Next Generation Sequencing to Define Prokaryotic and Fungal Diversity in the Bovine Rumen

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1–V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4–99.9% of OTUs represented by more than one read, using Good’s coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.

本研究采用桑格（Sanger）与454测序技术获得的小亚基rRNA基因座序列，对12头饲喂牧草日粮的奶牛瘤胃内的微生物多样性进行探究。基于16S rRNA基因V1-V3区的分析显示，当每头动物采集5520条读长（reads）时，观测到的细菌物种丰富度介于1903至2432个物种级操作分类单元（operational taxonomic unit, OTU）之间。其中80%的物种级OTU由梭菌目（Clostridiales）、拟杆菌目（Bacteroidales）、丹毒丝菌目（Erysipelotrichales）及未分类TM7类群的成员主导。普雷沃菌属（Prevotella）物种的丰度在12头受试个体间差异显著。
同样基于16S rRNA基因的古菌物种丰富度介于8至13个OTU之间，隶属于5个属。本研究鉴定的古菌OTU中，84%已在公共数据库中被报道，仅发现2个新OTU。
基于18S rRNA基因的瘤胃真菌物种丰富度介于21至40个OTU之间，经古德覆盖度（Good’s coverage）评估显示，98.4%至99.9%的OTU可被多于1条读长覆盖。对真菌群落的分析鉴定出多个新类群。
普雷沃菌属与坦纳菌属（Tannerella）在瘤胃液相组分中显著富集，而丁酸弧菌属（Butyrivibrio）与布劳特氏菌属（Blautia）则在瘤胃固相组分中显著富集。古菌与真菌的生物多样性在瘤胃液、固两相之间未观测到显著统计学差异。
本研究通过微生物群落调查与跨域相关性分析发现，牛瘤胃内的微生物多样性远超此前认知；下一代测序（next generation sequencing）技术有望揭示更多全新的微生物类群、互作关系及代谢通路，后续可通过对这些靶点的深入研究，进一步阐明瘤胃微生物群落结构与功能如何影响反刍动物饲料效率、生物燃料生产及环境影响。