SkIf: a tool for rapid identification of genes or regulators of interest

Briand, Martial; Gaborieau, Romain; Jacques, Marie‐Agnès; Barret, Matthieu; Boureau, Tristan; Gaillard, Sylvain; Wg, Nicolas Chen

doi:10.7490/f1000research.1112490.1

Cited by 4 publications

(1 citation statement)

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“…Specific sequences of clades and clusters were searched for in the genomic sequences using the SkIf_with_DSK tool (Briand et al 2016; https://iris.angers.inra.fr/galaxypub-cfbp).…”

Section: Identification Of Targets and Primer Designmentioning

confidence: 99%

Development of tools to detect and identify strains belonging to thePseudomonas syringaespecies complex responsible for vein clearing of zucchini

Lacault

Briand

Jacques

et al. 2023

Preprint

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Vein clearing of zucchini (VCZ) is a seed-borne bacterial disease that affects young plants of Cucurbita pepo subsp. pepo. VCZ agents are distributed into four phylogenetic clusters within the clades 2a and 2ba of the phylogroup 2 of Pseudomonas syringae species complex. Strains belonging to clades 2b and 2d are sometimes isolated from zucchini seeds but have not been associated with VCZ epidemics. Development of tools able to identify VCZ agents is important to better control the disease. Primers were designed to implement a seven-gene MLSA scheme on a collection of strains isolated from zucchini seeds. A clear predominance of strains with a host range on cucurbits limited to the genus Cucurbita (cluster 2ba-A) and the presence of VCZ strains in a fifth cluster (2ba-C) were evidenced. PCR tests were designed to characterize VCZ clusters and a multiplex qPCR test was proposed to distinguish strains having narrow cucurbit host range, associated to the presence of avrRpt2 and sylC, from broad host range strains associated to the presence of hopZ5 and sylC. Additional qPCR tests targeting clade 2b and 2d were also designed to gain insights on P. syringae strains that could be isolated from cucurbits. Specificity of these tools was evaluated in silico on the whole NCBI database and in vitro on a strain collection, showing a 100% inclusivity, except for the test dedicated to clade-2b strains, and an exclusivity ranging from to 96.7% to 100%. These different tools are intended to serve phylogenetic studies, epidemiological monitoring and seed testing.

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“…Specific sequences of clades and clusters were searched for in the genomic sequences using the SkIf_with_DSK tool (Briand et al 2016; https://iris.angers.inra.fr/galaxypub-cfbp).…”

Section: Identification Of Targets and Primer Designmentioning

confidence: 99%

Development of tools to detect and identify strains belonging to thePseudomonas syringaespecies complex responsible for vein clearing of zucchini

Lacault

Briand

Jacques

et al. 2023

Preprint

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Novel tetraplex qPCR assays for simultaneous detection and identification ofXylella fastidiosasubspecies in plant tissues

Dupas

Briand

Jacques

et al. 2019

Preprint

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AbstractXylella fastidiosa is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex qPCR assays for Xylella fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a dataset of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 103 cells.mL−1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.

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Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues

et al. 2019

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Xylella fastidiosa (Xf) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 10 3 cells.ml −1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.

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SkIf: a tool for rapid identification of genes or regulators of interest

Cited by 4 publications

References 0 publications

Development of tools to detect and identify strains belonging to thePseudomonas syringaespecies complex responsible for vein clearing of zucchini

Development of tools to detect and identify strains belonging to thePseudomonas syringaespecies complex responsible for vein clearing of zucchini

Novel tetraplex qPCR assays for simultaneous detection and identification ofXylella fastidiosasubspecies in plant tissues

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues

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