Fiber cell initiation and development affect cotton fiber yield and quality. Cotton fiber develops from the ovular epidermis of a seed, and approximately 25% to 30% of protodermal cells in each cotton ovule develop into fiber. However, the molecular basis for fiber cell development remains elusive. Here, we analyzed single-cell RNA-seq (scRNA-seq) data from over 40,000 cells during early stages of fiber cell development in Upland and Pima cotton and in a naked seed mutant. We found concerted expression changes of 900 to 1,700 genes in fiber-cell clusters involving gene expression, translation, and peptide biosynthesis, which were substantially delayed or absent in the mutant. Expression of ∼500 and ∼300 genes in Upland and Pima cotton, respectively, was distinguishably different and consistent with overrepresentation of the genes in transcriptional and translational regulation, implying their roles in fiber yield and quality traits. Gene coexpression network analysis of scRNA-seq along with scATAC-seq data revealed two modules of fiber gene coexpression networks. One module of the fiber coexpressed genes was associated with elevated chromatin accessibility for transcriptional regulation, whereas the other module of the genes was related to translational regulation and ribosome biogenesis. Indeed, expression of cotton putative translation factor genes was elevated in fiber cell clusters in both Upland and Pima cotton. Finally, cotton transgenic plants expressing promoter::GFP confirmed expression patterns of fiber-expressed GhRDL2_D5 during fiber cell initiation. These single-cell genomic resources provide insights into fiber cell development for breeding and biotechnological improvement of fiber yield and quality in Upland and Pima cotton.

Management of the reniform nematode (Rotylenchulus reniformis Linford and Oliveira) in upland cotton (Gossypium hirsutum L.) continues to be a challenge primarily due to inconsistent performance of resistant genotypes. We hypothesize that diversity in reproduction and virulence in R. reniformis has contributed to the inconsistent performance of resistant cotton genotypes. Rotylenchulus reniformis isolates from Tennessee (TN), Alabama (AL), Mississippi (MS), Texas (TX), Louisiana (LA), Arkansas (AR), Florida (FL), South Carolina (SC) and Georgia (GA) were assessed for their reproduction and virulence on a resistant cultivar DP 2141NR B3XF and a susceptible cultivar DP 2317 B3TXF. The AR isolate had the greatest reproduction factor (Rf) of 19.7, while the AL isolate had the least Rf of 3.3. Rfs of other isolates were intermediate. Up to 85% of reproductions were suppressed by the resistant cultivar relative to the susceptible cultivar. While plants inoculated with nematodes had lower root biomass than the non-inoculated control, the degree of root biomass reduction varied by isolate, suggesting differing levels of virulence among R. reniformis isolates. The AR isolate reproduced the most and was the most virulent. The AL isolate reproduced the least and was less virulent. The AR, MS, TN and LA isolates were the top four reproducing isolates, probably indicating a greater suitability of the Delta region for nematode reproduction. The correlation analysis indicated that up to 85% variation in plant biomasses was explained by nematode reproduction. These results suggest that resistance breeding programs in cotton must consider the diversity in R. reniformis.

Winter cover crops are commonly used as a source of nutrients in organic production. However, the impact of these crops on soil microbial functional traits at the genomic level is poorly understood.Here, we investigated the impacts of three-year inclusion of cereal rye (Secale cereale L), hairy vetch (Vicia villosa), and their mixture on soil microbial diversity and residue decomposition functional traits using 16 S rRNA gene and internal transcribed spacer (ITS) sequencing data and functional prediction tools. Integrating a mixture of rye and vetch increased bacterial richness and alpha diversity but had no impact on fungal diversity when compared to the control. The fungal community structure of the soils with either rye or vetch inclusion was significantly different from that of the mixture and the control. Compared to vetch, rye inclusion was predicted to have a significantly higher abundance of C1 compound utilization and assimilation pathways, but a lower abundance of amino acid degradation and carboxylic acid degradation pathways. Similarly, rye inclusion resulted in a significantly higher abundance of arbuscular mycorrhizae but a lower abundance of litter saprotrophs than vetch inclusion. Particularly, integrating vetch was predicted to have a significantly higher abundance of genes involved in cellulose and lignin decomposition than the rye inclusion. Five keystone cellulose and lignin bacterial decomposers were significantly more abundant with vetch inclusions than with rye inclusions. Nonlegume and legume winter cover crops imposed distinct effects on soil potential microbial C-cycling functional traits after three planting seasons. Legumes favored the growth of microbial decomposers compared to non-legumes.

The increasing prevalence of vector-borne diseases around the world highlights the pressing need for an in-depth exploration of the genetic and environmental factors that shape the adaptability and widespread distribution of mosquito populations. This research focuses on Culex tarsalis, a principal vector for various viral diseases including West Nile Virus. Through the development of a new reference genome and the examination of Restriction-Site Associated DNA sequencing (RAD-seq) data from over 300 individuals and 28 locations, we demonstrate that variables such as temperature, evaporation rates, and the density of vegetation significantly impact the genetic makeup of Cx. tarsalis populations. Among the alleles most strongly associated with environmental factors is a nonsynonymous mutation in a key gene related to circadian rhythms. These results offer new insights into the mechanisms of spread and adaptation in a key North American vector species, which is poised to become a growing health threat to both humans and animals in the face of ongoing climate change.

BACKGROUND:A nearly complete genome assembly consisting of 14 scaffolds, a total length of 969.6 Mb, and an N50 scaffold length of 99.88 Mb, was generated to better understand how transposable element activity has led to adaptive evolution in Bassia scoparia (kochia), an agronomically important weed. RESULTS:The nine largest scaffolds correspond to the nine chromosomes of the close relative, Beta vulgaris. From this assembly, 54 387 protein-coding gene loci were annotated. We determined that genes containing Far-Red Elongated Hypocotyl 3 (FHY3) or Far-Red Impaired Response 1 (FAR1) functional domains have undergone a large, kochia-specific gene family expansion. We discovered that putative Mutator Don-Robertson (MuDR) transposable elements with detectable FHY3/FAR1 domains were tightly associated with segmental duplications of 5-enolpyruvylshikimate-3-phosphate synthase subsequently conferring resistance to the herbicide glyphosate. Further, we characterized a new MuDR subtype, named here as 'Muntjac', which contributes to the evolution of herbicide resistance in kochia through the process of transduplication. CONCLUSION:Collectively, our study provides insights into the role FHY3/FAR1 genes as active transposable elements and contributes new perspectives on the interaction between transposons and herbicide resistance evolution. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Organic inputs are vital to sustainable agriculture because of their capacity to improve soil nutrients and nourish soil microbial communities. However, it is still not well known how organic inputs modify soil microbial functions. Here, we studied the effects of cover crop inclusion and manure compost amendment on maicrobial communities in sandy soils under organic vegetable production. Two manure composts (with and without) and four cover crop treatments, that is, cereal rye ( Secale cereale L.), hairy vetch ( Vicia villosa ), the mixture of the two, and no cover crop control, were fully crossed and established in the fields in 2020. After 2 years of repeated treatments, soils were collected for biogeochemical and microbial analyses in 2022. We found limited treatment effects on microbial alpha diversity, but both manure compost application and cover crop inclusion altered microbial community structure, in which cereal rye and hairy vetch had distinct effects. In addition, hairy vetch and cereal rye increased the abundances of dominant soil bacterial and fungal taxa, respectively. Organic inputs altered C and N-cycling extracellular enzyme activities (EA), which correlated with soil biogeochemical properties and microbial diversity. The changes in predicted microbial functions are likely to have a significant impact on long-term soil fertility.

Amaranthus palmeri (Palmer amaranth), Amaranthus retroflexus (redroot pigweed), and Amaranthus hybridus (smooth pigweed) are troublesome weeds that are economically damaging to several cropping systems. Collectively referred to as "pigweeds," these species are incredibly adaptive and have become successful competitors in diverse agricultural settings. The development of genomic resources for these species promises to facilitate the elucidation of the genetic basis of traits such as biotic and abiotic stress tolerance (e.g., herbicide resistance) and sex determination. Here, we sequenced and assembled chromosome-level genomes of these three pigweeds. By combining the haplotype-resolved assembly of A. palmeri with existing restriction site-associated DNA sequencing data, we identified an approximately 2.84 Mb region on chromosome 3 of Hap1 that is male-specific and contains 37 genes. Transcriptomic analysis revealed that two genes, RESTORER OF FERTILITY 1 (RF1) and TLC DOMAIN-CONTAINING PROTEIN (TLC), within the male-specific region were upregulated in male individuals across the shoot apical meristem, the floral meristem, and mature flowers, indicating their potential involvement in sex determination in A. palmeri. In addition, we rigorously classified cytochrome P450 genes in all three pigweeds due to their involvement in non-target-site herbicide resistance. Finally, we identified contiguous extrachromosomal circular DNA (eccDNA) in A. palmeri, a critical component of glyphosate resistance in this species. The findings of this study advance our understanding of sex determination in A. palmeri and provide genomic resources for elucidating the genetic basis and evolutionary origins of adaptive traits within the genus.

IntroductionPlants can adapt their growth to optimize light capture in competitive environments, with branch angle being a crucial factor influencing plant phenotype and physiology. Decreased branch angles in cereal crops have been shown to enhance productivity in high-density plantings. The Tiller Angle Control (TAC1) gene, known for regulating tiller inclination in rice and corn, has been found to control branch angle in eudicots. Manipulating TAC1 in field crops like cotton offers the potential for improving crop productivity.MethodsUsing a homolog-based methodology, we examined the distribution of TAC1-related genes in cotton compared to other angiosperms. Furthermore, tissue-specific qPCR analysis unveiled distinct expression patterns of TAC1 genes in various cotton tissues. To silence highly expressed specific TAC1 homeologs in the stem, we applied CRISPR-Cas9 gene editing and Agrobacterium-mediated transformation, followed by genotyping and subsequent phenotypic validation of the mutants.ResultsGene duplication events of TAC1 specific to the Gossypium lineage were identified, with 3 copies in diploid progenitors and 6 copies in allotetraploid cottons. Sequence analysis of the TAC1 homeologs in Gossypium hirsutum revealed divergence from other angiosperms with 1-2 copies, suggesting possible neo- or sub-functionalization for the duplicated copies. These TAC1 homeologs exhibited distinct gene expression patterns in various tissues over developmental time, with elevated expression of A11G109300 and D11G112200, specifically in flowers and stems, respectively. CRISPR-mediated loss of these TAC1 homeologous genes resulted in a reduction in branch angle and altered petiole angles, and a 5 to 10-fold reduction in TAC1 expression in the mutants, confirming their role in controlling branch and petiole angles. This research provides a promising strategy for genetically engineering branch and petiole angles in commercial cotton varieties, potentially leading to increased productivity.

Background: Palmer amaranth achieves resistance to glufosinate by overproducing the chloroplastic glutamine synthetase (GS2) protein, a result of the amplification and overexpression of its nuclear coding gene. This study examined how amplified GS2 copies are inherited, identified their physical location in the cell, and investigated the mechanism of GS2 amplification. Results: Segregation analysis revealed that inheritance of amplified GS2 copies deviates from classical Mendelian patterns, with poor correlation between plant level resistance and GS2 amplification. Fluorescence in situ hybridization revealed chromosomal insertions of GS2 and potential extrachromosomal circular DNA (eccDNA), and variability in GS2 amplification both among individual plants and within cells (not all cells in plants with high GS2 copy number showed GS2 amplification). The unpredictable inheritance patterns and distribution of GS2 copies across multiple chromosomes suggest a role for eccDNA in GS2 amplification. This was confirmed through eccDNA sequencing, which also identified multiple isoforms of GS2. Conclusion: This is the second documented case of herbicide resistance conferred by eccDNA-mediated target-site gene amplification in Palmer amaranth. ### Competing Interest Statement The authors Aimone Porri, Ingo Meiners and Jens Lerchl are affiliated with BASF. All other authors declare no conflict of interest.

Animals encounter diverse microbial communities throughout their lifetime, which exert varying selection pressures. Antimicrobial peptides (AMPs), which lyse or inhibit microbial growth, are a first line of defense against some of these microbes. Here we examine how developmental variation in microbial exposure has affected the evolution of expression and amino acid sequences of Defensins (an ancient class of AMPs) in the house fly (Musca domestica). The house fly is a well-suited model for this work because it trophically associates with varying microbial communities throughout its life history and its genome contains expanded families of AMPs, including Defensins. We identified two subsets of house fly Defensins: one expressed in larvae or pupae, and the other expressed in adults. The amino acid sequences of these two Defensin subsets form distinct monophyletic clades, and they are located in separate gene clusters in the genome. The adult-expressed Defensins evolve faster than larval/pupal Defensins, consistent with different selection pressures across developmental stages. Our results therefore suggest that varied microbial communities encountered across life history can shape the evolutionary trajectories of immune genes.

In vitro co-culture techniques that allow the growth of plants and pathogens under controlled environmental conditions are being used to re-create host plant infection. These approaches reduce infection times, promote reproducibility, and enable a rapid evaluation of plant-pathogen interactions. As a result, these systems have become essential in breeding programs aimed at developing plant resistance to diseases. In this study, we developed and validated an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting Prunus spp. This disease, caused by fungi Armillaria spp. and Desarmillaria caespitosa, posesa severe threat to the stone and nut fruit industry due to the susceptibility of most commercial rootstocks to infection and the lack of effective management options for its control. The system consists of a fiber-supported liquid approach in sterile plastic vessels that allows a fast and reproducible fungal infection under controlled environmental conditions. The floor of the vessels was covered with a polyester-fiber matte and a germination paper that served as an interface between the mycelia and the plant roots. The vessels were subjected to inoculation with Armillaria mellea and D. caespitosa, and three Prunus genotypes (‘Guardian‱’, ‘MP-29’, and Prunus cerasifera ‘14-4’) were co-cultured with both fungi. Disease progression and plant and fungal biomass were monitored during co-culture. The presented in vitro co-culture approach facilitates the concurrent growth of Armillaria/Desarmillaria spp. and Prunus spp., excluding most of the limitations associated with greenhouses and field experiments. This system provides consistent and reproducible conditions for investigating a prominent plant disease affecting Prunus spp.

Organic matter (OM) amendments are often encouraged in sustainable agriculture programs but can create heterogeneous soil environments when applied to perennial crops such as peaches (Prunus persica (L.) Batsch). To better understand the responses of peach roots to non-uniform soil conditions, transcriptomic analysis was performed in a split-root study using uniform soil (the same soil type for all roots) or non-uniform soil (different soil types for each half of the root system) from either (1) autoclaved sand (S), (2) autoclaved sand with autoclaved compost (A), or (3) autoclaved sand with compost which included inherent biological soil life (B). Each uniform soil type (S, A, and B) was grouped and compared by uniform and non-uniform soil comparisons for a total of nine treatments. Comparisons revealed peach roots had differentially expressed genes (DEGs) and gene ontology terms between soil groups, with the S and B groups having a range of 106–411 DEGs and the A group having a range of 19–94 DEGs. Additionally, six modules were identified and correlated (p > 0.69) for six of the nine treatment combinations. This study broadly highlights the complexity of how OM and biological life in the rhizosphere interact with immediate and distant roots and sheds light on how non-homogenous soil conditions can influence peach root gene expression.

Cover crops and manure application are two common nutrient management practices in organic production. However, their interactive influences on the biogeochemical properties of sandy Coastal Plains soils are not well-documented. Here, we investigated their interactive effects on nitrogen (N) availability and the abundance of selected N functional genes in organic vegetable production. An experiment was established in 2020 with a fully crossed treatment of manure compost application (2,365 and 0 kg ha-1) and cover crop inclusion (cereal rye, hair vetch, mixtures of rye and vetch, and no cover crop control). Two years after establishment, higher nitrate (NO3-) concentrations were found in the vetch plots when compared to the rye and control plots, while plots with manure application had higher ammonia (NH4+) concentrations than those without. Similarly, N-acetyl-β-D-glucosaminidase activities were higher in vetch plots than in rye and control plots, while leucine aminopeptidase activities were higher in manure plots than in the non-manure plots. In addition, the vetch plots had higher bacterial-associated ammonia-oxidizing gene (AOB amoA) abundance than other plots. Finally, manure application resulted in higher fungal abundance than no manure control plots. Integrating cover crops introduced higher N mineralization potentials, which, however, was not observed for manure application. Both cover crops and manure application increased N availability as expected but posed distinct short-term impacts on soil microbial communities.

In vitro co-culture techniques that allow the growth of plants and pathogens under controlled environmental conditions are being used to re-create host plant infection. These approaches reduce infection times, promote reproducibility, and enable a rapid evaluation of plant-pathogen interactions. As a result, these systems have become essential in breeding programs aimed at developing plant resistance to diseases. In this study, we developed and validated an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting Prunus spp. This disease, caused by fungi Armillaria spp. and Desarmillaria caespitosa, poses a severe threat to the stone and nut fruit industry due to the susceptibility of most commercial rootstocks to infection and the lack of effective management options for its control. The system consists of a fiber-supported liquid approach in sterile plastic vessels that allows a fast and reproducible fungal infection under controlled environmental conditions. The floor of the vessels was covered with a polyester-fiber matte and a germination paper that served as an interface between the mycelia and the plant roots. The vessels were subjected to inoculation with Armillaria mellea and D. caespitosa, and three Prunus genotypes ('Guardian®', 'MP-29', and Prunus cerasifera '14-4') were co-cultured with both fungi. Disease progression and plant and fungal biomass were monitored during co-culture. The presented in vitro co-culture approach facilitates the concurrent growth of Armillaria/Desarmillaria spp. and Prunus spp., excluding most of the limitations associated with greenhouses and field experiments. This system provides consistent and reproducible conditions for investigating a prominent plant disease affecting Prunus spp.

Cotton (Gossypium hirsutum L.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars (‘UGA230’, ‘UA48’ and ‘CSX8308’) and updating the ‘TM-1’ cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from ‘Pima’ cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability.

To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.

Reniform nematode (Rotylenchulus reniformis, Linford and Oliveira) is a sedentary, semi-endoparasite that infects a wide range of plant hosts and is one of the top three nematode pathogens affecting soybean in the southeastern United States. Previous studies have linked resistance to reniform nematode in soybean to two quantitative trait loci on chromosomes 11 and 18. A Kompetitive Allele-Specific PCR (KASP) assay was designed using SNP markers within these two regions to distinguish reniform nematode-resistant soybean based on genotype. A collection of 44 soybean plant introductions with resistant phenotype to reniform nematode and 40 susceptible soybean lines were genotyped at the two target loci to validate the KASP assay design. Of the 44 observed resistant lines, two carried the susceptible genotype; PI 438489B at the locus on chromosome 18 and PI 495017C on chromosome 11. Of the 40 observed susceptible soybean lines, only 25 had the expected susceptible genotype at the loci on chromosome 18 and 13 on chromosome 11. Our KASP assay was 68% accurate in predicting the phenotype of 84 soybean accessions based on their genotype at the SNP marker on chromosome 18 and 83% accurate at chromosome 11. These results indicate a moderate correlation of soybean SNP markers GlyREN18_46 and GlyREN11_190 with reniform nematode resistance. Further research is required to improve the accuracy of KASP assays to predict soybean response to reniform nematode, particularly host susceptibility.

Variable and uncontrollable environmental factors have a wide range of influence on crop field screening programs, with the potential to cause significant experimental errors in phenotypic data collection. These factors can be additive and negatively confound genetic studies. When field screening upland cotton for genetic resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4), the distribution and concentration of fungal inoculum in the field directly impact the disease severity, affecting the results of these studies. Variability among FOV4 screening fields and protocols has influenced the search for durable genetic resistance. To account for this spatial variability, rigorous use of check plots with predictable responses to FOV4 were planted throughout a screening nursery in Clint, TX, and scored for percent survival within each plot. The scores and locations were used to generate a predicted surface via kriging interpolation and conditional simulation, which estimate FOV4 inoculum pressure at every plot location in the field. These predictions created FOV4 pressure-adjusted disease severity ratings in F-3 generations of bi-parental crosses between FOV4-resistant and susceptible upland cotton lines. Environment-adjusted phenotypes allow breeders to consider the environmental variance associated with the heterogenous distribution of the pathogen in the field. The techniques presented here are transferrable to any field screening program that needs to account for spatial variation of environmental factors.

Flooding is becoming an increasing concern for soybean (Glycine max [L.] Merr.) production worldwide due to the sensitivity of most cultivars grown today to flood stress. Flooding can stunt plant growth and limit yield, causing significant economic loss. One sustainable approach to improve performance under flood stress is to develop flood-tolerant soybean cultivars. This study was conducted to evaluate soybean genotypes for the response to flood stress at three critical growth stages of production—germination, early vegetative growth (V1 and V4), and early reproductive growth (R1). The results demonstrated that stress imposed by flooding significantly affected soybean yield for each growth stage studied. The average germination rate over the various treatments ranged from 95% to 46%. Despite the poor germination rates after the extended flood treatments, the flood-tolerant genotypes maintained a germination rate of >80% after 8 h of flooding. The germination rate of the susceptible genotypes was significantly lower, ranging 58–63%. Imposing flood stress at the V1 and V4 growth stage also resulted in significant differences between the tolerant and susceptible genotypes. Genotypes with the highest level of flood tolerance continually outperformed the susceptible genotypes with an average 30% decrease in foliar damage based on visual scoring and a 10% increase in biomass. The yield of the tolerant genotypes was also on average 25% higher compared to the susceptible genotypes. These results suggest that breeding for flood tolerance in soybean can increase resiliency during crucial growth stages and increase yield under flood conditions. In addition, the genotypes developed from this research can be used as breeding stock to further make improvements to flood tolerance in soybean.

Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr®250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.