By designing specific primer pairs for bacterial 16S rDNA, the pollution-free sequencing analysis and quantitative detection of plant endophytes are realized

As the second set of plant genomes, plant-related microbiomes play an important role in plant nutrition, development, and stress resistance
.

The 16S amplicon sequencing technology has been widely used in the study of the microbiome
.

However, when this technology is applied to plant-related flora, especially plant endophytic flora, due to the high homology of plant organelle DNA (chloroplast 16S rDNA and mitochondrial 18S rDNA) and bacterial 16S rDNA, the amplicon library will There is a large amount of DNA contamination of organelles, which seriously interferes with the subsequent bio-information analysis
.

Recently, Zhang Lili and Fang Rongxiang’s team from the Institute of Microbiology of the Chinese Academy of Sciences and the Wu Jianxiang research team of Zhejiang University jointly published a research paper entitled "Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome" in Science China Life Sciences.
The specific primer pair realizes pollution-free sequencing analysis and qPCR quantitative detection of plant endophytic flora
.

In this study, the author designed three bacterial 16S rDNA-specific primers (not able to amplify chloroplast 16S rDNA or mitochondrial 18S rDNA) by setting certain base mismatch rules over the full length of 16S rDNA
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Combining the primer 799F designed to avoid chloroplast 16S rDNA, according to the read length limit of second-generation sequencing, two pairs of primer pairs are obtained.
Each pair of primers is one primer to avoid chloroplast 16S rDNA, and the other primer to avoid mitochondrial 18S.
rDNA
.

The two pairs of primers are 322F-Drs/796R and 799F/1107R, and the amplicon fragments are the V3V4 and V5V6 regions of 16S rDNA, respectively
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Online analysis and experimental evaluation results show that these two pairs of primers have good bacterial coverage
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When applied to the sequencing of bacterial populations in different tissues of rice, the proportion of plant organelle DNA in the amplicon library of the two pairs of primers is less than 5%
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Further, by experiments, two pairs of primers may be suitable for non-polluting sequencing of colonization in a variety of plants, such as Arabidopsis thaliana, tomatoes, citrus
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Since the proportion of plant organelle DNA in the amplicon library of these two pairs of primers is very low, the authors also used them for the qPCR quantitative analysis of endophytes in plant tissues
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In addition to the relative quantitative analysis of the bacterial content in different plant tissues based on the plant internal reference genes, the author also designed an experiment to produce a standard curve of the bacterial content of the plant tissue in the qPCR reaction and the Ct value of the qPCR reaction, and according to this standard curve, the plant rice is different The tissue was analyzed for absolute bacterial content
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 The 16S specific primer pair efficiently avoids the amplification of plant organelle DNA and is applied to the qPCR quantitative analysis of plant-related flora
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This research provides a convenient, economical and experimental technology platform for the sequencing and analysis of plant-related flora, especially endophytic flora, without plant-derived DNA contamination, and has realized the nucleic acid level of plant endophytic flora and plant-related flora.
The non-culture method is absolutely quantitative
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Liying Chen, College of Agriculture and Biotechnology, Zhejiang University, and Mengting Zhang, Institute of Microbiology, Chinese Academy of Sciences, are the co-first authors of this article, and researcher Zhang Lili and Academician Fang Rongxiang are corresponding authors
.

For details of the research, please read the original text▼[click the link below or read the original text] 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