanobacter_ & 0.95 & 1.44 & \(-\)0.49 \\  & 14 & Pro & _Rhizobacter_ & 0.60 & 1.14 & \(-\)0.54 \\  & 12 & Pro & _Plantatinospora_ & 0.35 & 0.90 & \(-\)0.55 \\  & 13 & Pro & _Polaromonas_ & 0.10 & 0.65 & \(-\)0.55 \\  & 11 & Pro & _Bradyrizobium_ & 0.75 & 1.34 & \(-\)0.59 \\  & 10 & Eu & _Pyrenochaeta_ & 0.15 & 0.79 & \(-\)0.64 \\  & 9 & Pro & _Crenobacter_ & 0.05 & 0.78 & \(-\)0.73 \\  & 8 & Pro & _Streroobacter_ & 0.05 & 0.84 & \(-\)0.79 \\  & 7 & Eu & _Vigna_ & - & 0.86 & \(-\)0.86 \\  & 6 & Pro & _Pseudarthrobacter_ & 0.55 & 1.44 & \(-\)0.89 \\  & 5 & Pro & _Pseudomonas_ & 0.05 & 1.27 & \(-\)1.22 \\  & & & _Braskoladeria-_ & & & \\  & & & _Caballeronia-_ & 1.05 & 2.70 & \(-\)1.65 \\  & & & _Parukholderia_ & & & \\  & & & _Candidatus_ & & & \\  & 4 & Pro & & Udaeobacter & & \\  & 2 & Eu & _Acari_ & - & 2.21 & \(-\)2.21 \\  & 1 & Eu & _Artemisia_ & - & 4.43 & \(-\)4.43 \\ \hline \hline \end{tabular} Gray highlight, prokaryote.  
\end{table} 
Table 2: _Cont._to analyze the diversity of biomarker candidates identified in this study in additional grassland topsoil samples in the future. In addition, there is a need to monitor biomarkers according to various land uses, such as agriculture and forestry, and to evaluate biodiversity in contaminated soils. In this study, eukaryotic 18S and prokaryotic 16S rDNA partial regions were used as eukaryotic and prokaryotic amplicons provided to NGS; a diversity analysis tool mainly used for biomarker discovery. It was difficult to analyze the benefits of eukaryotes for plants; eukaryotes are expected to contribute to the circulation of materials or nutrients in high-quality soils or act as plant pathogens in low-quality soil. For eukaryotic gene identification, in this study, we used the 18S universal primer mix, which is commonly used. As the identification of eukaryotes is more complicated than that of prokaryotes, it could be difficult to identify short genetic fragments. In addition, the eukaryotes that showed differences according to soil quality might be suitable for monitoring the genetic diversity of eukaryotes in grassland topsoil, but they may not be ideal for the identification of genetic fragments compared to fungi. As the proportion of fungi could decrease with the inclusion of these genes, the discovered soil quality markers will only target fungi, requiring a replacement with the ITS region. As _Penicillium_ can be commonly found in both soil and air [57], its potential as a marker would require identification at the species level.