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.
严婷婷, 赵艳, 王超霞, 汪成. 水稻种子内生细菌16S rDNA基因高通量测序PCR引物筛选和菌群结构分析[J]. 农业生物技术学报, 2021, 29(2): 316-326.
YAN Ting-Ting, ZHAO Yan, WANG Chao-Xia, WANG Cheng. 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. 农业生物技术学报, 2021, 29(2): 316-326.
[1] 陈泽斌, 李冰, 高熹, 等. 2017. 铁皮石斛不同部位内生细菌群落高通量分析[J].河南农业科学, 46(11): 98-102. (Chen Z B, Li B, Gao X, et al.2017. High throughput analysis of the endophytic bacterial community in different parts of Dendrobium candidum[J]. Journal of Henan Agricultural Sciences, 46(11): 98-102.) [2] 姜晓宇, 高菊生, 徐凤花, 等. 2013. 水稻种子内生细菌多样性及其分泌植物生长素能力的测定[J]. 微生物学报, 53(3): 269-275. (Jiang X Y, Gao J S, Xu F H, et al.2013. Diversity of endophytic bacteria in rice seeds and their secretion of indole acetic acid[J]. Acta Microbiologica Sinica, 53(3): 269-275.) [3] 李南南, 黎妮, 赵燃, 等. 2017. 优异杂交水稻亲本灌浆期种子内生细菌多样性研究[J]. 微生物学杂志, 37(2): 20-25. (Li N N, Li N, Zhao R, et al.2017. Diversity of endophytic bacterial communities in parental seeds of outstanding hybrid rice (Oryza sativa L.) in the milk[J]. Journal of Microbiology, 37(2): 20-25. [4] 沙月霞. 2018. 不同水稻组织内生细菌的群落多样性[J]. 微生物学报, 58(12): 2216-2228. (Sha Y X.2018. Diversity of bacterial endophytic community in different rice tissues[J]. Acta Microbiologica Sinica, 58(12): 2216-2228.) [5] 史玉倩, 赵艳. 2016. 水稻种子内生泛菌促进小球藻生长和油脂积累[J]. 中国农业科学, 49(8): 1429-1442. (Shi Y Q, Zhao Y.2016. Growth and lipid accumulation promotion of chlorella by endophytic Pantoea sp. from rice seeds[J]. Scientia Agricultura Sinica, 49(8): 1429-1442.) [6] 许艳蕊, 方志军, 卢晓平, 等. 2017. 基于高通量测序技术分析使它隆对玉米土壤细菌多样性的影响[J]. 微生物学报, 57(7): 985-993. (Xu Y R, Fang Z J, Lu X P, et al.2017. Effects of starane on maize soil bacterial diversity analyzed by high-throughput sequencing technology[J]. Acta Microbiologica Sinica, 57(7): 985-993.) [7] 邹媛媛, 刘琳, 刘洋, 等. 2012. 不同水稻品种种子固有细菌群落的多样性[J]. 植物生态学报, 36(8): 880-890. (Zhou Y Y, Liu L, Liu Y, et al.2012. Diversity of indigenous bacterial communities in Oryza sativa seeds of different varieties[J]. Journal of Plant Ecology, 36(8): 880-890.) [8] Bates S T, Donna B L, Caporaso J G, et al.2011. Examining the global distribution of dominant archaeal populations in soil[J]. The Isme Journal, 5(5): 908-917. [9] Beckers B, Beeck M O D, Thijs S, et al.2016. Performance of 16s rDNA primer pairs in the study of rhizosphere and endosphere bacterial microbiomes in metabarcoding studies[J]. Fronitiers in Microbiology, 7(650): 1-15. [10] Chakraborty U, Chakraborty B, Basnet M.2006. Plant growth promotion and induction of resistance in Camellia sinensis by Bacillus megaterium[J]. Journal of Basic Microbiology, 46(3): 186-195. [11] Chelius M K,Triplett E W.2001. The diversity of archaea and bacteria in association with the roots of Zea mays L.[J]. Microbial Ecology, 41(3):252-263. [12] Dussart-Baptista L, Bodilis J, Barray S, et al2007. Recurrent recovery of Pseudomonas oryzihabitans strains in a karstified chalk aquifer[J]. Water Research, 41(1): 111-117. [13] Edgar R C, Haas B J, Clemente J C, et al.2011. Uchime improves sensitivity and speed of chimera detection[J]. Bioinformatics, 27(16): 2194-2200. [14] Feng Y, Shen D, Song W.2006. Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates[J]. Journal of Applied Microbiology, 100(5): 938-945. [15] Ghyselinck J, Pfeifer S, Heylen K, et al.2013. The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies[J]. PLOS ONE, 8(8): 1-14. [16] Haas D, Défago G.2005. Biological control of soil-borne pathogens by Fluorescent pseudomonods[J]. Nature Reviews Microbiology, 3(4): 307-319. [17] Herlemann D P, Labrenz M, Jürgens K, et al.2011. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea[J].The Isme Journal, 5(5): 1571-1579. [18] Liu Y, Bai F R, Li N, et al.2017. Identification of endophytic bacterial strain RSE1 from seeds of super hybrid rice Shenliangyou 5814 (Oryza sativa L.) and evaluation of its antagonistic activity[J]. Plant Growth Regulation, 82(3): 403-408. [19] Liu Y, Xu P P,Yang F Z, et al.2018. Composition and diversity of endophytic bacterial community in seeds of super hybrid rice 'Shenliangyou 5814' (Oryza sativa L.) and its parental lines[J]. Plant Growth Regulation, 87(2): 257-266. [20] Logares R, Sunagawa S, Salazar G, et al.2014. Metagenomic 16S rDNA Illumina tags are a powerful alter native to amplicon sequencing to explore diversity and structure of microbial communities[J]. Environmental Microbiology, 16(9): 2659-2667. [21] Mano H, Tanaka F, Watanabe A, et al.2006. Culturable surface and endophytic bacterial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy field[J]. Microbes and Environments, 21(2): 86-100. [22] Pitta D W, Parmar N, Patel A K, et al.2014. Bacterial diversity dynamics associated with different diets and different primer pairs in the rumen of Kankrej cattle[J]. PLOS ONE, 9(11): 1-15. [23] Schwarz Z, Kssel H.1980. The primary structure of 16S rRNA from Zea mays chloroplast is homologous to E. coli 16S rRNA[J]. Nature, 283(5749): 739-742. [24] Spencer D F, Schnare M N, Gray M W.1984. Pronounced structural similarities between the small subunit ribosomal RNA genes of wheat mitochondria and Escherichia coli[J]. Proceedings of the National Academy of Sciences of the USA, 81(2): 493-497. [25] Sun L, Qiu F, Zhang X, et al.2008. Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis[J]. Microbial Ecology, 55(3): 415-424. [26] Trivedi P, Pandey A.2008. Plant growth promotion abilities and formulation of Bacillus megaterium strain B 388 (MTCC6521) isolated from a temperate Himalayan location[J]. Indian Journal of Microbiology, 48(3): 342-347. [27] Vasileiadis S, Puglisi E, Arena M, et al.2012. Soil bacterial diversity screening using single 16S rRNA gene V regions coupled with multi-million read generating sequencing technologies[J]. PLOS ONE, 7(8): 1-11. [28] Videira S S, De Araujo J L S, Da Silva Rodrigues L, et al.2009. Occurrence and diversity of nitrogenfixing Sphingomonas bacteria associated with rice plants grown in Brazil[J]. FEMS Microbiology Letters, 293(1): 11-19. [29] Xiong J B, Liu Y Q, Lin X G, et al.2012. Geographic distance and PH drive bacterial distribution in alkaline lake sediments across Tibetan Plateau[J]. Environmental Microbiology, 14(9): 2457-2466. [30] Yamamoto N, Kimura M, Matsuki H, et al.2010. Optimization of a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter[J]. Journal of Fermentation and Bioengineering, 109(1): 83-88. [31] Yu J, Zhou X F, Yang S J, et al.2013. Design and application of specific 16S rDNA-targeted primers for assessing endophytic diversity in Dendrobium officinale using nested PCR-DGGE[J]. Applied Microbiology and Biotechnology, 97(22): 9825-9836. [32] Zhang J Y, Liu Y X, Zhang N, et al.2019. NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice[J]. Nature Biotechnology, 37(6): 676-684. [33] Zhang Y Y, Zhang C W, Yang J, et al.2019. Insights into endophytic bacterial community structures of seeds among various Oryza sativa L. rice genotypes[J]. Journal of Plant Growth Regulation, 38: 93-102.
