Preparation method of endophytic bacterial diversity 16SrDNA amplicon

Abstract

The invention provides a set of primers for high-throughput sequencing. The primers comprise a primer pair 799F / 1193R in allusion to V5-V7 variable fragments of an endophytic bacterium 16S rDNA and Barcode sequences connected with the 5' ends of the primer pair 799F / 1193R, wherein the sequence of 799F is shown in SEQ ID NO: 1, the sequence of 1193R is shown in SEQ ID NO: 4, the Barcode sequence connected with the 5' end of the 799F is selected from sequences shown in SEQ ID NO: 5-28, and the Barcode sequence connected with the 5' end of the 1193R is selected from sequences shown as SEQ ID NO: 29-52. V5-V7 variable regions of the endophytic bacterium 16S rDNA are specifically amplified through two rounds of nested PCR, and the amplicon having the Barcode sequences at the 5' ends and applicable to the current second-generation high-throughput sequencing platform is obtained. After the adoption of the method provide by the invention for researching the endophytic bacterial diversity, the amplification efficiency of the endophytic bacterium 16S rDNA can be significantly improved, the proportion of host data in sequencing data is greatly reduced and the research cost is reduced.

Description

technical field [0001] The invention relates to the technical field of high-throughput sequencing of microbial diversity, in particular to the research on the diversity of plant endophytic bacteria. Background technique [0002] Plant endophytic bacteria (Endophytic bacteria) are a class of microorganisms that can colonize the intercellular space or intracellular space of healthy plants and establish a harmonious relationship with the host plant. They can provide mineral nutrition to plants and indirectly protect plants from pathogenic bacteria. However, in the process of studying plant endophytic bacteria, it is generally difficult to isolate and identify endophytic bacteria. Combining 16S rDNA-specific fragment PCR amplification and second-generation high-throughput sequencing technology for identification of plant endophytic bacteria is a popular method in recent years, but the total DNA extracted contains a large amount of host DNA, including a large number of mitochond...

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Problems solved by technology

However, when the inventors used this primer to conduct 16S rDNA sequencing research on plant endophytic bacterial diversity, they found that the proportion of host chloroplast data was still relatively high, refer to Table 1

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