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BackgroundPCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved to increase genotyping efficiency. A new assay, rhAmp, based on RNase H2-dependent PCR (rhPCR) combined with a universal reporter system attempts to reduce error rates from primer/primer and primer/probe dimers while lowering costs compared to existing technologies. Before rhAmp can be widely adopted, more experimentation is required to validate its effectiveness versus established methods.ResultsThe aim of this study was to compare the accuracy, sensitivity and costs of TaqMan, KASP, and rhAmp SNP genotyping methods in sugar beet (Beta vulgaris L.). For each approach, assays were designed to genotype 33 SNPs in a set of 96 sugar beet individuals obtained from 12 parental lines. The assay sensitivity was tested using a series of dilutions from 100 to 0.1 ng per PCR reaction. PCR was carried out on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The call-rate, defined as the percentage of genotype calls relative to the possible number of calls, was 97.0, 97.6, and 98.1% for TaqMan, KASP, and rhAmp, respectively. For rhAmp SNP, 24 of the 33 SNPs demonstrated 100% concordance with other two technologies. The genotype concordance with either technologies for the other 9 targets was above 99% (99.34–99.89%).ConclusionThe sensitivity test demonstrated that TaqMan and rhAmp were able to successfully determine SNP genotypes using as little as 0.2 ng DNA per reaction, while the KASP was unable to ascertain SNP states below 0.9 ng of DNA per reaction. Comparative cost per reaction was also analyzed with rhAmp SNP offering the lowest cost per reaction. In conclusion, rhAmp produced more calls than either TaqMan or KASP, higher signal to NTC data while offering the lowest cost per reaction.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0295-6) contains supplementary material, which is available to authorized users.

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The beet-cyst nematode (Heterodera schachtii\ud Schmidt) is one of the major pests of sugar beet. The identification\ud of molecular markers associated with nematode tolerance\ud would be helpful for developing tolerant varieties. The\ud aim of this study was to identify single nucleotide polymorphism\ud (SNP) markers linked to nematode tolerance from the\ud Beta vulgaris ssp. maritima source WB242. A WB242-\ud derived F2 population was phenotyped for host-plant nematode\ud reaction revealing a 3:1 segregation ratio of the tolerant and\ud susceptible phenotypes and suggesting the action of a gene\ud designated as HsBvm-1. Bulked segregant analysis (BSA)\ud was used. The most tolerant and susceptible individuals were\ud pooled and subjected to restriction site associated DNA sequencing\ud (RAD-Seq) analysis, which identified 7,241 SNPs.\ud A subset of 384 candidate SNPs segregating between bulks\ud were genotyped on the 20 most-tolerant and most-susceptible\ud individuals, identifying a single marker (SNP192) showing\ud complete association with nematode tolerance. Segregation of\ud SNP192 confirmed the inheritance of tolerance by a single\ud gene. This association was further validated on a set of 26\ud commercial tolerant and susceptible varieties, showing the\ud presence of the SNP192 WB242-type allele only in the tolerant\ud varieties. We identified and mapped on chromosome 5 the first\ud nematode tolerance gene (HsBvm-1) from Beta vulgaris ssp.\ud maritima and released information on SNP192, a linked marker\ud valuable for high-throughput, marker-assisted breeding of nematode\ud tolerance in sugar beet