State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China.
Nat Protoc. 2021 Feb;16(2):988-1012. doi: 10.1038/s41596-020-00444-7. Epub 2021 Jan 13.
Cultivating native bacteria from roots of plants grown in a given environment is essential for dissecting the functions of the root microbiota for plant growth and health with strain-specific resolution. In this study, we established a straightforward protocol for high-throughput bacterial isolation from fresh root samples using limiting dilution to ensure that most cultured bacteria originated from only one microorganism. This is followed by strain characterization using a two-sided barcode polymerase chain reaction system to identify pure and heterogeneous bacterial cultures. Our approach overcomes multiple difficulties of traditional bacterial isolation and identification methods, such as obtaining bacteria with diverse growth rates while greatly increasing throughput. To facilitate data processing, we developed an easy-to-use bioinformatic pipeline called 'Culturome' ( https://github.com/YongxinLiu/Culturome ) and a graphical user interface web server ( http://bailab.genetics.ac.cn/culturome/ ). This protocol allows any research group (two or three lab members without expertise in bioinformatics) to systematically cultivate root-associated bacteria within 8-9 weeks.
从特定环境中生长的植物根部培养本土细菌对于解析根微生物组在植物生长和健康方面的功能至关重要，因为这样可以实现基于菌株的特异性解析。在本研究中，我们建立了一种从新鲜根样本中进行高通量细菌分离的简单方案，使用限制稀释法确保大多数培养的细菌仅源自一个微生物。随后，我们使用双面条形码聚合酶链反应系统对菌株进行特征描述，以鉴定纯培养和异质培养的细菌。我们的方法克服了传统细菌分离和鉴定方法的多个难题，例如在大大提高通量的同时获得具有不同生长速度的细菌。为了便于数据处理，我们开发了一个名为“Culturome”的易于使用的生物信息学管道（https://github.com/YongxinLiu/Culturome）和一个图形用户界面网络服务器（http://bailab.genetics.ac.cn/culturome/）。该方案允许任何研究小组（两个或三个不具备生物信息学专业知识的实验室成员）在 8-9 周内系统地培养根系相关细菌。
