Bigdyetm terminator v3.1 cycle sequencing kit

The DNA was amplified by polymerase chain reaction (PCR) using specific primers for rs7975232, rs1544410, rs731236, and rs2228570. Primer sequence descriptions of the four single nucleotide polymorphisms (SNPs) are shown in Table S1. The PCR reaction system included 0.5 µL primer, 2.5 µL PCR buffer, 0.75 µL MgCl₂, 2.0 µL dNTP, 0.2 µL Taq polymerase, 1.0 µL DNA, and 17.55 µL water to a final reaction volume of 25 µL. PCR was carried out at Thermo Fisher Scientific Veriti^TM Thermal Cycler(Thermo Fisher Scientific Inc., Wilmington, DE, USA) using the following procedures: 1 cycle of 95 °C denaturation for 10 min, 35 cycles of 95 °C denaturation for 15 s, annealing temperature adjusted by primer for 30 s, and 72 °C extension for 1 min 30 s. Thereafter, this PCR product went through sequencing reaction using BigDye^TM Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Wilmington, DE, USA) in ABI PRISM 3130 Genetic Analyzer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The produced nucleotide sequences were analyzed with the Sequencing Analysis Software v5.2 (Thermo Fisher Scientific Inc., Wilmington, DE, USA).

Fully expanded cotyledons were inoculated using L. maculans isolate 87-41 (isolate-inoculated) or water (mock-inoculation). Four days after inoculation, cotyledon samples from eight individual plants per sample were pooled and ground in liquid nitrogen. Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Toronto, ON, Canada). RNA quality was determined using 1% agarose gel and Nanodrop. cDNA was synthesized using total RNA and the SuperScript^TM III kit following manufacturer’s protocol (Thermo Fisher Scientific, Toronto, ON, Canada). Then, primers (Table 1) were used to amply the full-length cDNA of the candidate gene of BLMR2 and cloned into the TA cloning vector with the TOPO^® TA Cloning^® Kit (Thermo Fisher Scientific, Toronto, ON, Canada). Positive clones were selected to extract plasmid DNA using the standard mini preparation protocol. The plasmid DNA was sequenced using the BigDye^TM terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific, Toronto, ON, Canada). Full length cDNA sequence was assembled using SeqMan software.

For the taxonomic identification of the Midgardsormr38 host, DNA from an overnight culture acquired from a single phage-susceptible bacterial colony was obtained using MagaZorb^® DNA Mini-Prep (Promega) in accordance with the manufacturer’s instructions. Classical PCR using 27F and 1492R primers followed by native agarose gel electrophoresis was performed (Weisburg et al., 1991 (link); Janda and Abbott, 2007 (link)).
The band containing a product of approximately 1,450 bp in length was extracted using GeneJET Gel Extraction kit (Thermo Fisher Scientific). 16s rRNA gene sequencing of the purified product was performed using the same aforementioned primers according to the BigDye^TM Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) protocol. The acquired partial sequence of the 16s rRNA gene was further compared to the 16s rRNA BLAST, EzBioCloud, and RDB Project databases (Altschul et al., 1990 (link); Cole et al., 2014 (link); Yoon et al., 2017 (link)). It was revealed that the Midgardsormr38 susceptible bacterial isolate belongs to the genus Erwinia and is a strain of E. persicina, sharing only minor nucleotide differences with E. persicina-type strain NBRC 102418 (GenBank: BCTN01000053).