Study the genetic variation of Polymyxa betae using targeted amplicon sequencing.

Abstract

Polymyxa betae, a plasmodiophorid protist, is the vector of the soilborne virus beet necrotic yellow vein virus (BNYVV), the cause of rhizomania disease and the most economically important virus of sugar beet. Molecular studies of these organisms are difficult due to their biotrophic nature; thus, there is still much to be learned about them. In this study, we attempted to develop molecular methods to analyze the genetic variation in seventeen specific regions of the P. betae genome using the Illumina MiSeq platform with a two-step PCR approach. The target-specific regions, which contain single nucleotide polymorphisms and insertions/deletions, were identified previously through comparative analysis of genome assemblies from ten isolates recovered from soil samples collected from sugar beet growing areas across the U.S.A. The variation in those target-specific regions was previously validated using a set of twelve genotyping markers. In this preliminary assay, we used P. betae DNA extracted from sugar beet roots and soil, to compare the total sequencing output using different DNA sources. Read mapping and variant calling allowed the identification of the SNP and INDEL observed previously using the genotyping markers. The total reads count for each of the seventeen target-specific amplicons for the soil samples were slightly lower than for sugar beet roots, but had no impact on the type or frequency of variants detected as they were similar in both types of samples. The pilot study demonstrated the capability of using the newly developed genotyping markers in MiSeq analysis to study the genetic variation of P. betae. Currently, we are evaluating the technique on soil samples collected from a sugar beet production area in Imperial Valley, CA.