Genome Chromosome Research Articles

The burst of satellite DNA in Leptidea wood white butterflies and their putative role in karyotype evolution.

Satellite DNAs (satDNAs) are abundant components of eukaryotic genomes, playing pivotal roles in chromosomal organization, genome stability and evolution. Here, we combined cytogenetic and genomic methods to characterize the satDNAs in the genomes of Leptidea butterflies. Leptidea is characterized by the presence of a high heterochromatin content, large genomes and extensive chromosomal reshuffling as well as the occurrence of cryptic species. We show that, in contrast to other Lepidoptera, satDNAs constitute a considerable proportion of Leptidea genomes, ranging between 4.11%-11.05%. This amplification of satDNAs, together with the hyperactivity of transposable elements, contributes to the substantial genome expansion in Leptidea. Using chromosomal mapping, we show that, particularly LepSat01-100 and LepSat03-167 satDNAs, are preferentially localized in heterochromatin exhibiting variable distribution that could influence in the highly diverse karyotypes within the genus. The satDNAs also exhibit W-chromosome accumulation, suggesting their involvement in sex chromosome evolution. Our results provide insights into the dynamics of satDNAs in Lepidoptera genomes and highlight their role in genome expansion and chromosomal organization, which could influence the speciation process. The high proportion of repetitive DNAs in the genomes of Leptidea underscores the complex evolutionary dynamics revealing the interplay between repetitive DNAs and genomic architecture in the genus.

Genome-wide analysis of the SWEET gene family and its response to powdery mildew and leaf spot infection in the common oat (Avena sativa L.)

The nutritional quality and yield of oats (Avena sativa) are often compromised by plant diseases such as red leaf, powdery mildew, and leaf spot. Sugars Will Eventually be Exported Transporters (SWEETs) are newly identified sugar transporters involved in regulating plant growth and stress responses. However, the roles of SWEET genes in biotic stress responses remain uncharacterized in oats. In this study, 13 AsSWEET genes were identified across nine chromosomes of the oat genome, all of which were predicted to contain seven transmembrane regions. Phylogenetic analysis revealed four clades of AsSWEET proteins, with high homology to SWEET proteins in the Poaceae family. Collinearity analysis demonstrated strong relationships between oat and Zea mays SWEETs. Using subcellular localization prediction tools, AsSWEET proteins were predicted to localize to the plasma membrane. Promoter analysis revealed cis-acting elements associated with light response, growth, and stress regulation. Six AsSWEET proteins were predicted to interact in a network centered on AsSWEET1a and AsSWEET11. Gene expression analysis of two oat varieties, ‘ForagePlus’ and ‘Molasses’, indicated significant expression differences in several AsSWEET genes following infection with powdery mildew or leaf spot, including AsSWEET1a, AsSWEET1b, AsSWEET2b, AsSWEET3a, AsSWEET11, and AsSWEET16. These SWEET genes are potential candidates for disease resistance in oats. This study provides a foundation for understanding the regulatory mechanisms of AsSWEET genes, particularly in response to powdery mildew and leaf spot, and offers insights for enhancing oat molecular breeding.

Leuconostoc koreense sp. nov., isolated from kimchi, a fermented vegetable.

The novel strain CBA3628T was isolated from kimchi, a Korean fermented vegetable. CBA3628T is a cocci-shaped, Gram-stain-positive, catalase- and oxidase-negative and facultatively anaerobic bacterium. The results of phylogenetic analysis based on 16S rRNA gene sequencing indicated that CBA3628T represented a member of the genus Leuconostoc of the family Leuconostocaceae. CBA3628T has a circular chromosomal genome and three plasmids of 1 864 558 bp (37% DNA G+C content), containing 1,887 genes, 1,762 predicted protein-coding genes, 4 complete rRNA loci and 70 tRNA genes. The cells were non-haemolytic, non-motile and non-spore forming. The optimal growth of CBA3628T occurred at 30 °C, pH 6.0 and with 0-2% (w/v) NaCl. The major polar lipids of CBA3628T were diphosphatidylglycerol and phosphatidylglycerol. The major fatty acids (>10%) of CBA3628T were C16 : 0, C20 : 0 and C19 : 0 cyclo ω8c. CBA3628T contained A3α-type peptidoglycans. CBA3628T was most closely related to Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293T, L. mesenteroides subsp. dextranicum DSM 20484T and L. suionicum DSM 20241T with 99.52% 16S rRNA gene sequence similarity. However, the average nucleotide identities of 91.9%, 91.7% and 91.1% and the digital DNA-DNA hybridisation values of 45.6%, 45.4% and 45.4% indicated that the novel isolate represented a distinct species. Phylogenetic analyses of both the 16S rRNA gene and genome sequences revealed that CBA3628T formed a distinct phylogenetic lineage within the genus Leuconostoc and was most closely related to Leuconostoc litchii MB7T. The ANI and dDDH values between CBA3628T and L. litchii MB7T were 84.9 and 22.8%, respectively. Functional genes belonging to COG categories E, J and K were enriched in the genome of CBA3628T (>7.9%). On the basis of its physiological, chemotaxonomic, phylogenetic and genomic properties, strain CBA3628T represents a novel species from the genus Leuconostoc, for which we propose the name Leuconostoc koreense sp. nov., with the type strain CBA3628T (= KACC 23049T = DSM 116836T).

Widespread mutagenesis and chromosomal instability shape somatic genomes in systemic sclerosis

Systemic sclerosis is a connective tissue disorder characterized by excessive fibrosis that primarily affects women, and can present as a multisystem pathology. Roughly 4-22% of patients with systemic sclerosis develop cancer, which drastically worsens prognosis. However, the mechanisms underlying systemic sclerosis initiation, propagation, and cancer development are poorly understood. We hypothesize that the inflammation and immune response associated with systemic sclerosis can trigger DNA damage, leading to elevated somatic mutagenesis, a hallmark of pre-cancerous tissues. To test our hypothesis, we culture clonal lineages of fibroblasts from the lung tissues of controls and systemic sclerosis patients and compare their mutation burdens and spectra. We find an overall increase in all major mutation types in systemic sclerosis samples compared to control lung samples, from small-scale events such as single base substitutions and insertions/deletions, to chromosome-level changes, including copy-number changes and structural variants. In the genomes of patients with systemic sclerosis, we find evidence of somatic hypermutation or kategis (typically only seen in cancer genomes), we identify mutation signatures closely resembling the error-prone translesion polymerase Polη activity, and observe an activation-induced deaminase-like mutation signature, which overlaps with genomic regions displaying kataegis.

Exposure to benzyl butyl phthalate (BBP) leads to increased double-strand break formation and germline dysfunction in Caenorhabditis elegans.

Benzyl butyl phthalate (BBP), a plasticizer found in a wide range of consumer products including vinyl flooring, carpet backing, food packaging, personal care products, and children's toys, is an endocrine-disrupting chemical linked to impaired reproduction and development in humans. Despite evidence that BBP exposure perturbs the integrity of male and female gametes, its direct effect on early meiotic events is understudied. Here, using the nematode Caenorhabditis elegans, we show that BBP exposure elicits a non-monotonic dose response on the rate of X-chromosome nondisjunction measured using a high-throughput screening platform. From among the range of doses tested (1, 10, 100 and 500 μM BBP), we found that 10 μM BBP elicited the strongest effect on the germline, resulting in increased germ cell apoptosis and chromosome organization defects. Mass spectrometry analysis shows that C. elegans efficiently metabolizes BBP into its primary metabolites, monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP), and that the levels of BBP, MBP, and MBzP detected in the worm are within the range detected in human biological samples. Exposure to 10 μM BBP leads to germlines with enlarged mitotic nuclei, altered meiotic progression, activation of a p53/CEP-1-dependent DNA damage checkpoint, increased double-strand break levels throughout the germline, chromosome morphology defects in oocytes at diakinesis, and increased oxidative stress. RNA sequencing analysis indicates that BBP exposure results in the altered expression of genes involved in xenobiotic metabolic processes, extracellular matrix organization, oocyte morphogenesis, meiotic cell cycle, and oxidoreduction. Taken together, we propose that C. elegans exposure to BBP leads to increased oxidative stress and double-strand break formation, thereby compromising germline genomic integrity and chromosome segregation.

A Haplotype-resolved, Chromosome-scale Genome for Malus domestica Borkh. 'WA 38'.

Genome sequencing for agriculturally important Rosaceous crops has made rapid progress both in completeness and annotation quality. Whole genome sequence and annotation gives breeders, researchers, and growers information about cultivar specific traits such as fruit quality and disease resistance, and informs strategies to enhance postharvest storage. Here we present a haplotype-phased, chromosomal level genome of Malus domestica, 'WA 38', a new apple cultivar released to market in 2017 as Cosmic Crisp®. Using both short and long read sequencing data with a k-mer based approach, chromosomes originating from each parent were assembled and segregated. This is the first pome fruit genome fully phased into parental haplotypes in which chromosomes from each parent are identified and separated into their unique, respective haplomes. The two haplome assemblies, 'Honeycrisp' originated HapA and 'Enterprise' originated HapB, are about 650 Megabases each, and both have a BUSCO score of 98.7% complete. A total of 53,028 and 54,235 genes were annotated from HapA and HapB, respectively. Additionally, we provide genome-scale comparisons to 'Gala', 'Honeycrisp', and other relevant cultivars highlighting major differences in genome structure and gene family circumscription. This assembly and annotation was done in collaboration with the American Campus Tree Genomes project that includes 'WA 38' (Washington State University), 'd'Anjou' pear (Auburn University), and many more. To ensure transparency, reproducibility, and applicability for any genome project, our genome assembly and annotation workflow is recorded in detail and shared under a public GitLab repository. All software is containerized, offering a simple implementation of the workflow.

Distinct patterns of satDNA distribution in holocentric chromosomes of spike-sedges (Eleocharis, Cyperaceae).

Eleocharis R. Br. (Cyperaceae) species are known for having holocentric chromosomes, which enable rapid karyotype differentiation. High intra- and interspecific variations in chromosome numbers and genome sizes are documented for different Eleocharis species, frequently accompanied by fluctuations in the repetitive DNA fraction. However, a lack of detailed analysis has hampered a better understanding of the interplay between holocentricity and repetitive DNA evolution in this genus. In our study, we confirmed the holocentricity of Eleocharis chromosomes by immunostaining against the kinetochore protein KNL1 and the cell-cycle dependent posttranslational modifications histone H2AThr121ph and H3S10ph. We further studied the composition and chromosomal distribution of the main satellite DNA repeats found in the newly sequenced species E. maculosa, E. geniculata, E. parodii, E. elegans, and E. montana. Five of the six satellites discovered were arranged in clusters, while EmaSAT14 was distributed irregularly along the chromatid length in a line-like manner. EmaSAT14 monomers were present in a few copies in few species across the Eleocharis phylogenetic tree. Nonetheless, they were accumulated within a restricted group of Maculosae series, subgenus Eleocharis. The data indicates that the amplification and line-like distribution of EmaSAT14 along chromatids may have occurred recently within a section of the genus.

The updated mouse universal genotyping array bioinformatic pipeline improves genetic QC in laboratory mice.

The MiniMUGA genotyping array is a popular tool for genetic quality control of laboratory mice and genotyping samples from most experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve the array's performance and the associated report's usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA. Here, we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for classical inbred strains and substrains, and increase the number of constructs reliably detected with MiniMUGA. In addition, we have implemented key changes in the informatics pipeline to identify and quantify the contribution of specific genetic backgrounds to the makeup of a given sample, remove arbitrary thresholds, include the Y Chromosome and mitochondrial genome in the ideogram, and improve robust detection of the presence of commercially available substrains based on diagnostic alleles. Finally, we have updated the layout of the report to simplify the interpretation and completeness of the analysis and added a section summarizing the ideogram in table format. These changes will be of general interest to the mouse research community and will be instrumental in our goal of improving the rigor and reproducibility of mouse-based biomedical research.

Triple-color FISH chromosome constitution and genome size analyses confirmed the ploidy of newly developed ten Hibiscus sp. cultivars with different breeding history

Triple-color FISH chromosome constitution and genome size analyses confirmed the ploidy of newly developed ten Hibiscus sp. cultivars with different breeding history

Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

The first Chromosomal-level genome assembly of Sageretia thea using Nanopore long reads and Pore-C technology

Sageretia thea, a notable species within the mock buckthorn genus, is recognized for its intriguing biogeographical distribution and diverse medicinal properties. Despite this significance, genomic studies on S. thea are still in the nascent stages. We present the first chromosome-level genome assembly of S. thea that was generated using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies complemented by Pore-C chromatin conformation capture. The genome assembly had a size of 197.8 Mb with 12 chromosomal scaffolds and a scaffold N50 length of 15.9 Mb. A total of 25,434 protein-coding genes were identified and functionally annotated, and the gene model indicated 96.5% complete eukaryotic BUSCOs. Additionally, orthologous gene profiling and synteny analysis were performed to elucidate the evolutionary relationships within the Rhamnaceae family and Rosales. This high-quality chromosomal genome is the first genomic view of S. thea, which will serve as the basis for future studies on its biological and medicinal properties, and evolutionary history.

Application of CRISPR/Cas9 system to knock out GluB gene for developing low glutelin rice mutant

The nutritional quality improvement is among the most integral objective for any rice molecular breeding programs. The seed storage proteins (SSPs) have greater role to determine the nutritional quality of any cereal grains. Rice contains relatively balanced amino acid composition and the SSPs are fractioned into albumins (ALB), globulins (GLO), prolamins (PRO) and glutelins (GLU) according to differences in solubility. GLUs are further divided into subfamilies: GluA, GluB, GluC, and GluD depending on resemblance in amino acid. The GLU protein accounts for 60–80% of total protein contents, encoded by 15 genes located on different chromosomes of rice genome. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was employed to knockout Glu-B (LOC-Os02g15070) gene in non-basmati rice PK386 cultivar. The mutant displayed two base pair and three base pair mutation in the targeted regions. The homozygous mutant plant displayed reduction for both in total protein contents and GLU contents whereas, elevation in GLO, ALB and PRO. Moreover, the mutant plant also displayed reduction in physio-chemical properties e.g., total starch, amylose and gel consistency. The agronomic characteristics of both mutant and wild type displayed non-significant differences along with increase in higher percentage of chalkiness in mutant plants. The results obtained from scanning electron microscopy showed the loosely packed starch granules compared to wild type. The gene expression analysis displayed the lower expression of gene at 5 days after flowering (DAF), 10 DAF, 15 DAF and 20 DAF compared to wild type. GUS sub-cellular localization showed the staining in seed which further validated the results obtained from gene expression. Based on these findings it can be concluded Glu-B gene have significant role in controlling GLU contents and can be utilized in breeding programs to enhance the nutritional quality of rice, and may serve as healthy diet for patient allergic with high GLU contents.

Molecular characterization of cassava zinc finger-homeodomain (ZF-HD) transcription factors reveals their role in disease resistance

The production of cassava (Manihot esculenta Crantz) is constantly threatened by cassava bacterial blight (CBB), caused by Xanthomonas phaseoli pv. manihotis (Xpm). Zinc finger-homeodomain (ZF-HD) belongs to a family of homozygous heterotypic cassette genes widely implicated in various developmental and physiological processes in plants. Despite their importance, a comprehensive analysis of ZF-HD genes, particularly those involved in disease resistance, has not been performed for cassava. In the present study, we utilized bioinformatics methods to identify 21 ZF-HD genes distributed across 11 chromosomes of cassava genome, with the majority exhibiting gene structure without introns. Phylogenetic analysis categorized these genes into two major groups (MIF and ZHD) with five subgroups. We observed fourteen pairs of duplicated genes, suggesting that segmental duplication has likely facilitated the expansion of the cassava ZF-HD gene family. Comparative orthologous analyses between cassava and other plant species shed light on the evolutionary trajectory of this gene family. Promoter analyses revealed multiple hormone- and stress-related elements, indicative of a functional role in stress responses. Expression profiling through RNA-seq and quantitative real-time PCR (qRT-PCR) demonstrated that certain cassava ZF-HD genes are up-regulated in response to Xpm infection, suggesting their involvement in defense mechanisms. Notably, MeZHD7 gene was identified via virus induced gene silencing (VIGS) as potentially crucial in conferring resistance against CBB. Results from subcellular localization experiments indicated that MeZHD7 was localized in the nucleus. The Luciferase reporter assay demonstrated an interaction between MeZHD7 and MeMIF5. These findings may lay the foundation for further cloning and functional analyses of cassava ZF-HD genes, particularly those associated with pathogen resistance.

Chromosome-specific painting reveals the Y genome origin and chromosome rearrangements of the St genome in Triticeae.

Karyotypes provide key cytogenetic information on phylogenetic relationships and evolutionary origins in related plant species. The St genome of Pseudoroegneria contributes to eight alloploid genera, representing over half of the species that are highly valuable for wheat (Triticum aestivum) breeding and for understanding Triticeae species evolution. However, St chromosome characterization is challenging due to limited cytogenetic markers and DNA information. We developed a complete set of St genome-specific chromosome painting probes for identification of the individual chromosomes 1St to 7St based on the genome sequences of Pse. libanotica and wheat. We revealed the conservation of St chromosomes in St-containing species by chromosome painting, including Pseudoroegneria, Roegneria, Elymus, and Campeiostachys. Notably, the Y genome showed hybridization signals, albeit weaker than those of the St genome. The awnless species harboring the Y genome exhibited more intense hybridization signals compare to the awned species in Roegneria and Campeiostachys, yet weaker than the hybridization signals of the St genome in autotetraploid Pse. strigosa. Although awnless species were morphologically more similar to each other, phenotypic divergence progressively increased from awnless to awned species. Our results indicate that the Y genome originated from the St genome and shed light on the possible origin of the Roegneria and Campeiostachys species, enhancing our understanding of St-genome-containing species evolution.

Telomere-to-telomere genome assembly of Eleocharis dulcis and expression profiles during corm development

Eleocharis dulcis (Burm. f.) Trin. ex Hensch., commonly known as Chinese water chestnut, is a traditional aquatic vegetable in China, and now is widely cultivated throughout the world because of its high nutritional value and unique tastes. Here, we report the assembly of a 493.24 Mb telomere-to-telomere (T2T) genome of E. dulcis accomplished by integrating ONT ultra-long reads, PacBio long reads and Hi-C data. The reference genome was anchored onto 111 gap-free chromosomes, containing 48.31% repeat elements and 33,493 predicted protein-coding genes. Whole genome duplication (WGD) and inter-genomic synteny analyses indicated that chromosome breakage and genome duplication in E. dulcis possibly occurred multiple times during genome evolution after its divergence from a common ancestor with Rhynchospora breviuscula at ca. 35.6 Mya. A comparative time-course transcriptome analysis of corm development revealed different patterns of gene expression between cultivated and wild accessions with the highest number of differentially expressed genes (DEGs, 15,870) at the middle swelling stage and some of the DEGs were significantly enriched for starch metabolic process.

Whole-genome sequencing of 13 Arctic plants and draft genomes of Oxyria digyna and Cochlearia groenlandica

To understand the genomic characteristics of Arctic plants, we generated 28–44 Gb of short-read sequencing data from 13 Arctic plants collected from the High Arctic Svalbard. We successfully estimated the genome sizes of eight species by using the k-mer-based method (180–894 Mb). Among these plants, the mountain sorrel (Oxyria digyna) and Greenland scurvy grass (Cochlearia groenlandica) had relatively small genome sizes and chromosome numbers. We obtained 45 × and 121 × high-fidelity long-read sequencing data. We assembled their reads into high-quality draft genomes (genome size: 561 and 250 Mb; contig N50 length: 36.9 and 14.8 Mb, respectively), and correspondingly annotated 43,105 and 29,675 genes using ~46 and ~85 million RNA sequencing reads. We identified 765,012 and 88,959 single-nucleotide variants, and 18,082 and 7,698 structural variants (variant size ≥ 50 bp). This study provided high-quality genome assemblies of O. digyna and C. groenlandica, which are valuable resources for the population and molecular genetic studies of these plants.

The smallest angiosperm genomes may be the price for effective traps of bladderworts.

Species of the carnivorous family Lentibulariaceae exhibit the smallest genomes in flowering plants. We explored the hypothesis that their minute genomes result from the unique mitochondrial cytochrome c oxidase (COX) mutation. The mutation may boost mitochondrial efficiency, which is especially useful for suction-bladder traps of Utricularia, but also increase DNA-damaging reactive oxygen species, leading to genome shrinkage through deletion-biased DNA repair. We aimed to explore this mutation's impact on genome size, providing insights into genetic mutation roles in plant genome evolution under environmental pressures. We compiled and measured genome and mean chromosome sizes for 127 and 67 species, respectively, representing all three genera (Genlisea, Pinguicula, and Utricularia) of Lentibulariaceae. We also isolated and analyzed COX sequences to detect the mutation. Through phylogenetic regressions and Ornstein-Uhlenbeck models of trait evolution, we assessed the impact of the COX mutation on the genome and chromosome sizes across the family. Our findings reveal significant correlations between the COX mutations and smaller genome and chromosome sizes. Specifically, species carrying the ancestral COX sequence exhibited larger genomes and chromosomes than those with the mutation. This evidence supports the notion that the COX mutation contributes to genome downsizing, with statistical analyses confirming a directional evolution towards smaller genomes in species harboring these mutations. Our study confirms that the COX mutation in Lentibulariaceae is associated with genome downsizing, likely driven by increased reactive oxygen species production and subsequent DNA damage requiring deletion-biased repair mechanisms. While boosting mitochondrial energy output, this genetic mutation compromises genome integrity and may potentially affect recombination rates, illustrating a complex trade-off between evolutionary advantages and disadvantages. Our results highlight the intricate processes by which genetic mutations and environmental pressures shape genome size evolution in carnivorous plants.

Mosquitocidal toxin-like islands in Bacillus thuringiensis S2160-1 revealed by complete-genome sequence and MS proteomic analysis

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC–MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

Chromosome genome assembly of the Camphora longepaniculata (Gamble) with PacBio and Hi-C sequencing data.

Camphora longepaniculata, a crucial commercial crop and a fundamental component of traditional Chinese medicine, is renowned for its abundant production of volatile terpenoids. However, the lack of available genomic information has hindered pertinent research efforts in the past. To bridge this gap, the present study aimed to use PacBio HiFi, short-read, and highthroughput chromosome conformation capture sequencing to construct a chromosome-level assembly of the C. longepaniculata genome. With twelve chromosomes accounting for 99.82% (766.69 Mb) of the final genome assembly, which covered 768.10 Mb, it was very complete. Remarkably, the assembly's contig and scaffold N50 values are exceptional as well-41.12 and 63.78 Mb, respectively-highlighting its excellent quality and intact structure. Furthermore, a total of 39,173 protein-coding genes were predicted, with 38,766 (98.96%) of them being functionally annotated. The completeness of the genome was confirmed by the Benchmarking Universal Single-Copy Ortholog evaluation, which revealed 99.01% of highly conserved plant genes. As the first comprehensive assembly of the C. longepaniculata genome, it provides a crucial starting point for deciphering the complex pathways involved in terpenoid production. Furthermore, this excellent genome serves as a vital resource for upcoming research on the breeding and genetics of C. longepaniculata.

Comprehensive analyses of the ARF gene family in cannabis reveals their potential roles in regulating cannabidiol biosynthesis and male flower development.

Cannabidiol (CBD), as an important therapeutic property of the cannabis plants, is mainly produced in the flower organs. Auxin response factors (ARFs) are play a crucial role in flower development and secondary metabolite production. However, the specific roles of ARF gene family in cannabis remain unknown. In this study, various bioinformatics analysis of CsARF genes were conducted using online website and bioinformatics, quantitative real time PCR technology was used to investigate the expression patterns of the CsARF gene family in different tissues of different cannabis varieties, and subcellular localization analysis was performed in tobacco leaf. In this study, 22 CsARF genes were identified and found to be unevenly distributed across 9 chromosomes of the cannabis genome. Phylogenetic analysis revealed that the ARF proteins were divided into 4 subgroups. Duplication analysis identified one pair of segmental/whole-genome duplicated CsARF, and three pairs of tandemly duplicated CsARF. Collinearity analysis revealed that two CsARF genes, CsARF4 and CsARF19, were orthologous in both rice and soybean. Furthermore, subcellular localization analysis showed that CsARF2 was localized in the nucleus. Tissue-specific expression analysis revealed that six genes were highly expressed in cannabis male flowers, and among these genes, 3 genes were further found to be highly expressed at different developmental stages of male flowers. Meanwhile, correlation analysis between the expression level of CsARF genes and CBD content in two cultivars 'H8' and 'Y7' showed that the expression level of CsARF13 was negatively correlated with CBD content, while the expression levels of six genes were positively correlated with CBD content. In addition, most of CsARF genes were responsive to IAA treatment. Our study laid a foundation for the further studies of CsARFs function in cannabis, and provides candidate genes for breeding varieties with high CBD yield in cannabis production.