Targeted amplicon sequencing for genotyping isolates of Polymyxa betae

Authors: CAMELO, VIVIANA - Forest Service (FS); Wintermantel, William - Bill; Martin, Frank

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/28/2022
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Polymyxa betae is a vector of several soilborne viruses in sugar beet, in an effort to develop methods for its population characterization, a genotyping assay using the Illumina MiSeq platform with a two-step PCR approach was developed to analyze the genetic variation in specific regions of the P. betae genome. The target-specific regions containing single nucleotide polymorphisms and insertions/deletions were identified previously through comparative analysis of sequences obtained with a set of twelve genotyping markers. In this pilot study, we used DNA extracted from sugar beet roots and from soil infested with isolate 17-1 of P. betae (Moorhead, MN). In the first PCR amplification products between 351 and 379-bp were generated using seventeen target-specific primers with the Fluidigm common oligos CS1/CS2 fused at their 5' ends. After confirming amplification of each target sequence the pooled amplicons were subject to a secondary PCR reaction amplified with dual indexed, Illumina compatible primers which target the Fluidigm CS1/CS2 oligomers. The sequencing was carried out on an Illumina MiSeq platform in a 2×250-bp paired-end format using a MiSeq v2 500 cycle reagent cartridge. We obtained sequence data for samples from roots and soil. Read mapping and variant calling allowed identification of the SNP and INDEL observed previously for the 17-1 isolate using the genotyping markers and calculation of the sequencing error rate. Currently, we are adjusting the technique to allow analysis of a larger number of samples for population studies in sugar beet production areas.