Screening of PCR Primers Targeted 16S rDNA Gene for High Throughput Sequencing of Endophytic Bacteria in Rice (Oryza sativa) Seeds and Analysis of Bacterial Community Structure
YAN Ting-Ting, ZHAO Yan*, WANG Chao-Xia, WANG Cheng
College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China
Abstract Plant endophytic bacteria are important microbial resource. The universal primer pairs for bacteria 16S rDNA gene amplification show similarity with plant DNA sequence from organelles such as chloroplast and Mitochondria, which brings great challenge to the high-throughput sequencing of endophytic bacteria. The aim of present study is to eliminate the interference of non-target DNA to high throughput sequencing of rice(Oryza sativa) endophytic bacteria, screen out of the optimal primer pair for analysis of the endophytic bacteria in rice seeds and describe their community structure. Seeds of japonica rice variety Nipponbare were used as material. Seven universal primer pairs targeted bacterial 16S rDNA gene for high throughput sequencing were selected as candidate primers. The results of bioinformatics analysis revealed that the primer pairs dP1, dP3 and dP7 showed low homology with plant organelle DNA sequences. They were less likely to amplify the gene fragments of rice chromosomes and organelles.Thus, they were selected as the initial screening primers and tested by 16S rDNA gene high throughput sequencing using Illumina MiSeq pyrosequencing. The three selected primers produced effective DNA fragments, except that the dP7 primer pairs need to use high fidelity Taq enzyme. After quality control (QC), decontamination treatment and organelle_filtered, the number of residual good reads from dP7 products were 11.7 and 5.3 times of that from dP1 and dP3, which could reduce the interference of rice organelle gene sequences to a certain extent, and produced the most OTUs (632). So dP7 was selected as the optimal primer pair. Furthermore, rice seeds diverse bacterial endophytic which were divided into 5 Phylum, 8 class, 13 order, 32 family, and 51 genera. Among them, the first dominant bacterium genera were Bacillus and Lactococcus, and their relative abundance were 64.46% and 15.07%, respectively. The second sub-dominant bacterium genera were Pantoea and Exiguobacterium, and their relative abundance were 4.27% and 3.61%. In conclusion, different primers have great influence on the results of high throughput sequencing of plant-associated bacterial communities. Among all the tested primer pairs, dP7 was the optimal primer pair for the analysis, with which more diversity endophytic bacterial communities were obtained in rice seeds. The study provides basic data for the future research on the microbial resources and the interaction mechanism between endophytic bacteria and rice host.
YAN Ting-Ting,ZHAO Yan,WANG Chao-Xia, et al. Screening of PCR Primers Targeted 16S rDNA Gene for High Throughput Sequencing of Endophytic Bacteria in Rice (Oryza sativa) Seeds and Analysis of Bacterial Community Structure[J]. 农业生物技术学报, 2021, 29(2): 316-326.
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