Synergetic effect of non-complementary 5’ AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis

Adriana Larrea-Sarmiento; Anne M. Alvarez; James P. Stack; Mohammad Arif

A total of forty-three partial sequences belonging to the Chromosomal initiation factor gene, dnaA, were used to perform the phylogenetic analysis. Generated sequences from thirty-two strains of C. michiganensis were submitted to NCBI GenBank nucleotide database (S1 Table) . DNA sequences from ten Gram-positive plant bacteria were retrieved from GenBank. Accession The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. , which also served as the outgroup. All the sequences were aligned using muscle algorithm [21] plugged in MEGA 7.0.25 [22] . For single gene dnaA, the best evolution model was determined, and maximum likelihood (ML) method with 1,000 bootstrap values to evaluate node support was used to infer the phylogenetic tree. A color-coded matrix showing pairwise identity was generated using Sequence Demarcation Tool version 1.2 [23] .