Application of GSS in Analysis of Endophytic Bacterial Diversity in Ligusticum chuanxiong
10.13422/j.cnki.syfjx.20240111
- VernacularTitle:GSS高通量测序在川芎内生细菌群落多样性分析中的应用
- Author:
Hai WANG
1
;
Dongmei HE
2
;
Hongyang LYU
2
;
Guiqi HAN
1
;
Zhuyun YAN
2
Author Information
1. College of Medical Technology， Chengdu University of Traditional Chinese Medicine， Chengdu 611137， China
2. State Key Laboratory of Characteristic Chinese Medicine Resources in Southwest China， Chengdu 611137， China
- Publication Type:Journal Article
- Keywords:
Green Shield Sequencing （GSS）;
endophytic bacteria;
high-throughput sequencing;
host gene contamination;
Ligusticum chuanxiong
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2024;30(20):151-159
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveHost mitochondrial DNA （mtDNA） and chloroplast DNA （cpDNA） contamination severely affects high-throughput sequencing of endophytic bacteria in plants. This study aims to explore and evaluate a novel strategy of inhibiting host gene amplification in high-throughput sequencing of endophytic bacteria in medicinal plants. MethodGreen Shield Sequencing （GSS） was introduced in the 16S rDNA polymerase chain reaction （PCR） of endophytic bacteria to shield the non-target amplification of genes in the host （Ligusticum chuanxiong）. The performance was compared between GSS-PCR and conventional PCR in the high-throughput sequencing of endophytic bacteria and rhizosphere soil bacteria. ResultCompared with conventional PCR， GSS-PCR significantly reduced the amplification of mtDNA and cpDNA in L. chuanxiong in high-throughput sequencing， decreasing the non-target genes by more than 60%. Moreover， this strategy significantly increased the diversity of endophytic bacteria and multiplied the species without compromising the extraction of the information about the dominant bacteria. The GSS amplification of 16S rDNA V4 region of L. chuanxiong showed lower host contamination rate and higher endophytic bacterial diversity than that of V3-V4 regions. ConclusionGSS can significantly reduce host gene contamination in the high-throughput sequencing of L. chuanxiong endophytic bacteria and improve the accuracy of endophytic bacterial diversity analysis at the same sequencing depth， thus improving the high-throughput analysis quality of endophytic bacteria in plants. Accordingly， this strategy improves the feasibility and reliability of high-throughput sequencing for the 16S rDNA V3-V4 and V4 regions of endophytic bacteria. GSS used in this study provides a method reference for studying the endophytic bacteria in other medicinal plants.
