Genomic tip 100 g

The relevant data for all X. translucens strains used in this study are listed in Table 1. The genome sequence of X. translucens pv. translucens strain DSM 18974 was retrieved from the National Center for Biotechnology Information (NCBI) GenBank database (accession number LT604072). Strains LMG 726, LMG 727, LMG 728, LMG 730, LMG 843, and UPB458 were grown at 28°C on YDC agar medium (2% dextrose, 1% yeast extract, 2% CaCO3, 1.5% agar) for 48 h. Bacteria were then dissolved in 10 ml washing buffer (50 mM TRIS-HCl pH 8.0, 50 mM Ethylenediaminetetraacetic acid (EDTA) pH 8.0, 150 mM NaCl). The genomic DNA was then extracted with the NucleoSpin ^® Microbial DNA kit (Macherey Nagel, Duren, Germany), according to the manufacturer’s recommendations. Strains CFBP 2055, CFBP 2539, CFBP 2541, and CFBP 8304 were grown at 28°C on PSA medium (0.5% peptone, 2% sucrose, 1.5% agar) for 24 h. Bacteria were then resuspended in 10 mM MgCl₂ and diluted to an optical density at 600 nm of 1.0. Cells from 2 ml were harvested by centrifugation and washed once with 10 mM MgCl₂, and genomic DNA was isolated using QIAGEN Genomic tip 100/G (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The genomic DNA from strain ICMP 16317 was extracted following a standard phenol/chloroform method (Booher et al., 2015 (link)).

Bacteria were streaked out from the culture stock and grew on 2, 3, 5-triphenyltetrazolium chloride (TZC) agar medium (dextrose 5 gL⁻¹, peptone 10 gL⁻¹, 0.001% sterilized TZC, and agar 18 gL⁻¹) at 28°C for 2 days. Bacterial genomic DNA was isolated from pure culture using QIAGEN Genomic-tip 100/G, following the manufacturer’s instruction (QIAGEN, Valencia, CA, USA). The Seqwell plexWell LP384 Library Preparation Kit and Native Barcoding Kit 24 V14 (SQK-NBD112.24) were used for barcode-indexed whole genome sequencing with Illumina NovaSeq system (Illumina San Diego, CA, USA) and Oxford Nanopore MinIoN Mk1C device (Oxford Nanopore Technologies, ONT, Oxford, UK), respectively. ONT long reads were base called and demultiplexed using basecaller and barcoder of GUPPY v6.3.2 on MANA, a high-performance computing cluster at the University of Hawaii at Manoa.

All molecular biology procedures (cloning, DNA amplification, purification and manipulation) were performed according to standard protocols43 . Purification of genomic bacterial DNA was carried out using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). High-molecular-weight gDNA required for transformation experiments was prepared using QIAGEN Genomic-tip 100/G and QIAGEN Genomic-tip 500/G (QIAGEN).

The MT, KD, and PM trees whose materials were used in this study were maintained at Chanthaburi horticultural research center, Chanthaburi, Thailand. They grow in the non-flooded area and receive 150 liters/tree/day of water supply. Fertilizers and pesticides are supplied regularly. For this study, healthy leaves were collected, immediately frozen in liquid nitrogen, and stored at −80°C. DNA was extracted and purified using the QIAGEN Genomic-tip 100/G following the manufacturer’s protocol (Qiagen, Germany). DNA quality was assessed using 0.75% pulsed-field gel electrophoresis and the concentration was tested with Qubit^® dsDNA BR Assay Kits (Thermo Fisher Scientific) and NanoDrop (Thermo Fisher Scientific).
Total RNA was extracted from healthy leaves using CTAB buffer and 25:24:1 phenol:chloroform:isoamyl alcohol. Contaminated DNA was removed by using DNA-free™ DNA Removal Kit (Invitrogen™). The quality and quantity of RNA were evaluated with the fragment analyzer machine (Agilent). Poly(A) mRNAs were enriched from total RNA samples using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Leaf tissues from a mature C. zippeliana (12°31′33.6″N 102°05′45.8″E) in the International Mangrove Botanical Garden Rama IX (Chantaburi, Thailand) was collected, flash-frozen and stored in liquid nitrogen until use. High molecular weight genomic DNA were extracted using the QIAGEN Genomic-tip 100/G following the manufacterer’s protocol (Qiagen, Hilden, Germany). The integrity of the DNA samples was evaluated with the Pippin Pulse Electrophoresis System (Sage Science, Beverly, MA, USA) prior to the 10x Genomics library preparation.
For transcriptome sequencing, we isolated total RNA from leaf tissues collected from the same individuals used for genome sequencing following the protocol in Pootakham et al. (2021b (link)). Briefly, total RNA was isolated using the CTAB buffer (2% CTAB, 1.4M NaCl, 2% PVP, 20 mM EDTA pH 8.0, 100 mM Tris-HCl pH 8.0, 0.4% SDS). RNA was extracted from the aqueous phase 3 times using 25:24:1 phenol: chloroform: isoamylalcohol and precipitated overnight in ¼ volumes of 8M LiCl. The pellets were washed with 70% ethanol, air-dried and resuspended in RNase-free water. Poly(A) mRNA was enriched using the Dynabeads mRNA Purification Kit (ThermoFisher Scientific, Waltham, MA, USA). Prior to the MGISEQ library construction, the integrity of the RNA samples was assessed on the Fragment Analyzer System (Agilent, Santa Clara, CA, USA).

Genomic DNA for SMRT library construction was preparated from leaf material with a modified CTAB-DNA extraction method followed by QIAGEN Genomic-tip 100/G (Qiagen, Hilden, Germany) cleaning and filtering. The sugar beet DH plants of the sequenced genotype KWS2320 [17 (link)] were provided by KWS SAAT SE.
Plants were grown in the greenhouse under long day conditions on soil for 6 weeks. Reduction of starch content was performed by etiolation for 4 days prior to harvest. About 2.5 g young tissue was ground under liquid nitrogen and mixed with 20 ml prewarmed modified Carlson-buffer [18 (link)] containing 3 % CTAB, 3 μl/ml 2-ME and 0.2 mg RNAse. The homogenate was incubated at 74 °C for 30 min with inverting every 5 min. The DNA was than extracted with 1 Vol. chloroform:isoamylalcohol (24:1) and centrifuged with 17,000 rpm at RT. The aqueous phase was diluted with 1 Vol. H₂O and adjusted to pH 7.0 prior to Genomic-tip 100/G purification and precipitation. The final pellet was resuspended in 500 μl sterile distilled water, the DNA concentration determined and the purity and integrity visualized on an agarose gel.

A. hydrophila strains were cultured overnight at 28°C in LB medium. Genomic DNA was extracted using the QIAGEN Genomictip 100/G (Qiagen, CA, USA). The Qubit 4.0 Fluorometer for double-strand-DNA high-sensitivity assay kit (Thermo Fisher Scientific, MA, USA) was used to quantified the concentrations of DNA. Whole-genome sequencing was performed on the PacBio RS II platform (Pacific Biosciences, CA, USA). The sequencing reads were assembled using Falcon version 0.7.0 and FALCON-Unzip. The presence of single-nucleotide polymorphisms (SNPs) and other genomic variations were analysed using CLC Genomic Workbench 5.5.1 probalistic variant detection software.