Kapa hifi hs readymix

Bacterial genomic DNA was extracted from each hemolytic colony using a heat extraction method⁶⁸ . The 16S rRNA genes of each hemolytic bacterium were amplified using KAPA HiFi HS ReadyMix (Kapa Biosystems), according to the manufacturer’s instructions. A polymerase chain reaction (PCR) amplification was performed using the following universal bacterial primers: 16S_27f (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 16S_1492r (5′-GGT TAC CTT GTT ACG ACT T -3′)^{69 (link)}. The PCR products were gel purified using the FastGene Gel/PCR Extraction Kit (Nippon Genetics, Japan) and the sequences were confirmed via a commercial sequencing service (Fasmac, Kanagawa, Japan). The sequences of the PCR products were compared with known 16S rRNA gene sequences in GenBank, and the hemolytic bacterial species were identified.

For genomic DNA extraction, target sequences were PCR-amplified with KAPA HiFi HS ReadyMix (Kapa Biosystems, Cat. No. KK2602), amplicons were treated with ExoSAP-IT Express reagent (Thermo Fischer Scientific, Cat. No. 75001) for enzymatic cleanup, and Sanger sequencing was prepared with the BigDye Terminator v3.1 CS Kit (Thermo Fischer Scientific, Cat. No. 4337456). Reactions were then purified by ethanol precipitation and acquired on a 3130xl Genetic Analyzer (Applied Biosystems). Sequence alignments were analyzed with Snapgene (GSL Biotech LLC), and sequence trace files with low base calling confidence were excluded from analyses. Genotyping primers are listed in Supplementary Table 9.

The amplicon PCR targeted the V3-V4 regions of bacterial 16S ribosomal RNA (rRNA) gene. Sequencing libraries of the V3-V4 region were generated according to 16S Metagenomic Sequencing Library Preparation instructions (Illumina, San Diego, CA, USA). In brief, V3-V4 regions of 16S bacterial rRNA gene were amplified using a two-step polymerase chain reaction (PCR) protocol (Illumina). The amplicon PCR used KAPA HiFi HS ReadyMix (Kapa Biosystems, Wilmington, MA, USA) and V3-V4 region primers (forward, 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′ and reverse, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′). The index PCR used KAPA HiFi HS ReadyMix and Nextera XT index kit (Illumina). Libraries were purified using AMPure XP (Beckman Coulter, Indianapolis, IN, USA) and quantified using a Qubit 3 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The library was diluted, mixed with PhiX (Illumina) and then subjected to an Illumina MiSeq system for sequencing with a MiSeq reagent kit v3 (600 cycles, Illumina). The sequencing was set up with MiSeq Control Software (Illumina).

The procedure used is the same as that previously described (Kitamura et al., 2016 (link)).
Eubacterial 16S rRNA genes 586 bp in length were amplified using the primers 341F (5′-CCTACGGGAGGCAGCAG-3′) and 907R (5′-CCGTCAATTCCTTTRAGTTT-3′), targeting the V3, V4, and V5 regions, as performed in previous studies (Muyzer et al., 1993 (link); Schmalenberger et al., 2001 (link); Chakravorty et al., 2007 (link)). PCR was conducted using the KAPA HiFi HS Ready Mix (KAPA Biosystems) under the following conditions: initial denaturation at 95°C for 3‍ ‍min, 28 cycles of denaturation at 98°C for 30‍ ‍s, annealing at 62°C for 15‍ ‍s, and extension at 72°C for 15‍ ‍s, with a final extension step at 72°C for 3‍ ‍min. PCR products were visualized with SYBR Green I after 1% (w/v) agarose gel electrophoresis at 100‍ ‍V for 25‍ ‍min with 470‍ ‍nm blue light illuminated by a Dark Reader transilluminator (Clare Chemical Research); purified DNA was excised from a gel band by Takara RECOCHIP (Takara Bio).
The purified PCR products of the eubacterial 16S rRNA gene were quantified using the Invitrogen Quant-iT PicoGreen dsDNA Reagent (Life Technologies). Ten nanograms of PCR products at a final concentration of 200 pg mL^–1 was used for library preparation. DNA libraries were produced using the TruSeq ChIP DNA Sample Prep Kit (Illumina).

DNA was extracted from fresh floral buds (180 mg per sample) using innuPREP Plant DNA Kit (AnalytikJenaInd, Berlin, Germany) following the manufacturer’s instructions. Restriction-site-Associated DNA (RAD) libraries were prepared following the protocol according to Sakaguchi et al. (2015) (link). In brief, we performed an enzymatic digestion using BglII and EcoRI and ligation with Y-shaped adaptors followed by a PCR with KAPA HiFi HS ReadyMix (KAPA BIOSYSTEMS). Size selection for fragments of approximately 350 base pairs (bp) was carried out using the E-Gel size select (Life technologies, CA, United States). We conducted a single-end sequencing with a read length of 51bp with the Illumina HiSeq 2500 platform (Illumina, CA, United States).