Chloroplast excluding primers for metagenomic analysis of bacteria in plant tissues

Plant microbiomes are responsible for the growth, health, nutrition, and resistance to biotic and abiotic stress. Moreover, some plant microbiome members, especially the Enterobacteriaceae family, can act as human pathogens. In recent years, metagenomic analysis of amplified 16S rRNA gene sequenced on Illumina second generation sequencers became the most widely used method for bacterial microbiome studies. The 16S r RNA gene of the bacterium is phylogenetically similar to the 16S genes of chloroplasts and mitochondria since they have common prokaryotic ancestors. Thus, plant microbiome analysis is affected by unwanted non-target chloroplast and mitochondrial sequences. The solution to this contamination uses specially designed primer pairs that exclude chloroplasts and mitochondria and provide undistorted information. In this study, we analyzed chloroplast excluding primer 799R (reverse complement of 799F) which contains 4 mismatches of nucleotides against the chloroplast sequence modified for use with Illumina sequencers. We used primer 799R with universal bacterial forward primers 341F and 515F and compared the results obtained with 799R to the results obtained with the universal 806R primer. Microbiomes of 12 samples from 4 distinct parts of the tomato plants (Solanum lycopersicon L.) leaves, fruits, roots, and rhizosphere, were amplified using all combinations of these primers. The combination of universal forward primers 515F or 341F with the universal bacterial primer 806R amplified 60-85% of chloroplast sequences in samples of plant tissue. The 799R primer effectively reduced the number of chloroplast sequences to less than 1%. However, some sequences derived from mitochondria remained, with a higher proportion using the 341F primer (66-72%) than 515F (21-28%). The ability to describe microbial population diversity using 799R was similar to 806R, although there is a clear difference in the proportions of amplified microorganism groups using these primers. Analysis revealed the highest frequency of Enterobacteriaceae when primer pair 515F+799R was used. Based on the results, the combination of primers 515F+799R was the most appropriate, met all parameters, and can be recommended for routine analysis of plant microbiomes using second generation sequencers.