Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.06.02.657502

Oligo-FISH Validates Genome Assemblies and Delivers the Most Precise Karyotype for Lens Mill. Species

Silvestrini, A. J. A.·Ramsay, L.·von Wettberg, E. B.·Bett, K.
View original source ↗Export metadata ↓

Abstract

Chromosome structural rearrangements play a significant role in karyotype evolution and speciation. These rearrangements pose challenges for precise karyotyping, leading to asymmetric chromosomes and complicating the assembly of a genus pan-genome for crops and their wild relatives. Lens culinaris, an important cool-season legume primarily cultivated in India and Canada, is the cultivated species among six wild relatives. All seven species of Lens face significant challenges due to chromosomal rearrangements, ranging from introgression issues to difficulties in developing a precise karyotype and advancing genomic studies. Using the gene synteny analysis between the cultivated Lens species and six wild relatives, we developed cross-species oligo-FISH (Fluorescent in situ hybridization) probes aiming to further attest to the genome assembly and synteny analysis of Lens species. Roots of seven Lens spp. accessions were harvested and used for chromosome spread preparations. Those slides were then used for Oligo-FISH experiments, where the DNA present in the slides was denatured, and a set of red and green oligo probes was hybridized to the chromosomes. Pictures were taken using a fluorescence microscope. The combination of both oligo sets/probes resulted in a distinct pattern for each Lens spp. chromosome, allowing the inference of the most precise karyotype to date for six Lens species. The number of oligo probe signals reflects the species phylogenetic proximity, while the distribution of those signals changed drastically within the same gene pool. The karyotyping of Lens confirmed the proper assignment of chromosomes in the genome assemblies and validated the rearrangements detected in the synteny analysis. Differences in the assembly probe prediction and the oligo-FISH results were used to improve the assemblies. The results attest to a higher sequence-level similarity among the closest related species despite the occurrence of several structural changes among them. The oligo-FISH probes can be used in conjunction with plant genome assembly projects, supporting the delivery of a precise representation of their physical chromosomes.

Explore related subjects

genomicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Silvestrini, A. J. A., Ramsay, L., von Wettberg, E. B., Bett, K.. 2025-06-04. Oligo-FISH Validates Genome Assemblies and Delivers the Most Precise Karyotype for Lens Mill. Species. https://doi.org/10.1101/2025.06.02.657502

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Integrative Nanopore and Illumina sequencing reveals age-associated tRNA modification and CCA-tail dynamics in yeast

Aging is characterized by a progressive loss of proteostasis. Transfer RNAs (tRNAs) are essential regulators of translation, yet their dynamics during aging remain poorly understood due to challenges in sequencing highly modified RNAs. Here we present a benchmarked Nanopore direct RNA sequencing (RNA004 chemistry) resource that profiles the Saccharomyces cerevisiae tRNAome during replicative aging at single-molecule resolution. Using in vitro transcribed tRNA controls, we establish modification detection thresholds and validate key findings with orthogonal Illumina sequencing. While overall tRNA abundance remains largely stable, our resource reveals age-associated terminal A cleavage at the 3' CCA tail of mature tRNAs, targeted T-loop and anticodon modification changes, and single-molecule evidence of modification co-occurrence. This dataset provides a resource for exploring tRNA regulation, translation fidelity, and longevity.

genomics↗
bioRxiv2026

A hydrogen-producing mitochondrion in an anaerobic eukaryotrophic rhizarian

Diverse eukaryotes thrive under low oxygen conditions, in part through highly modified mitochondrion-related organelles (MROs) that use alternate metabolic pathways to support ATP production and cofactor recycling. Anaerobic lifestyles have evolved repeatedly across the eukaryotic tree of life, each providing an independent opportunity to understand how eukaryotes adapt to life in low oxygen conditions. Here, we use single-cell transcriptomics to reconstruct the MRO metabolism of PCE SSF, a benthic eukaryotrophic flagellate and the first cultivated representative of Novel Clade 12 (NC12; Rhizaria), an independently anaerobic rhizarian lineage. PCE SSF possesses an anaerobic hydrogen-producing mitochondrion capable of hydrogenosome-type substrate-level phosphorylation. It also retains a nearly complete but likely branched tricarboxylic acid pathway that lacks citrate synthase and malate dehydrogenase. The function of citrate synthase may instead be fulfilled by the typically cytosolic ATP citrate lyase, previously reported in this context only in the anaerobic cercozoan, Brevimastigomonas motovehiculus. Unlike B. motovehiculus, however, PCE SSF retains only Complex II and the NuoE/NuoF subunits of the electron transport chain and lacks a mitochondrial genome. Together, these features indicate an atypical and reduced mitochondrial metabolism, highlighting the diversity of evolutionary solutions to anaerobic energy metabolism in eukaryotes.

genomics↗
bioRxiv2026

Targeted CRISPRi screening reveals unexpected resilience across the RNA polymerase III transcriptome

Increased RNA polymerase III (Pol III) activity and tRNA abundance are widely linked to cancer cell growth, yet the functional requirement for individual Pol III genes and core components remains unclear, in part due to the difficulty of achieving gene-specific perturbation of highly conserved loci. Here, we developed an inducible CRISPR interference platform and a custom single-guide RNA (sgRNA) library enabling gene-specific targeting of Pol III-transcribed genes and Pol III machinery. Genome-wide screening identified several Pol III dependencies in diploid fibroblasts and HEK293T cells, including multiple initiator methionine tRNA genes among the strongest fitness dependencies. Unexpectedly, glioblastoma models remained largely insensitive to repression of both individual Pol III genes and core Pol III components, despite efficient target repression. These findings establish a general strategy for gene-specific interrogation of conserved Pol III genes and indicate that glioblastoma models tolerate extensive perturbation of Pol III genes and machinery.

genomics↗