DNA Research

Pacific herring (Clupea pallasii) serve as a critical trophic link between plankton and many marine species targeted by fisheries. With a broad distribution throughout the North Pacific Ocean, from the Arctic to temperate latitudes, herring hold ecological, economic, and cultural importance. Despite this importance, genomic resources for this species, such as reference genome sequences, have only recently become available. To date, only one scaffold-level reference genome, representing a specimen from the Gulf of Alaska (Vancouver; 1,379 scaffolds), has been published to NCBI. Addressing this data gap, we produced a high quality 795Mb genome sequence organized into 26 chromosomes combining long read sequencing with short read sequencing of proximity ligation libraries. Our assembly is highly complete (BUSCO score of 97.7%) and contiguous (922 contigs, N50 = 7,338,470, L50 = 38; 26 scaffolds, N50 = 31,494,017; L50 = 12). Pacific herring south of the Aleutian Islands and the Alaska Peninsula are genetically differentiated from those in the Bering Sea, making a reference genome from the eastern Bering Sea an important addition to the Pacific herrings genomic toolbox.

Cnidarians are important models for the studying the evolution of animal development, regeneration, cell type differentiation, and allorecognition. The marine hydrozoan Podocoryna americana is related to the well-established model species Hydractinia symbiolongicarpus. Although both species possess a sessile polyp stage, P. americana differs in that it also has a free-swimming medusa (jellyfish) stage in its life cycle. We used a combination of PacBio CLR long-read and Illumina Hi-C short-read genome sequencing to produce a chromosome-level genome assembly for P. americana. The final assembly is 327 Mbp in total length with 17 chromosome-scale scaffolds representing 98% of the assembly. Comprehensive functional annotation with BRAKER3 generated a total of 19,085 predicted protein-coding genes in this assembly, covering 91.2% of the metazoan BUCSO gene set. Comparison of the P. americana genome to other chromosome-level cnidarian genome assemblies revealed a high degree of macrosynteny conservation, and ortholog identification and gene family evolution analysis identified 522 expanded and 1,026 contracted gene families in P. americana. This high-quality, chromosome-level genome assembly of P. americana will be an invaluable resource for researchers studying the evolution of development, regeneration, and allorecognition in cnidarians and other metazoans.

Cowpea (Vigna unguiculata (L.) Walp.) is a globally important legume crop. However, the scarcity of efficient molecular markers has hindered molecular breeding efforts and the protection of plant breeders rights. In this study, we employed double-digest restriction-site associated DNA sequencing (ddRAD-seq) to characterize the genetic diversity of 19 core cowpea germplasm accessions. We generated 791,621 SNPs, from which 13,469 high-quality SNPs were filtered. Population structure and phylogenetic analyses revealed that these accessions cluster into three distinct groups. To facilitate cost-effective and rapid genotyping, we developed a panel of KASP (Kompetitive Allele Specific PCR) markers. Through rigorous screening for polymorphism and stability, we identified six core KASP markers located in exonic regions. These six markers alone were sufficient to discriminate all 19 accessions. Based on these core markers, we constructed a unique DNA fingerprinting profile and assigned specific QR codes for each accession. This study demonstrates that extracting core KASP markers from ddRAD-seq data is a powerful strategy for germplasm identification. The developed fingerprinting system provides a robust, low-cost tool for seed purity testing, variety authentication, and marker-assisted selection in cowpea breeding programs.

Common buckwheat (Fagopyrum esculentum Moench) is a globally cultivated pseudocereal with a high nutritional quality and economic value. Due to its self-incompatibility, common buckwheat exhibits a high level of heterozygosity, making genome assembly challenging. Consequently, reference-level haplotype-resolved assemblies of common buckwheat are scarce, hindering research and genomics-assisted breeding. Here, we present a near-complete, chromosome-level, haplotype-resolved assembly of a common buckwheat F1 genotype (named Tuka), generated using a trio-binning approach that integrated parental Illumina short-read data with PacBio HiFi and Hi-C data from Tuka. The Tuka assembly comprises two haplomes, Tuka_h1 and Tuka_h2, both showing high contiguity (contig N50 of 76.68 Mb and 84.57 Mb, respectively), high completeness (assembly sizes of 1.28 Gb and 1.23 Gb with BUSCO scores of 96.9% and 96.8%, respectively), high base-level accuracy (QV of 59.08 and 63.03, respectively), and few gaps (35 and 30, respectively). This near-complete assembly of Tuka serves as a valuable genomic resource for common buckwheat, enabling advanced genomic analyses and accelerating research and breeding using state-of-the-art genomic tools.

European pear (Pyrus communis) is the most widely cultivated Pyrus species outside of Asia; however, production has been declining in Europe, Oceania, and North America. Most European pear cultivars are over 100 years old and face pressure from disease, a changing climate, and challenging postharvest characteristics, while demand for increased production efficiency rises. To generate germplasm with desirable characteristics to address these concerns, a mutation breeding approach was chosen, with crosses made between four economically significant cultivars ( Bartlett, dAnjou, Abbe Fetel, and Comice) using gamma-irradiated pollen. This resulted in 49 viable offspring, of which 37 have survived at least 10 years. Nanopore whole-genome sequencing was used to test the success of this approach and screen for variants of interest. Sequence reads were mapped to both a lightweight, purpose-built pangenome derived from assemblies of parental haplotypes and a linear reference genome, enabling the high-quality discovery of variants of all sizes, ranging from single-base substitutions to megabase-scale deletions, with the overwhelming majority being small variants. The overall rate of mutation was 153 novel small variants and 0.228 novel structural variants per Gray of absorbed gamma radiation. Alternate ploidy levels were detected in four lines, which included three triploids and one tetraploid. While the resulting individuals appear incapable of floral development, they may be of utility as rootstock cultivars and a valuable genetic resource for understanding the underlying basis of structural traits.

Garcinia binucao (Blanco) Choisy is an indigenous species endemic to the Philippines. Its fruit is traditionally used as a souring agent in local cuisine and has been reported to possess nutritional and medicinal properties. Despite its ethnobotanical significance and promising bioactive properties, the species remains underutilized. To date, no genomic resources have been published for G. binucao, limiting its application in food systems, genetic studies, and conservation programs. This study reports the first complete chloroplast genome of G. binucao from an accession conserved at the Institute of Plant Breeding, University of the Philippines Los Banos. The assembled plastome is circular with a length of 156,570 base pairs (bp). It displays the typical quadripartite structure of most angiosperms, consisting of a large single-copy (LSC) region (85,357 bp), a small single-copy (SSC) region (17,129 bp), and a pair of inverted repeats (IR), each 27,042 bp in size. A total of 128 genes were annotated, including 83 protein-coding genes, 37 transfer RNAs (tRNAs), and eight ribosomal RNAs (rRNAs), consistent with the majority of Garcinia species. Of the protein-coding genes, 45 are involved in photosynthesis, 28 genes for self-replication, five genes with conserved open reading frames, and five genes are associated with other functions. The GC content was 36.2%. Leucine (10.6%) was the most abundant amino acid, with a codon usage bias toward UUA. Additionally, 98 simple sequence repeats (SSRs) were detected, 88.78% consisting of A/T motifs. Phylogenomic analysis based on assembled plastome and publicly available cpDNA sequences of 17 other species in the order Malpighiales revealed that G. indica is the closest relative of G. binucao. These findings provide a framework for future research on the species, including its conservation and potential use as a genetic resource.

Fusion of gametes possessing meiotically reduced (haploid) chromosome complements is the main pathway of propagation among eukaryotes. However, duckweeds, the smallest angiosperms, propagate mainly vegetatively, and meiosis has not yet been documented in detail for this plant family. The more surprising was the recent evidence of rather frequent interspecific hybrids and triploid clonal accessions which became obvious by genome size measurements, genomic in situ hybridization (GISH) and combined plastid and nuclear DNA markers. These observations indicated sexual propagation involving reduced as well as unreduced male and female gametes in Lemna minor and L. turionifera leading to allodiploid and allotriploid hybrids (MT, MMT, MTT) and autotriploid L. minor (MMM) accessions. Here, we i) documented the meiotic stages of Lemna species for the first time; ii) provided evidence of unreduced male gametes through fluorescent in situ hybridization (FISH) with single locus probes; iii) determined their abundance in different individuals and iv) hypothesized about the reasons of unreduced male gamete formation. These findings open new insights into the modes of sexual reproduction and evolution of duckweeds which may be useful for future breeding efforts in this emerging crop.

We present a genome assembly from a specimen of Quercus canariensis (Fagaceae; Fagales; Magnoliopsida). The assembly was generated using PacBio HiFi long reads with an approximate sequencing depth of 39X and scaffolded using a reference-guided approach. The genome sequence has a total length of 816.0 megabases for haplotype 1 and 804.8 megabases for haplotype 2. The two haplotypes are each resolved into 12 chromosomal pseudomolecules, with only 3.48% and 1.36% of sequences remaining unplaced in haplotypes 1 and 2, respectively. Assembly completeness is supported by BUSCO scores of 98.3% and 98.2% complete genes for haplotypes 1 and 2, respectively. Structural annotation identified 51,882 and 46,482 protein-coding genes in haplotypes 1 and 2, respectively. This genome assembly provides the first chromosome-scale reference genome for Q. canariensis, laying the base for future genomic and evolutionary studies in this understudied species of the hybridizing white oak species complex. TaxonomyLineage cellular organisms; Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae; rosids; fabids; Fagales; Fagaceae; Quercus EBI:txid568684 Quercus canariensis Willd. 1809 (Willdenow)

Natural history and agricultural collections, which contain hundreds of millions of specimens classified in terms of time, space, and taxonomy, are valuable resources for diverse fields of research. Since the first success of ancient DNA (aDNA) isolation in the 1980s, these repositories, including herbaria for plants, have been intensively used to support studies in taxonomy, macroevolution, and genetic responses to anthropogenic activities over the past centuries. Two major challenges of aDNA research are environmental contamination and DNA degradation. For herbarium specimens, aDNA is usually extracted from leaf samples. It is highly fragmented (typically length of 50 to 100 bp) with a higher breakdown rate than that in most bone remains. To optimise the amount of data retrieved and minimise destructive sampling, we isolated DNA from an unconventional plant tissue type - seed embryos. We carried out whole-genome sequencing and compared sequenced DNA quality between embryo and leaf tissue. We evaluated endogenous DNA proportion, median fragment length, damage fraction per site ({lambda}), decay rates, nucleotide misincorporations, and library complexity for three species: cultivated rice Oryza sativa, wild rice O. rufipogon, and wild barley Hordeum spontaneum. In O. sativa, embryos exhibited significantly higher endogenous content and median fragment length than leaves, while in O. rufipogon only median fragment length was higher. The superior DNA preservation was likely due to the protective role of the seed husk, which might play an important role in DNA preservation in plants collected in the tropics. By contrast, in temperate H. spontaneum, tissue type had minimal impact on DNA quality. Despite the minuscule size of the embryos, all derived genomic libraries were highly complex, sufficient for deep whole genome sequencing. These results highlight seed embryos as a promising alternative aDNA source for millions of herbarium specimens, and enable effective genomic analyses of other historical plant collections, such as economic botany and anthropological museum collections.

Characterizing genomic properties such as genome size, ploidy level, heterozygosity, and repetitive DNA proportion and composition without relying on genome assembly is crucial for profiling the genomes of non-model species. Little is known about the nuclear genome of the large neotropical orchid genus Epidendrum. This study compares genome profiles of Epidendrum anisatum and Epidendrum marmoratum, using flow cytometry and k-mer analysis approaches, as well as bioinformatics ploidy level estimation and repeatome characterization. Multiple depths of coverage, k values, and k-mer-based tools for genome size estimation were explored and contrasted with cytometry genome size estimations. Cytometry and k-mer analyses yielded a consistently higher genome size for E. anisatum (mean 1C genome size = 2.59 Gb) than E. marmoratum (mean 1C genome size = 1.13 Gb), which represents a 2.3-fold genome size difference. Both species were identified as diploid with no evidence of strict partial endoreplication. The most important aspects to be taken into account to improve genome size estimation were heterozygosity, depth of coverage, and the maximum k-mer coverage. The genomes of both species were found to be highly repetitive (63-73%) and heavily dominated by Ty3-gypsy retrotransposons, particularly those of the Ogre family. Additionally, the genome of E. anisatum was characterized by the presence of a 172 bp satellite (AniS1), which represented 11% of the genome size. Together, both Ty3-gypsy transposons and AniS1 shape the genome size difference between the two genomes. This study provides the first genome profiling for species in the genus Epidendrum, but also highlights the importance of using flow cytometry, cytogenetic approaches and bioinformatics techniques in combination for genome profiling.

Chloroplast genomes are invaluable resources for plant genomic research, providing insights into genome evolution and molecular adaptation. With the growing scientific and economic interest in Adansonia digitata, a comprehensive characterization of its chloroplast is timely and necessary. A complete chloroplast genome of A. digitata was assembled, annotated, and characterized. Comparative structural analysis was conducted against other Adansonia species, and the assembly was validated through phylogenetic placement within Malvaceae. The assembled genome exhibits the canonical quadripartite organization, spanning 160,061 bp with a GC content of 36.88%, 79 protein-coding genes, 32 tRNAs, and 4 rRNAs. Repeat analysis identified 100 simple sequence repeat motifs, predominantly A/T-rich mononucleotide types (76%), alongside 50 long sequence repeats dominated by forward (26) and palindromic (17) repeats. Comparative analysis with other Adansonia species revealed conserved genome structure, with minor IR boundary shifts involving the ndhF gene, and ycf1 duplication in A. gregorii and A. grandidieri. Average nucleotide identity exceeded 99% across all Adansonia species, with near-complete similarity (ANI {approx} 99.96%) observed with the putative A. kilima. All predicted RNA editing events were nonsynonymous, dominated by C[->]U conversions (55.02%). Codon usage showed non-random synonymous preferences biased toward A/U-ending codons, driven primarily by mutational pressure with detectable gene-specific translational selection. Nucleotide diversity ({pi}) was higher in intergenic spacers (0.00490 {+/-} 0.00574) than in coding regions (0.00167 {+/-} 0.00199), with the majority of genomic regions showing no sequence variation ({pi} = 0). Substitution patterns indicated pervasive purifying selection, with relatively high but insignificant signals in matK, ycf1, accD, and rpoB. Phylogenomic analyses placed the assembled A. digitata chloroplast genome within the Adansonia lineage, consistent with its established systematic position. This study provides detailed insight into the chloroplast genome of A. digitata, and the findings will contribute towards advancing its genomic research.

The common pine sawfly, Diprion pini, is a widespread defoliator of pine forests across Europe and Asia, with outbreaks causing substantial ecological and economic damages. However, genomic resources for this species have been limited, hindering advances in molecular ecology or pest management. Here, we present a near chromosome-level reference genome for D.pini, generated using PacBio HiFi reads, Oxford Nanopore MionION long reads, and 10x Genomics linked reads. The final assembly is organized into mostly chromosome-sized scaffolds. It spans a length of 268 Mb, comprises 81 scaffolds, and has a scaffold N50 of 18.7 Mb. BUSCO analysis (hymenoptera_odb10) indicates a high genome completeness of 97.2%. With 22,7 kb the mitochondrial genome is unusually large due to an extended non-coding control region (6,874 bp). Gene prediction identified 26,335 protein-coding genes, of which 12,769 were functionally annotated. Comparative analyses with other sawflies and Apocrita identified 2,472 proteins unique to D. pini, some of which are putatively associated with the processing of plant secondary metabolites. Notably, our genome assembly highlights that, when a closely related, high-quality reference genome is available, chromosome-scale assemblies can be generated without the need of Hi-C sequencing. The genome provides a valuable foundation for the development of improved monitoring and management strategies for D. pini outbreaks and contributes to advancing fundamental research on Hymenoptera evolution.

Continuous developments in sequencing technologies have led to the generation of chromosome-scale genome assemblies across the whole tree of life, but our ability to annotate genomes has lacked behind. One major problem consists in the fact that typically not all genes are expressed at detectable levels at any given life stage or environment. Therefore, available transcriptome data needs to be complemented by gene prediction programs and protein homology evidence. However, how to optimally combine these different data types is not well understood. Here, we present a case study, where we community curated gene annotations of the Pristionchus pacificus strain RSC011. By incorporation of new Iso-seq and RNA-seq data and genome-wide screening, we identified and corrected more than 7,500 ([~]24%) gene models. While the improved gene annotation for the RSC011 strain will be useful for the P. pacificus community, our study reveals several gene annotation problems that may affect data from other species. Among these, we identified assembly errors, artificial transcript fusions resulting from overlapping genes and polycistronic RNAs, falsely called open reading frames, and error propagation based on homology data as frequent sources of gene annotation errors. Thus, our findings may be helpful in guiding future efforts to annotate genomes across different taxonomic groups.

The acidic blackwaters of Southeast Asias peat-swamp forests represent some of the most extreme freshwater environments on Earth. Despite their very low pH values, limited nutrients, and hypoxic conditions, these blackwater habitats harbor a remarkable diversity of freshwater fishes, including multiple lineages that have independently adapted to these extreme conditions and, in some cases, exhibiting extreme body miniaturization. These replicate evolutionary lineages therefore provide a powerful comparative framework to investigate adaptation to extreme environments and the genomic basis of miniaturization. Here, we present high-quality, annotated reference genomes for four cypriniform species endemic to these peat-swamp forest ecosystems: Paedocypris sp., Sundadanio atomus, Boraras brigittae, and Rasbora kalochroma. The first two are progenetic miniatures, including Paedocypris, comprising the smallest known fish, while B. brigittae represents a proportioned dwarf and R. kalochroma a non-miniature taxon. Genome sizes ranged from 401-1,290 Mb and heterozygosity from 0.34-1.7%. All genome assemblies achieved pseudo-chromosome-level contiguity, high k-mer completeness (>99%), and high BUSCO completeness (94.5-98.9%). Repeat analyses revealed lineage-specific differences in transposable element landscapes and abundances, while gene annotation identified notable intron length reduction in progenetic miniatures.

Choanoflagellate genetics has undergone rapid and impactful developments in the last decade. Currently, the primary method for genetic modification of choanoflagellates relies on proprietary nucleofection reagents to deliver transgenes for ectopic expression or CRISPR-Cas9 ribonucleoprotein complexes for targeted genome editing. The acquisition of proprietary buffers required for nucleofection can hamper advances in choanoflagellate research due to costs, shipping limitations, and restrictions that prevent buffer components from being optimized for understudied organisms. Therefore, we test whether a low-cost in-house electroporation buffer developed for other systems can replace the proprietary buffer currently used for choanoflagellate transfection. Here, we present an in-house buffer with transfection efficiency comparable to that of the previously established proprietary buffer. This work increases the accessibility of choanoflagellate genetics and can broaden research participation in investigating animal origins.

Tropaeolum tuberosum is a tetraploid tuber-forming crop with agroecological and agronomic potential, yet genomic resources for this species remain scarce and limit genetic and functional studies. To address this gap, we generated a reference genome assembly for T. tuberosum using PacBio HiFi reads with an estimated genome size of 418 Mb based on k-mer analysis. The final assembly spans 1.32 Gb with 2,189 contigs (contigs N50 = 32.2 Mb, longest contig = 60 Mb) and recovers 79 % of the estimated genome size. We assessed assembly completeness and accuracy using Benchmarking Universal Single-Copy Orthologs (BUSCO), which detected 98.8 % complete genes (21.6 % single-copy, 77.1 % duplicated), 0.5 % fragmented, and 0.7 % missing, demonstrating near-complete gene space recovery consistent with a high-quality tetraploid reference genome. Repetitive sequences account for 71.6 % of the genome, and we annotated 87,927 protein-coding genes using Helixer. This reference genome assembly represents the first genome-scale resource for T. tuberosum and will enable studies of evolution, domestication and comparative genomics, and support breeding, conservation, and functional genomics in this species and related taxa.

Genetic mosaicism can arise from in vivo CRISPR-Cas9 gene editing, especially in the embryos. This study evaluates the extent of genetic mosaicism resulted from CRISPR-Cas9-mediated knockout for 11 genes in the freshwater microcrustacean Daphnia magna, D. pulex, and D. sinensis. Based on extensive genotyping data of the asexually produced progenies of successfully edited females, we find strong evidence of mosaicism in 9 of these genes. The genotyping data also suggest the gene editing activity can take place as early as the one-cell embryo stage and extends into the 32-cell and later stages. This study establishes genetic mosaicism as an important feature of Cas9-mediated gene editing in Daphnia.

The scaly-sided merganser, Mergus squamatus, is an Endangered piscivorous duck which has been declining since the late 1900s due to habitat loss, over-hunting, and climate change. Despite being a species of global conservation concern and subject to ex- and in-situ conservation efforts, genomic research has been limited, hindering our understanding of its population genetic status and evolutionary history. In this study, we present the first fully annotated, chromosome-level genome for the scaly-sided merganser, generated using Oxford Nanopore long reads, Illumina short reads, and Hi-C sequencing. The final assembly spans 1.1 Gb across 307 scaffolds, 64 of which are anchored into 35 chromosomes, covering 99.5% of the genome. The assembly shows high contiguity (N50 = 84.3Mb) and completeness, with a Benchmarking Universal Single-Copy Ortholog (BUSCO) score of 98%. Repeat sequences comprise 9.55% of the genome. Homology-based gene annotation identified [~]15,200 protein-coding genes. A complete 16,624 bp mitochondrial genome was also assembled and annotated. Synteny analysis revealed strong chromosomal conservation across the wider Anatidae family, with evidence of lineage-specific rearrangements. Pairwise Sequential Markovian Coalescence modelling indicates recent stability in the effective population size of the species, with past declines coinciding with Pleistocene glacial cycles. Our high-quality genome provides an essential resource for conservation genomic and evolutionary studies of the scaly-sided merganser, supporting ongoing efforts to manage and protect this threatened species.

Carmenere is a widely cultivated and internationally recognized grapevine cultivar in Chile, yet genetic variation among its clones remains poorly characterized. Early studies based on SSR and AFLP markers detected limited polymorphism, but these approaches interrogate only a small fraction of the genome, leaving the extent of clonal diversity unresolved. Here, we generated an improved chromosome-scale diploid genome assembly of Carmenere FPS clone 02 and characterized clonal genomic diversity by sequencing 36 biological replicates representing 12 clones maintained in Chile, including heritage selections rescued from old producer vineyards by Vina Santa Carolina as part of its Bloque Herencia conservation program, and commercial nursery-derived clones. Focusing on low-frequency variants and using replicate-aware consensus calling, we identified more than 9,000 private single nucleotide variants (SNVs) and small indels per clone, providing high-resolution markers for clonal identification. Although most variants were located in repetitive or intergenic regions, a subset affected coding sequences, with genes involved in plant-pathogen interactions, transport, and secondary metabolism most frequently impacted. While variant-affected genes associated with wine anthocyanin content, TA, pH, and alcohol percentage were identified, broader phenotypic characterization will be required to assess their biological significance. Overall, this study provides a genome-wide characterization of extant clonal diversity in Carmenere, with implications for clonal selection and genetic resource conservation.

Gene Model for Insulin-like peptide 4 (Ilp4) in the D. simulans DsimGB2 assembly (GCA_000754195.3). The characterization of this ortholog was carried out as part of a larger, ongoing dataset designed to explore the evolution of the insulin/insulin-like growth factor signaling (IIS) pathway across the genus Drosophila, utilizing the Genomics Education Partnership gene annotation protocol within Course-based Undergraduate Research Experiences.