Diversity Of Archaea In Mila Mountain Meadow Soil Of Tibet Estimated By 16S RRNA And AmoA Genes

Archaea could not only survive under extreme conditions, but also exist in normal environments. With its huge abundance, Archaea is playing a highly important role in the biogeochemistry processes on the Earth. Nitrification in soil plays an important role in the global nitrogen cycle. Although bacteria were thought to be solely responsible for catalyzing the rate-limiting step of this process, several recent studies have suggested that Crenarchaeota are capable of performing ammonia oxidation. This area is a sensitive key region to the global warming. Alpine meadow is the key plant community type, which covered most area of Tibetan Plateau. Here we examine the diversity of archaea and ammonia-oxidizing archaea (AOA) within the soil of MP (at the entrance of Mila Mountain where the grass cover below 50%) and GY (at the Gongbo'gyamda County where the grass cover above 50%) of Mila Mountain. The four clone libraries were named ARC-MP, ARC-GY, AOA-MP and AOA-GY respectively. DNA was extracted from 10 replicate subsamples from each of the two soil samples. For each library, three replicate PCRs were conducted per soil DNA template (for a total of 30 replicate PCRs per library) using group-specific primers. To verify whether the method has the properties of good repeatability and high reliability or not, the archaea 16S rRNA and amoA gene libraries were generated for the soil of MP again.Phylogenetic analysis revealed that archaea in the three archaeal 16S rRNA clone libraries of Mila Mountain including the Crenarchaeota and unclassified_Archaea phyla, not found the Euryarchaeota. All the Crenarchaeota belong to the Thermoprotei. Mila Mountain belonged to the kingdom Crenarchaeota. The archaea and AOA have high similar with the sequences of protected soil.The sequences of archaea and AOA were divided into operational taxonomic units (OTUs) according to the 97% similarity threshold for OTU assignment was performed using the software program DOTUR. Archaea species composition from the three archaeal 16S rRNA gene clone libraries of ARC-MP, ARC-MPr and ARC-GY included 54, 51 and 51 OTUs respectively. AOA species composition from the three archaeal amoA gene clone libraries of ARC-MP, ARC-MPr and ARC-GY included 51, 44 and 23 OTUs respectively. The MP has roughly the same abundance of archaea as the GY, but the AOA abundance of MP are more than those in the GY.In order to compare the microbial populations present in the soil samples from two sites with statistic test, P values obtained by∫-Libshuff and TreeClimber. The P value between ARC-MP and ARC-GY clone libraries are 0.0000 (P <0.05), there have significant differences between the two libraries. The P value between AOA-MP and AOA-GY clone libraries are 0.0000 (P <0.05), there is significant differences between the two libraries. It means that the microbial populations of MP and GY are significant differences. It shows that the composition of the community of the AOA of MP and GY that related to the nitrogen cycle are significant change. The P value between ARC-MP and ARC-MPr clone libraries are above 0.05 (P >0.05), The P value between ARC-MP and ARC-MPr clone libraries are above 0.05 (P >0.05). It means that the microbial populations of the soil from same site have no significant difference. So conclude that the method used contruct libraries has the properties of good repeatability and high reliability.To compare the memberships and structures of two communities at a particular operational taxonomic units (OTUs) definition and implement nonparametric estimators for the fraction and richness of OTUs shared between clone libraries. Community overlap of inter and inner sample comparisons for OTUs obtained by the program SONS. For the archaeal 16S rRNA gene libraries of two samples (ARC-MP and ARC-GY), there are 36.5% overlapped community and for the archaeal amoA libraries of two samples (ARC-MP and ARC-GY), there are 31.26%overlapped community, which reconfirmed the microbial populations of MP and GY is significant differences. For the archaeal amoA libraries of two samples (ARC-MP and ARC-MPr), there are 98.29% overlapped community, and for the archaeal amoA libraries of two sites (ARC-MP and ARC-GY), there are 84.10%overlapped community. The overlapped communities between reconstruction libraries both for archaeal 16S rRNA and amoA genes are obviously large, which reconfirmed the method we used has the properties of good repeatability and high reliability.In a conclusion, to analysis the diversity of Archaeal 16S rRNA and amoA genes in Mila Mountain meadow soil of Tibet, the program DOTUR,∫-Libshuf, TreeClimber and SONS were used. Reconfirmed the microbial populations of MP and GY is significant differences. These findings show prolific archaeal diversity in the alp prairie soil of Mila Mountain, where they may be actively involved in nitrification.