Table 1.
List of isolate name, plant host, and SNP discrimination by sequencing, HRM and Taqman-SNP Genotyping assay.
Table 2.
Discriminative detection of SNI and SRB by HRM and TaqMan SNP assays in single and artificial mixed samples at different dilutions.
Fig 1.
Schematic representation of TSWV NSm gene sequence showing primers and probes localization.
(a) the single nucleotide polymorphism (in bold) determining the different pathogenic properties of the SNI and SRB TSWV biotypes. Boxed in orange, the sequence segment chosen for TaqMan probe hybridization. (b) Position of primers (in light blue) and probes (in orange) used in the Custom TaqMan™ SNP Genotyping Assays. In pink, the primer pair used for reverse-transcription-PCR and NSm gene sequencing. (c) Position of primers (green) used in the high-resolution melting assays.
Fig 2.
High resolution melting and custom TaqMan SNP Genotyping assays for the detection and discrimination of Sw-5 non infecting (SNI) and Sw-5 resistant breaking (SRB) alleles.
(a) The temperature-shifted difference curves discriminated the two reference samples and their mixture in three separated clusters. The two lines correspond to two technical replicates for each sample. (b) Scatter plot of fluorescence data from Custom TaqMan SNP Genotyping assays. Each dot denotes individual replicates (four technical replicates per sample) and corresponds to FAM and VIC fluorescence levels associated to SNI- and SRB-specific probes, respectively. Allele-specific boundaries defined four non-overlapping areas corresponding to SNI, SRB, SNI/SRB mix and no template control (NTC).
Fig 3.
Co-detection and discrimination of SNI and SRB alleles in artificial mixtures by high resolution melting and custom TaqMan SNP Genotyping assays.
(a) The normalized temperature-shifted melt curves grouped the artificial mixtures in three clusters: blue, SNI allele (mixtures 3 and 6); green, SNI/SRB alleles (mixtures 1, 2, 4 and 5); red, SRB allele (mixtures 7 and 8). (b) The scatter plot from Custom TaqMan SNP Genotyping assays. SNI and SRB (samples 15.9 and Borgo 1, respectively) were included as the the reference panel samples. Only mixtures 1 and 5 fell within the SNI/SRB boundary. Mixture 9 did not provide amplification signal in both assays.
Fig 4.
Detection and discrimination of SNI and SRB alleles among different TSWV isolates from tomato and artichoke samples by high resolution melting and custom TaqMan SNP Genotyping assays.
(a) The normalized temperature-shifted melt curves relative to HRM analysis assigned with a high percent confidence ranging from 99.8 to 100%, the eighteen samples to three clusters according to reference panel genotypes: SNI (blue), SRB (red) and an additional cluster (orange). (b) The scatter plot from Custom TaqMan SNP Genotyping assays displayed the two biotypes into two boundaries according to the panel reference samples, specifically assigning field isolates to either the SNI allele (within blue boundary) or the SRB allele (red) with a call rate of 100%. Four NTC were included in the analysis.
Fig 5.
Detection and discrimination of SNI and SRB alleles from crude leaf extracts of tomato plants collected in field conditions by HRM and custom TaqMan SNP Genotyping assays.
(a) The temperature shifted melt curves relative to HRM analysis assigned the samples to two clusters according to reference panel genotypes, SNI and SRB, whose colours are associated to reference panel samples like in the previous figures. (b) The scatter plot from Custom TaqMan SNP Genotyping assays identified three boundaries corresponding to SNI, SRB and NTC. All samples were correctly amplified and assigned to the proper biotype.
Table 3.
Primers and probes used in the described experimental procedures.