DNA Research

As an important cultivated red alga, Gracilariopsis lemaneiformis has great economic and ecological value. However, its existing genome assembly is highly fragmented and inadequately annotated. In this study, we constructed the first high-quality chromosome-level genome of Gp. lemaneiformis using PacBio long reads, Illumina short reads and Hi-C sequencing data. The assembled genome was approximately 86.66 Mb and the assembled sequences were anchored to 28 pseudo-chromosomes with lengths ranging from 1.70 to 7.81 Mb. 99.91% of the PacBio reads could be mapped to our assembly. In total, 8,664 genes were annotated, and the repeat elements identified in Gp. lemaneiformis constituted 65.04% of the whole genome, including 2.24% tandem repeat sequences and 62.81% interspersed repeats. We also established a high-evidence phylogenetic tree from 19 representative algae species, with the main aim to calculate their divergence times. This high-quality genome of Gp. lemaneiformis provides a crucial foundation for understanding genetic characteristics, investigating the genomic evolution, and facilitating molecular breeding.

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.

The Japanese quail (Coturnix japonica) is a widely used model organism in developmental biology, genetics, and agriculture. Here, we present new, haplotyped, high-quality genome assemblies of the Japanese quail, generated using a combination of state-of-the-art sequencing technologies, including PacBio HiFi long reads, Oxford Nanopore sequencing, and Hi-C scaffolding. This assembly has a total length of 1.19 Gb, 80% of which is included in chromosomes, and is highly complete (BUSCO score aves_odb10: 97.3). Assembly metrics show a marked improvement in contiguity, with a significantly higher scaffold N50 and a lower number of contigs compared to the reference genome assembly. Remarkably, the assembly extends previously truncated chromosome ends, with 31 telomeres detected. In addition, we merged the existing Ensembl and Refseq annotations and obtained a combined set of 26,102 genes, of which 25,038 genes were successfully mapped on the improved assembly haplotype 1 (Cjap1.hap1). Together, these new genome assemblies and their enriched annotation provide a robust genomic framework for future research. They enhance our ability to investigate developmental processes, genetic and epigenetic inheritance, and host-pathogen interactions. Furthermore, they offer valuable insights for conservation genetics and sustainable breeding programs. This resource represents a critical step forward in leveraging the full potential of the Japanese quail as a model species in both basic and applied research.

As one of the earliest-diverging multicellular eukaryotic lineages, the bladed Bangiales (Rhodophyta) possess a deep evolutionary history with a central role in the multi-billion-dollar global seaweed aquaculture industry. Although North Atlantic representatives are emerging candidates for regional mariculture, the scarcity of high-quality genomic resources for these taxa hinders both fundamental research and commercial optimization. To address this, we present the first chromosome-level genome assemblies for two native European species: Porphyra dioica (150.44 Mbp) and Porphyra linearis (95.22 Mbp). By integrating Oxford Nanopore Technologies (ONT) long-read sequencing with Hi-C proximity ligation, we generated highly contiguous nuclear genomes resolved into five chromosomes. Structural gene models were predicted through the BRAKER3 pipeline, identifying 12,548 and 10,382 protein-coding genes for P. dioica and P. linearis, respectively. Subsequent homology-based functional annotation characterized 57.4% and 59.8% of these predicted proteins. Supplemented by circularized organellar genomes, these reference genomes provide a critical framework for future research, enabling comparative studies of Atlantic-Pacific divergence and facilitating the development of selective breeding programs for sustainable European aquaculture.

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.

The worlds largest and rarest eagle, the Philippine Eagle (Pithecophaga jefferyi), also known as the monkey-eating eagle, is the national bird of the Philippines. This raptor species is endemic to the Philippine archipelago, with populations on the islands of Luzon, Leyte, Samar, and Mindanao. It is critically endangered, with an average estimated population of 392 potentially breeding pairs or 784 mature individuals. In this paper, we describe a reference genome of the Philippine Eagle (Pithecophaga jefferyi) from a female juvenile from the province of Nueva Ecija on the island of Luzon. We generated a de novo genome assembly with high contiguity and completeness, comprising 178 contigs totaling 1.345 Gbp. The genome was sequenced at a coverage of 75.2x, and Benchmarking Universal Single-Copy Orthologs (BUSCO)/Compleasm analysis yielded a BUSCO score of 99.92% (aves_odb12), corresponding to 99.7% single-copy, 0.21% duplicated, and 0.08% fragmented genes. A consensus mitogenome sequence of 19,377 bp was also generated. The genome assembly included 23,847 putative genes, and our annotation estimated that 15.78% of the genome consisted of repetitive elements. Genome heterozygosity (H) was estimated to be 0.020%, in comparison to other birds with genome heterozygosity values ranging from 0.0103% to 0.923%. Whole-genome comparisons with publicly available genomes suggest that the Philippine eagle belongs to the snake-eagle subfamily (Circaetinae) rather than the harpy-eagle subfamily (Harpiinae). Pairwise sequentially Markovian coalescent (PSMC) analysis suggests that the effective population size was around 4,000 individuals from about 100 KYA to about 1 KYA. Finally, we constructed a minimum spanning network, which revealed that our mitogenome from the northern island of Luzon occupies a peripheral position, separated from the dominant haplotype cluster found in the southern island of Mindanao by multiple mutational steps, indicating substantial mitochondrial divergence.

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.

Holcosus orcesi, the Orces Blue Whiptail, is a Critically Endangered lizard endemic to the upper Jubones River basin in southern Ecuador. Restricted to a narrow elevational range within semi-arid Andean shrublands, it represents one of the few montane members of a predominantly lowland lineage. Here we present the first high-quality reference genome for H. orcesi, generated using Oxford Nanopore Technologies long-read sequencing. The assembly spans 1.68 Gb across only 91 contigs, with an N50 of 76.2 Mb and a BUSCO completeness of 96.8%, making it among the most contiguous and complete squamate genomes to date. Structural annotation predicted 25,682 genes, of which 85% showed homology to known proteins and 45% were assigned Gene Ontology terms. Repetitive elements accounted for 46.3% of the genome, with LINEs representing the predominant class. This genome provides a foundational resource for future evolutionary, comparative and conservation-genomic research of H. orcesi and other mountain reptiles, enabling studies of population genomics, local adaptation, and genomic erosion in isolated populations. By expanding the genomic representation of tropical montane reptiles, this work helps address longstanding phylogenetic and geographic gaps in global biodiversity genomics and provides a foundation for evidence-based conservation of H. orcesi and related taxa.

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.

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)

Gene model for the ortholog of Lst8 (Lst8) in the May 2011 (WUGSC dyak_caf1/DyakCAF1) Genome Assembly (GenBank Accession: GCA_000005975.1) of Drosophila yakuba. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

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.

Wide hybridization between related species and genera provides valuable opportunities for broadening genetic diversity and introducing desirable traits. In the tribe Maleae (Rosaceae), Malus (apple) and Pyrus (pear) are phylogenetically closely related, and apple x pear hybrids represent promising materials such as for disease-resistance breeding. However, the effective utilization of such hybrids in breeding programs is constrained by long juvenile period. In this study, we established a tissue culture-based regeneration and genetic transformation platform for apple x pear hybrids. Key stages affecting adventitious shoot regeneration were optimized, and appropriate ranges of antibiotic selection pressure and bacterial elimination conditions were systematically evaluated. Regeneration capacity was predominantly genotype-dependent and became further restricted under Agrobacterium infection, necessitating precise balancing between regeneration competence and selection pressure. Using the highly competence line and the established transformation system, MdFT1 gene was successfully introduced and over-expressed in intergeneric hybrids, resulting in transgenic plants exhibiting floral bud initiation approximately six months after infection under in vitro conditions. This study provides a practical and efficient regeneration-transformation framework for apple x pear hybrids and demonstrates its applicability for FT-mediated early flowering. The established system offers technical support for accelerated breeding strategies and facilitates the utilization of novel resources in genetic improvement of pome fruit.

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.

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.

The sugar kelp Saccharina latissima is a promising candidate for sustainable aquaculture in the North Atlantic and North-East Pacific but genetic improvement has been hindered by limited understanding of the genetic basis of economically important traits. We conducted the first genome-wide association study (GWAS) for this species using 202 self-fertilised pseudo-F1 individuals derived from 12 populations spanning northern and southern European genetic clusters. Individuals were genotyped with ddRAD-seq-derived SNP markers and phenotyped in a common garden experiment for four morphological traits (blade length, blade width, blade area, stipe length) and six metabolic traits related to nitrogen metabolism. We identified 26 significant marker-trait associations, with phenotypic variance explained (PVE) ranging from 0.65% to 52.44%. Major-effect loci were detected for blade width (52.44% PVE) and blade area (45.22% PVE) and a locus on chromosome 17 influenced both blade length and blade area. Marker-based heritability estimates ranged from 0.75 to 0.99 for morphological traits and from 0.00 to 0.99 for metabolic traits, though with large standard errors. Cross-validation of genomic selection models yielded predictive abilities of 0.21-0.59 across traits. Our findings reveal a mixed genetic architecture with major-effect loci suitable for marker-assisted selection and polygenic traits amenable to genomic selection, providing a foundation for genomics-assisted breeding programs in kelp aquaculture.

The nematode Caenorhabditis elegans was the first metazoan to have its genome completely sequenced and assembled. Since that time, researchers have continuously updated the reference genome and manually curated its approximately 20,000 genes. The closely related species, Caenorhabditis briggsae, has served as a comparative model because of its similar morphology, mode of reproduction, and patterns of intra-species genetic variation. However, the genomic resources for C. briggsae lag behind C. elegans, hindering comparative genomics studies between the species. Decades of experimentation have been performed in the AF16 reference strain genetic background, so we obtained high-coverage long-read sequencing and high-throughput chromosome conformation capture data to create an updated reference genome for an isogenic derivative of AF16, named CGC2. The CGC2 genome is vastly improved relative to the existing AF16 assemblies, with no unplaced sequence, no gaps, and telomere-to-telomere contiguity. To provide genomic resources for CGC2, we exploited deep RNA-seq libraries from all developmental stages to predict protein-coding gene annotations comparable in accuracy and completeness to the existing AF16 gene models. We also performed lift-over of 108 validated insertion-deletion variants to the updated coordinate system of the CGC2 genome to facilitate future mappings of mutations. In summary, we present an updated reference genome for the canonical AF16 reference strain with improved genomic resources to enable high-quality intra- and inter-species comparative studies.

Nanopore sequencers have the potential to liberate DNA sequencing from centralized core facilities to distributed analytical nodes. Until now, Oxford Nanopore Technologies (ONT) has been the sole manufacturer of a portable nanopore sequencer, but analogous platforms are in production. Nanopore sequencers from Qitan Technology (QT) are widely used in China but have been unavailable outside that nation and lack independent performance testing. Enabled by early access to QTs least expensive sequencer and flow cell, the QNome-3841 and QCell-384, we tested whether they could generate accurate DNA barcodes cost-effectively. In several tests involving amplicon pools from 95 to 9,120 specimens, QT recovered valid DNA barcodes from nearly as many specimens (98%) as ONT. QT sequences had slightly lower fidelity than their ONT counterparts and QT frequently failed to resolve the correct length of G/C homopolymers. However, barcode sequences from the two platforms were nearly indistinguishable after bioinformatic treatment. QTs wash kit performed well, enabling a QCell to sequence eight amplicon pools with zero carryover between runs and minimal degradation of the flow cell. Its ultra-fast protocol allowed library preparation in a single step that could be completed in 15 minutes, but this came at the cost of lower quality data. Once widely available, QT devices will be well-suited for supporting DNA barcode analysis.

Museum collections of Coleoptera contain genetic material of potential interest to biotechnology, and non-destructive DNA extraction enables the preservation of important specimens with concomitant release of mitochondrial and genomic DNA. Mini-barcoding of regions of the mitochondrial cytochrome oxidase subunit I (MT-COI) gene helps identify and eliminate known species from further investigation. Here we identify a novel luciferase gene, using Consensus Degenerate Hybrid Oligonucleotide (CODEHOP) primers targeting the region of the luciferase gene spanning the fourth exon, intron, and fifth exon to detect luciferase gene content and eliminate samples containing known luciferase sequences. Biotinylated luciferase gene probes from the firefly Photinus pyralis enabled the enrichment of potential luciferase gene fragments for next-generation sequencing. A bioinformatic analysis suite was then used to identify a luciferase gene sequence from a previously unidentified firefly originally collected in Costa Rica in 2012. We demonstrate that this newly discovered luciferase, termed CRLuc, catalyses a bioluminescent reaction and we determined its emission spectra, Km for the substrates ATP and D-luciferin, and pH stability.

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.