Abi3130 1 genetic analyzer

To investigate the effect of the mprF mutation (L291I), identified in DAP^R isolates of each patient, on drug susceptibility, gene replacement was performed using the pKOR1 plasmid^{30 (link),80 (link)}. In brief, mprF genes were amplified from each H-1 (DAP^S) and H-5 (DAP^R) strain with primer sets listed in Supplemental Table 3. The PCR fragments were individually cloned into the pKOR1 plasmid using Gateway BP Clonase II enzyme mix (Thermo Scientific, USA), and recombinant plasmids were selected through CcdB-based positive selection system in Escherichia coli DH5α. The plasmid-carrying wild-type mprF gene was then introduced into DAP^R strain H-5, while the mutated mprF gene was transformed into DAP^S strain H-3. This was achieved by electroporation using NEPA21 electroporator (NEPAGENE, Japan) following the parameters reported previously^{81 (link)}. Chromosomal gene replacement involved single-crossover plasmid integration at 43°C followed by overnight incubation in drug-free medium at 37°C to eliminate the plasmid. Anhydrotetracycline was used to select for non-plasmid-carrying mutants. The presence of gene mutations was confirmed by PCR and targeted gene sequencing with an ABI3130 × 1 Genetic Analyzer (Applied Biosystems, USA).

RT-PCR products were purified using a Qiagen Purification Kit (Qiagen) and cloned using an InsTAclone PCR Cloning Kit (Thermo Fisher Scientific). Both strands of 12–20 clones of each accession were sequenced using an ABI 3130 × 1 Genetic Analyzer (Applied Biosystems Inc., CA) and an ABI BigDye Kit according to a standard protocol. Similarity searches between the obtained rye CENH3 sequences and their orthologous from other species were carried out using the TBLASTN software^{39 (link)} in the NCBI database (http://blast.ncbi.nlm.nih.gov/Database/). Multiple alignments of amino acids and coding sequences were performed online using Clustal Omega^{40 (link)} (http://www.ebi.ac.uk/Tools/msa/clustalo). Alignments were further refined manually and used for downstream analysis with the aid of statistical, phylogenetic programs and for visualization^{41 (link)} (http://www.jalview.org). The deduced protein sequences were examined for potential posttranslational modifications using NetPhos 2.0 (www.cbs.dtu.dk/services/NetPhos).

RTPCR products were purified using a Qiagen Purification Kit (Qiagen) and cloned using an InsTAclone PCR Cloning Kit (Thermo Fisher Scientific). Both strands of each of 15–20 clones of each parental variety and addition line were sequenced using an ABI 3130×1 Genetic Analyzer (Applied Biosystems Inc., CA) and an ABI BigDye Kit according to a standard protocol. Similarity searches between the CENH3 sequences and their orthologs in other species were carried out using the TBLASTN software (Altschul et al. 1990 (link)) in the NCBI database (http://blast.ncbi.nlm.nih.gov/Database/).