Pavel Beran

Xanthomonas arboricola pv. juglandis ( Xaj ) causes walnut bacterial blight (WBB), significantly impacting walnut ( Juglans regia L.) production. This work aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, and specific detection of Xaj . Utilizing next-generation sequencing data, we identified a unique 524 nt region in Xaj and designed three primer sets. The Xaj_K13041 primer set demonstrated 100% specificity, detecting Xaj DNA at concentrations as low as 0.1 pg/µL (2 copies per tube) within 40 min, outperforming existing PCR methods. The LAMP assay was validated on DNA from infected walnut fruits, showing good performance compared to established methods and effective detection for future field conditions. The assay’s reliability, speed and low cost make it suitable for phytosanitary services. This work presents the first LAMP-based assay for Xaj detection, providing a valuable tool for early and accurate identification, essential for managing WBB and ensuring stable walnut crop production.

Motivation: Efficient and collision-free hashing of DNA sequences is essential for accuracy and performance in bioinformatics applications such as genome assembly, sequence alignment, and metagenomic classification. Traditional hashing methods often result in collisions, impacting the precision and/or performance of downstream analyses. Thus, it is highly advantageous to have hashing functions that guarantee collision-free mappings for DNA sequences, particularly for k-mers up to length 16, where practical limits for 32-bit hashing are reached. In this study, we evaluate genCRC32 as a hashing primitive, reporting collision behavior, bucket balance, sensitivity to single-base changes, and speed to inform its potential use in downstream tools. Evaluation within specific software tools is outside the scope of this paper and is planned as future work. Results: We present genCRC32, an innovative hashing method that integrates a straightforward preprocessing step (gen32) with CRC32 hashing, specifically identifying eight CRC32 polynomials that ensure collision-free hashing for all DNA k-mers up to 16 nucleotides in length. Through extensive empirical evaluations, genCRC32 demonstrated zero collisions for these k-mers, achieving a one-to-one mapping without auxiliary data structures. Benchmark tests confirmed minimal computational overhead introduced by preprocessing, maintaining hashing performance comparable to established methods such as MurmurHash3 and xxHash32. Availability and implementation: The source code for genCRC32 is publicly available at: https://github.com/berybox/genCRC32. The implementation is provided in Go (version 1.24) and leverages only standard libraries, ensuring portability and ease of integration into existing bioinformatics workflows.

Edible insects, particularly Tenebrio molitor (Linnaeus) (mealworm) and Zophobas morio (Fabricius) (superworm), have drawn increasing attention as alternative protein sources. This study aims to develop an accurate molecular detection method for T. molitor, an EU-approved food species, and to differentiate it from Z. morio, which remains unapproved for human consumption in the EU. The process enables precise and sensitive identification methods by optimizing singleplex and duplex PCR techniques targeting 16S rRNA and COI gene regions. The DNA of T. molitor was detected in various food matrices, including pastries, chocolate, and porridge, while avoiding cross-reactivity with Z. morio, Gryllus asimilis, and Locusta migratoria. The detection limit for both singleplex and duplex PCR was 10 pg of DNA, ensuring robustness against inhibitory effects from complex food matrices. The developed approach ensures reliable detection and compliance with EU regulations regarding insect-based foods, providing a critical tool for food authentication and preventing adulteration. The key advancements of this approach lie in its improved specificity and sensitivity, allowing for the ability to detect complex food matrices. An applied perspective was evaluated using real commercial food products.

Microsatellite markers were used for the assessment of genetic diversity and genetic structure in a germplasm collection of yellow mustard, Sinapis alba L. The comprehensive collection of genetic resources represented 187 registered varieties, landraces, and breeding materials. Microsatellites generated 44 polymorphic alleles in 15 loci. Eleven of them were medium to highly polymorphic, and the high levels of observed heterozygosity (0.12–0.83) and Nei’s gene diversity index (0.11–0.68) indicated a high level of polymorphism. Based on PCoA and neighbor joining analyses, the genetic resources were divided into two groups. The range of genetic dissimilarity in the analysed collection was in the range of 0.00–1.00. The high level of dissimilarity between the accessions was documented by the high WAM value (33.82%). Bayesian clustering algorithms were performed in the STRUCTURE 2.3.4 software. The number of clusters was estimated at K = 2. The accessions were classified according to Q1/Q2 values. The low average values of the parameters Fst_1 (0.3482), Fst_2 (0.1916), and parameter alpha (0.0602) indicated substantial mating barriers between varieties and reproductive isolation due to the limited exchange of genetic resources between breeders. These results demonstrated the importance of extensive collections of genetic resources for the maintenance of genetic diversity and indicated considerable genetic differentiation among accessions.

Xanthomonas euvesicatoria and X. vesicatoria are two economically important causal agents of bacterial spot (BS) of tomato and pepper. Management of BS in the field requires rapid and accurate detection. Therefore, this work aimed to develop a pipeline to design a simple, fast, and reliable assay for the detection of X. euvesicatoria and X. vesicatoria by loop-mediated isothermal amplification. In total, 109 publicly available whole genomic sequences of 24 different species of bacterial pathogens were used to design primers that would amplify the DNA of the two target species. Laboratory testing of the assay was performed on pure bacterial cultures and artificially infected plants, and amplification was conducted with both a sophisticated laboratory instrument and a simple mobile platform. The testing of the assay confirmed its specificity with a sensitivity reaching 1 pg µl⁻¹ for both pathogens with an assay duration of 40 min on a mobile detection platform. Our diagnostics development pipeline enables the easy and fast design of a reliable detection assay in the genomics age. By validating the pipeline with X. euvesicatoria and X. vesicatoria pathogens, we have simultaneously developed an assay with high specificity, sensitivity, and speed, which will allow it to be deployed, contributing to successful management of BS.

Searching for amino acid or nucleic acid sequences unique to one organism may be challenging depending on size of the available datasets. K-mer elimination by cross-reference (KEC) allows users to quickly and easily find unique sequences by providing target and non-target sequences. Due to its speed, it can be used for datasets of genomic size and can be run on desktop or laptop computers with modest specifications. KEC is freely available for non-commercial purposes. Source code and executable binary files compiled for Linux, Mac and Windows can be downloaded from https://github.com/berybox/KEC. Supplementary data are available at Bioinformatics online.

Summary: ABIF Manager is simple application for low-level access to ABIF formatted files. Those are produced by Data Collection software based on the data, collected from capillary sequencing machine. The application shows all the data as readable text, which can be easily edited. Availability and implementation: The program source code and compiled executable is freely available at: https://sourceforge.net/projects/abifmanager/ Contact:pavel.beran@centrum.cz