Biology subjects

Jankowicz-Cieslak, J.

Publications and source records attributed to Jankowicz-Cieslak, J..

3 recordsLinked to original sources

bioRxiv2024

Rapid and Sensitive Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 Using a RPA-DETECTR Assay

Early and prompt detection of banana wilt pathogen Fusarium oxysporum f. sp. cubense (Foc), tropical race 4 (Foc TR4), causing global banana crop losses is crucial to curtail the disease spread, minimize damage and implement quarantine measures. We report the first successfully developed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) assay for the highly sensitive and specific detection of Foc TR4, validated across several isolates. Notably, specific crRNA spacer sequences and recombinant polymerase amplification (RPA) primers designed and evaluated for the DETECTR assay exhibited exceptional sensitivity, detecting Foc TR4 with genomic DNA as low as 0.005 ng, and demonstrated a very high specificity, and reproducibility. The RPA-DETECTR assay was validated using a diverse panel of samples, including both target and non-target pathogens confirming its robustness and reliability across different types of samples, ensuring practical application under varied conditions. In terms of throughput, the RPA-DETECTR assays enabled faster detection of Foc TR4 than other molecular diagnostics including PCR, quantitative PCR (qPCR) and droplet digital PCR (ddPCR) analyses, making this assay suitable for point-of-care (POC) detection of Foc TR4, and facilitating rapid decision-making and immediate response. This rapid detection capability is crucial in restricting any potential transboundary movement as part of ongoing disease management efforts.

pathology↗

bioRxiv2020

Deep sampling and pooled amplicon sequencing reveals hidden genic variation in heterogeneous rye accessions

Loss of genetic variation negatively impacts breeding efforts and food security. Genebanks house over 7 million accessions representing vast allelic diversity that is a resource for sustainable breeding. Discovery of DNA variations is an important step in the efficient use of these resources. While technologies have improved and costs dropped, it remains impractical to consider resequencing millions of accessions. Candidate genes are known for most agronomic traits, providing a list of high priority targets. Heterogeneity in seed stocks means that multiple samples from an accession need to be evaluated to recover available alleles. To address this we developed a pooled amplicon sequencing approach and applied it to the out-crossing cereal rye (Secale cereale). Ninety-five rye accessions of different improvement status and worldwide origin, each represented by a pooled sample comprising DNA of 96 individual plants, were evaluated for sequence variation in six target genes involved in seed quality, biotic and abiotic stress resistance. Seventy-four predicted deleterious variants were identified using multiple algorithms. Rare variants were recovered including those found only in a low percentage of seed. A large extent of within-population heterogeneity was revealed, providing an important point for consideration during rye germplasm conservation and utilization efforts. We conclude that this approach provides a rapid and flexible method for evaluating stock heterogeneity, probing allele diversity, and recovering previously hidden variation.

genomics↗

bioRxiv2018

Fragmentation of pooled PCR products for highly multiplexed TILLING

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING, where whole genome approaches become cost prohibitive. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multidimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Thirty-two PCR products were produced from genomic DNA pools representing 265 pooled barley mutant lines. Mutant lines were produced with the chemical mutagen ethyl methanesulfonate. Samples were subjected to 2x300PE illumina sequencing. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach.

plant biology↗