Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome

16S rRNA gene sequencing has been widely used to decipher bacterial communities. The bacterial 16S contains nine hypervariable regions (V1–V9) along with highly conserved sequences between these regions. The 16S amplicon sequencing (16S-seq) is used to decipher the composition of bacterial communities through universal primers that amplify the 1–3 hypervariable regions of 16S. The sequence identities among plant mitochondrial 18S rRNA (Mt18S), chloroplast 16S rRNA (Ct16S), and bacterial 16S exceed 70% overall and are higher in conserved regions. Thus, the contamination of plant Mt18S and Ct16S has been a major problem in the analysis of plant endophytic bacterial communities by 16S-seq.

 “There are very little plant DNA contamination, lower than 5%, in the 16S amplicon libraries produced by the primer sets we designed.” said Miss. Liying Chen, the first author for this work. This result indicated that the newly designed primer sets can effectively avoid plant-DNA contamination. “The primer sets we designed show good bacterial coverage by both in silico and experimental analysis, and are applicable to various plants, such as rice, Arabidopsis, tomato and so on”, said Chen. The advantage of these primer sets is the simple operation of 16S amplification, low cost and extensive applications. Given the numerous advantages, this plant DNA-free 16S profiling method is a good choice to decipher plant bacterial communities. “Compared with other methods excluding plant DNA contamination, like PCR clamping by PNA or LNA and Cas-16S-seq method by engineering CRISPR/Cas9, the highlight of our method is the direct PCR without auxiliary elements. Thus, our primer sets can be applied to the quantitation of plant bacterial communities by qPCR. The combination of quantitation and 16S-seq can display the plant bacterial community more precisely than the 16S-seq alone does”, said Professor Lily Zhang, the co-corresponding author.

This research developed plant DNA-free bacterial 16S amplification methods and quantitated plant endophytic bacterial communities through non-culture method. These protocols are suitable for the analyses of various plants and will significantly promote the research on plant endo-bacteriome.

This work was supported by grants from the Major Program of the National Natural Science Foundation of China (No. 32090013) to LZ, the National Transgenic Major Project of China (2019ZX08010-004) to LZ, and the National Natural Science Foundation of China (No. 31871932) to LZ.

See the article:

Chen, L., Zhang, M., Liu, D., Sun, H., Wu, J., Huo, Y., Chen, X., Fang, R., and Zhang, L. (2021). Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome. Sci China Life Sci 64, https://doi.org/10.1007/s11427-021-1953-5.

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