Zhou Xue, Liu Xiaoyin, Liu Meiyu, Liu Weixuan, Xu Junzeng, Li Yawei
College of Agricultural Science and Engineering, Hohai University, Nanjing, China.
Jiangsu Province Engineering Research Center for Agricultural Soil-Water Efficient Utilization, Carbon Sequestration and Emission Reduction, Hohai University, Nanjing, China.
Front Microbiol. 2024 Jul 15;15:1424795. doi: 10.3389/fmicb.2024.1424795. eCollection 2024.
Compared with 454 sequencing technology, short-read sequencing (e.g., Illumina) technology generates sequences of high accuracy, but limited length (<500 bp). Such a limitation can prove that studying a target gene using a large amplicon (>500 bp) is challenging. The ammonia monooxygenase subunit A () gene of ammonia-oxidizing archaea (AOA), which plays a crucial part in the nitrification process, is such a gene. By providing a full overview of the community of a functional microbial guild, 16S ribosomal ribonucleic acid (rRNA) gene sequencing could overcome this problem. However, it remains unclear how 16S rRNA primer selection influences the quantification of relative abundance and the identification of community composition of nitrifiers, especially AOA. In the present study, a comparison was made between the performance of primer pairs 338F-806R, 515F-806R, and 515F-907R to a shotgun metagenome approach. The structure of nitrifier communities subjected to different long-term organic matter amendment and water management protocols was assessed. Overall, we observed higher Chao1 richness diversity of soil total bacteria by using 515F-806R compared to 338F-806R and 515F-907R, while higher Pielou's evenness diversity was observed by using 515F-806R and 515F-907R compared to 338F-806R. The studied primer pairs revealed different performances on the relative abundance of Thaumarchaeota, AOB, and NOB. The Thaumarchaeota 16S rRNA sequence was rarely detected using 338F-806R, while the relative abundances of Thaumarchaeota detected using 515F-806R were higher than those detected by using 515F-907R. AOB showed higher proportions in the 338F-806R and 515F-907R data, than in 515F-806R data. Different primers pairs showed significant change in relative proportion of NOB. Nonetheless, we found consistent patterns of the phylotype distribution of nitrifiers in different treatments. Nitrosopumilales (NP) and Nitrososphaerales (NS) clades were the dominant members of the AOA community in soils subject to controlled irrigation, whereas Nitrosotaleales (NT) and NS clades dominated the AOA community in soils subject to flooding irrigation. Nitrospira lineage II was the dominant NOB phylotype in all samples. Overall, ideal 16S rRNA primer pairs were identified for the analysis of nitrifier communities. Moreover, NP and NT clades of AOA might have distinct environmental adaptation strategies under different irrigation treatments.
与454测序技术相比，短读长测序技术（如Illumina技术）能生成高精度序列，但序列长度有限（<500 bp）。这种局限性表明，使用大扩增子（>500 bp）研究目标基因具有挑战性。氨氧化古菌（AOA）的氨单加氧酶亚基A（ ）基因在硝化过程中起关键作用，就是这样一个基因。通过全面概述功能性微生物群落，16S核糖体核糖核酸（rRNA）基因测序可以克服这一问题。然而，尚不清楚16S rRNA引物选择如何影响硝化细菌，尤其是AOA相对丰度的定量和群落组成的鉴定。在本研究中，对引物对338F-806R、515F-806R和515F-907R与鸟枪法宏基因组方法的性能进行了比较。评估了不同长期有机物质改良和水分管理方案下硝化细菌群落的结构。总体而言，我们观察到，与338F-806R和515F-907R相比，使用515F-806R时土壤总细菌的Chao1丰富度多样性更高，而与338F-806R相比，使用515F-806R和515F-907R时观察到更高的Pielou均匀度多样性。所研究的引物对在奇古菌、氨氧化细菌（AOB）和亚硝酸盐氧化细菌（NOB）的相对丰度上表现出不同的性能。使用338F-806R很少检测到奇古菌16S rRNA序列，而使用515F-806R检测到的奇古菌相对丰度高于使用515F-907R检测到的。AOB在338F-806R和515F-907R数据中的比例高于在515F-806R数据中的比例。不同引物对显示NOB的相对比例有显著变化。尽管如此，我们发现在不同处理中硝化细菌的系统发育型分布模式是一致的。在控制灌溉的土壤中，亚硝化侏儒菌目（NP）和亚硝化球形菌目（NS）分支是AOA群落的主要成员，而在淹水灌溉的土壤中，亚硝化弧菌目（NT）和NS分支主导AOA群落。硝化螺菌谱系II是所有样品中主要的NOB系统发育型。总体而言，确定了用于分析硝化细菌群落的理想16S rRNA引物对。此外，AOA的NP和NT分支在不同灌溉处理下可能有不同的环境适应策略。
