Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola

Shefali Dobhal; Gamze Boluk; Brooke Babler; Michael J. Stulberg; John Rascoe; Mark Nakhla; Toni A. Chapman; Alex B. Crockford; Michael Melzer; Anne M. Alvarez; Mohammad Arif

Genomic DNA extraction, identification and phylogenetic analysis Extraction of genomic DNA from infected and healthy plant materials was performed using the Wizard Genomic DNA purification kit (Promega, Madison, WI); DNA was isolated from bacterial cultures using the Ultra Clean Microbial DNA Isolation kit (Mo Bio., Carlsbad, CA). For microbial . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/847590 doi: bioRxiv preprint DNA isolation, 3-4 loopfuls of bacterial cultures from plates were suspended in 300 μl of the microbead solution and vortexed gently to mix; Mini-BeadBeater 16 (Biospec products, Bartlesville, OK) was used to rupture the cell wall at maximum speed for 1 min; bacterial DNA was isolated following the manufacturer's instructions. DNA concentration and purity were measured using NanoDrop 2000/c spectrophotometers (Thermo Fisher Scientific Inc., Worcester, MA). The genomes of all Dickeya, and Pectobacterium species were aligned separately with progressive Mauve ( Pectobacterium Pec.dnaA-F1 (5'-CATACGTTTGATAACTTCGTTG-3') and Pec.dnaA-R1 (5'-GATGTCGTGGCTTTCTTCAC-3') primers were used to amplify the dnaA gene region of Pectobacterium species. The identity of other bacteria listed in the exclusivity panel was confirmed using primers P16s-F1 and P16s-R1 targeting the 16S ribosomal RNA region. The PCR conditions used for Dickeya and Pectobacterium dnaA gene amplifications were: Initial denaturation at 95 • C for 5 min followed by 35 cycles with 95 • C for 20 s, 58 • C for 1 min, 72 • C for 1 min and a final extension at 72 • C for 3 min. The amplified PCR products were cleaned by adding 2 µl ExoSAP-IT (Affymetrix Inc, Santa Clara, CA) in 5 µl of PCR product and incubated at 37 • C for 15 min followed by enzyme inactivation at 80 • C for 15 min. Sequencing was performed at the GENEWIZ facility (Genewiz, La Jolla, CA) for both sense and anti-sense strands. Obtained sense and antisense strands of each strain were aligned, manually edited and the error free consensus sequence was used to confirm identity by comparing the sequences of each strain against the NCBI GenBank nucleotide and genome databases using NCBI BLASTn tool. Sequences generated from each strain were deposited in the NCBI GenBank database and the accessions numbers are listed in Table 1 . Consensus sequences of 46 strains used in this study were aligned to generate a phylogenetic tree. Phylogenetic relationships among the strains was generated using the Neighbor-Joining method and the evolutionary distances were calculated using Tamura The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/847590 doi: bioRxiv preprint (NZ_CP006929), strain Ech586 (NC_013592), Pectobacterium carotovorum subspecies carotovorum strain PCC21 (NZ_018525), P. atrosepticum strain 21A (NZ_CP009125), P. wasabiae strain CFBP 3304 (NZ_CP015750), Erwinia amylovora strain CFBP1430 (NC_013961), Ralstonia solanacearum GMI 1000 (NC_003295), and Clavibacter sepedonicus strain ATCC33113 (NC_10407) were retrieved from NCBI GenBank Genome database (Supplementary Table 1 ). Whole genomes were aligned using progressive Mauve (2.4.0) and Geneious (10.2.3). Generated locally Collinear Blocks (LCBs) were analyzed to search unique and conserved regions for the genus Dickeya and D. dianthicola. The galactose/methy