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.</p

Rapid identification of tomato Sw-5 resistance-breaking isolates of <i>Tomato spotted wilt virus</i> using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques

<div><p>In tomato, resistance to <i>Tomato spotted wilt virus</i> (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50–70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.</p></div

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.

<p>(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).</p

Primers and probes used in the described experimental procedures.

<p>Primers and probes used in the described experimental procedures.</p

Co-detection and discrimination of SNI and SRB alleles in artificial mixtures by high resolution melting and custom TaqMan SNP Genotyping assays.

<p>(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.</p

List of isolate name, plant host, and SNP discrimination by sequencing, HRM and Taqman-SNP Genotyping assay.

<p>List of isolate name, plant host, and SNP discrimination by sequencing, HRM and Taqman-SNP Genotyping assay.</p

Discriminative detection of SNI and SRB by HRM and TaqMan SNP assays in single and artificial mixed samples at different dilutions.

<p>Discriminative detection of SNI and SRB by HRM and TaqMan SNP assays in single and artificial mixed samples at different dilutions.</p

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.

<p>(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.</p

Schematic representation of TSWV NSm gene sequence showing primers and probes localization.

<p>(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.</p

Data_Sheet_1_Comparative Genetic Analysis of Durum Wheat Landraces and Cultivars Widespread in Tunisia.xlsx

The durum wheat (Triticum turgidum L. ssp. durum Desf.) landraces constitute a useful natural germplasm to increase the genetic diversity in the modern durum cultivars. The Tunisian durum germplasm constitutes 28 accessions conserved in Genebank of Tunisia, which are still unexplored. In this study, a comparative genetic analysis was performed to investigate the relationships between the Tunisian durum lines and the modern cultivars and detect divergent loci involved in breeding history. The genetic diversity analyses carried out using nine morphological descriptors and the 25K single-nucleotide polymorphism (SNP) array allowed us to distinguish two groups of Tunisian landraces and one of durum cultivars. The analysis of molecular variance and diversity indices confirmed the genetic variability among the groups. A total of 529 SNP loci were divergent between Tunisian durum landraces and modern cultivars. Candidate genes related to plant and spike architecture, including FLOWERING LOCUS T (FT-B1), zinc finger CONSTANS, and AP2/EREBPs transcription factors, were identified. In addition, divergent genes involved in grain composition and biotic stress nucleotide-binding site and leucine-reach repeats proteins and disease resistance proteins (NBS-LRR and RPM) were found, suggesting that the Tunisian durum germplasm may represent an important source of favorable alleles to be used in future durum breeding programs for developing well-adapted and resilient cultivars.</p