16S rRNA seq of unparasitized Helianthus annuus
The extracted DNA was used to perform a nested PCR with chloroplast-excluding primers, 16S 799f (AACMGGATTAGATACCCKG) and 16S 1492r (TACGGHTACCTT- GTTCGACTT). A nested PCR was used to reduce the amplification of plastid sequences, improve consistency and minimize non-specific amplification from primer pairs. The 50 L PCR reaction consisted of 2 L (100 ng) of sample genomic DNA, 1 L of primer 799f, 1 L of primer 1492r, 28 L of sterile water and 10 L of GoTaq Buffer (contains MgCl2 at a concentration of 7.5mM for a final concentration of 1.5mM in the 1X reaction), 5 L MgCl2, 2.5 L of BSA (Bovine Serum Albumin), 1 L DNTPs, and 0.5 L GoTaq Polymerase.After PCR amplification was performed, an electrophoretic agarose gel was used to separate the PCR products. We expected to obtain a mitochondrial product of about 1000bp and a bacterial product of about 750bp. A challenge to PCR amplification is that most bacterial 16S rDNA primer pairs may also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. The bacterial product band was cut out and gel extracted using the ZymocleanTM Gel DNA Recovery Kit (Zymo Research, Irvine, California, USA). The extracted endophytic bacterial product was then PCR amplified a second time using the barcoded primer set 16S 799f and 16S 1115r (AGGGTTGCGCTCGTTG). The second PCR products were then run through gel electrophoresis on a 1% agarose gel and the 0.3-0.4 kb band was excised. The bacterial product bands were gel extracted a second time using the ZymocleanTM Gel DNA Recovery Kit. The cleaned PCR products were then sent to the Microbial Analysis, Resources, and Services (MARS) facility at the University of Connecticut (UCONN) for genomic sequencing. The MARS facility is a specialized next generation sequencing (NGS) laboratory. DNA sequencing was performed using the two-step amplicon-to-data approach on an Illumina MiSeq sequencer.