Functional Genomics Research Articles

Viral genome sequencing methods: benefits and pitfalls of current approaches.

Whole genome sequencing of viruses provides high-resolution molecular insights, enhancing our understanding of viral genome function and phylogeny. Beyond fundamental research, viral sequencing is increasingly vital for pathogen surveillance, epidemiology, and clinical applications. As sequencing methods rapidly evolve, the diversity of viral genomics applications and catalogued genomes continues to expand. Advances in long-read, single molecule, real-time sequencing methodologies present opportunities to sequence contiguous, haplotype resolved viral genomes in a range of research and applied settings. Here we present an overview of nucleic acid sequencing methods and their applications in studying viral genomes. We emphasise the advantages of different viral sequencing approaches, with a particular focus on the benefits of third-generation sequencing technologies in elucidating viral evolution, transmission networks, and pathogenesis.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against thebroad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

A G-quadruplex-binding platinum complex induces cancer mitochondrial dysfunction through dual-targeting mitochondrial and nuclear G4 enriched genome

BackgroundG-quadruplex DNA (G4) is a non-canonical structure forming in guanine-rich regions, which play a vital role in cancer biology and are now being acknowledged in both nuclear and mitochondrial (mt) genome. However, the impact of G4-based targeted therapy on both nuclear and mt genome, affecting mt function and its underlying mechanisms remain largely unexplored.MethodsThe mechanisms of action and therapeutic effects of a G4-binding platinum(II) complex, Pt-ttpy, on mitochondria were conducted through a comprehensive approaches with in vitro and in vivo models, including ICP-MS for platinum measurement, PCR-based genetic analysis, western blotting (WB), confocal microscope for mt morphology study, extracellular flux analyzer, JC1 and Annexin V apoptosis assay, flow cytometry and high content microscope screening with single-cell quantification of both ROS and mt specific ROS, as well as click-chemistry for IF study of mt translation. Decipher Pt-ttpy effects on nuclear-encoded mt related genes expression were undertaken via RNA-seq, Chip-seq and CUT-RUN assays.ResultsPt-ttpy, shows a highest accumulation in the mitochondria of A2780 cancer cells as compared with two other platinum(II) complexes with no/weak G4-binding properties, Pt-tpy and cisplatin. Pt-ttpy induces mtDNA deletion, copy reduction and transcription inhibition, hindering mt protein translation. Functional analysis reveals potent mt dysfunction without reactive oxygen species (ROS) induction. Mechanistic study provided first evidence that most of mt ribosome genes are highly enriched in G4 structures in their promoter regions, notably, Pt-ttpy impairs most nuclear-encoded mt ribosome genes’ transcription through dampening the recruiting of transcription initiation and elongation factors of NELFB and TAF1 to their promoter with G4-enriched sequences. In vivo studies show Pt-ttpy’s efficient anti-tumor effects, disrupting mt genome function with fewer side effects than cisplatin.ConclusionThis study underscores Pt-ttpy as a G4-binding platinum(II) complex, effectively targeting cancer mitochondria through dual action on mt and nuclear G4-enriched genomes without inducing ROS, offering promise for safer and effective platinum-based G4-targeted cancer therapy.Graphical

Transcriptome-wide identification of ARF gene family in medicinal plant Polygonatum kingianum and expression analysis ofPkARF members in different tissues.

Polygonatum kingianum holds significant importance in Traditional Chinese Medicine due to its medicinal properties, characterized by its diverse chemical constituents including polysaccharides, terpenoids, flavonoids, phenols, and phenylpropanoids. The Auxin Response Factor (ARF) is a pivotal transcription factor known for its regulatory role in both primary and secondary metabolite synthesis. However, our understanding of the ARF gene family in P. kingianum remains limited. We employed RNA-Seq to sequence three distinct tissues (leaf, root, and stem) of P. kingianum. The analysis revealed a total of 31,558 differentially expressed genes (DEGs), with 43 species of transcription factors annotated among them. Analyses via gene ontology and the Kyoto Encyclopedia of Genes and Genomes demonstrated that these DEGs were predominantly enriched in metabolic pathways and secondary metabolite biosynthesis. The proposed temporal expression analysis categorized the DEGs into nine clusters, suggesting the same expression trends that may be coordinated in multiple biological processes across the three tissues. Additionally, we conducted screening and expression pattern analysis of the ARF gene family, identifying 12 significantly expressed PkARF genes in P. kingianum roots. This discovery lays the groundwork for investigations into the role of PkARF genes in root growth, development, and secondary metabolism regulation. The obtained data and insights serve as a focal point for further research studies, centred on genetic manipulation of growth and secondary metabolism in P. kingianum. Furthermore, these findings contribute to the understanding of functional genomics in P. kingianum, offering valuable genetic resources.

Genomic Advancement in Wheat (Triticum aestivum L.): Harnessing Technological Breakthroughs for Future Strategies

Wheat can be greatly improved by identifying genes that are significant to agronomy. Although progress in wheat genetics and genomics has been limited by the genome’s vast size and complexity. Wheat has a high-quality genome sequence in light of recent developments in genome sequencing and sequence assembly. Here, we propose that wheat biology can benefit from the same approaches that have been used to define biological systems in model species, such as the generation and characterization of mutants, methods for gene cloning, and enhanced transgenic technology. These tactics will encourage the establishment of wheat breeding programs and hasten the field of wheat biology. We also summarize current developments in functional genomics of wheat. In order to contribute to global food security, we conclude by talking about the future of wheat functional genomics and the sensible design-based molecular breeding of new wheat varieties. We suggest that researchers studying wheat should embrace the methods utilized for functional genomic analysis in other model species. One method for doing this is using gene cloning to find the gene causing an intriguing phenotype, deciphering the biological role of genes through the analysis of their corresponding mutants, and creating a related wheat mutant from the proposed wild-type gene to confirm the target gene’s functionality.

Rethinking eco‐evo studies of gene expression for non‐model organisms in the genomic era

AbstractRecent advances in genomic technology, including the rapid development of long‐read sequencing technology and single‐cell RNA‐sequencing methods, are poised to significantly expand the kinds of studies that are feasible in ecological genomics. In this perspective, we review these new technologies and discuss their potential impact on gene expression studies in non‐model organisms. Although traditional RNA‐sequencing methods have been an extraordinarily powerful tool to apply functional genomics in an ecological context, bulk RNA‐seq approaches often rely on de novo transcriptome assembly, and cannot capture expression changes in rare cell populations or distinguish shifts in cell type abundance. Advancements in genome assembly technology, particularly long‐read sequencing, and improvements in the scalability of single‐cell RNA‐sequencing (scRNA‐seq) offer unprecedented resolution in understanding cellular heterogeneity and gene regulation. We discuss the potential of these technologies to enable disentangling differential gene regulation from cell type composition differences and uncovering subtle expression patterns masked by bulk RNA‐seq. The integration of these approaches provides a more nuanced understanding of the ecological and evolutionary dynamics of gene expression, paving the way for refined models and deeper insights into the generation of biodiversity.

A Simple and Low-Cost CRISPR/Cas9 Knockout System Widely Applicable to Insects

The CRISPR/Cas9 gene-editing system is a standard technique in functional genomics, with widespread applications. However, the establishment of a CRISPR/Cas9 system is challenging. Previous studies have presented numerous methodologies for establishing a CRISPR/Cas9 system, yet detailed descriptions are limited. Additionally, the difficulties in obtaining the necessary plasmids have hindered the replication of CRISPR/Cas9 techniques in other laboratories. In this study, we share a detailed and simple CRISPR/Cas9 knockout system with optimized steps. The results of gene knockout experiments in vitro and in vivo show that this system successfully knocked out the target gene. By sharing detailed information on plasmid sequences, reagent codes, and methods, this study can assist researchers in establishing gene knockout systems.

The Function of RNA Interference (RNAi) in Crop Enhancement- Mechanism and Applications: A Review

RNA interference (RNAi) is a conserved molecular mechanism that plays a critical role in post-transcriptional gene silencing across diverse organisms. This review delves into the role of RNAi in plant functional genomics and its applications in crop improvement, highlighting its mechanistic insights and practical implications. The overview starts with the fundamental finding of the mechanism of RNAi, following its journey from petunias to its ubiquitous presence in a wide range of species. The discovery of several kinds of regulatory non-coding small RNAs, including as phasiRNAs, miRNAs, and siRNAs, has broadened our knowledge of RNAi-mediated gene regulation beyond what is previously thought. These RNA types have an impact on gene expression through complex post-transcriptional and epigenetic mechanisms. RNA interference (RNAi) has become a potent tool for comprehending gene activities in the context of crop improvement. It has shown to be useful in determining the functions of genes for metabolic pathways, stress tolerance, and more. Furthermore, RNAi-based strategies have potential for sustainable agriculture and integrated pest management, supporting international efforts to ensure food security. The many uses of RNA interference (RNAi) are covered in this article, including improving crop nutrition, changing plant architecture, and increasing shelf life. The challenges and future prospects of RNAi technology are also explored. The worldwide geography of RNA interference research is emphasized, with notable contributions from North America, Europe, and China. In conclusion, RNA interference (RNAi) continues to be a flexible and essential tool in contemporary plant research, providing new pathways for understanding gene activities and enhancing crop characteristics. Its combination with other biotechnological techniques, such as gene editing, might influence how agriculture and sustainable food production develop in the future.

CRWN nuclear lamina components maintain the H3K27me3 landscape and promote successful reproduction in Arabidopsis.

Arabidopsis lamin analogs CROWDED NUCLEIs (CRWNs) are necessary to maintain nuclear structure, genome function, and proper plant growth. However, whether and how CRWNs impact reproduction and genome-wide epigenetic modifications is unknown. Here, we investigate the role of CRWNs during the development of gametophytes, seeds, and endosperm, using genomic and epigenomic profiling methods. We observed defects in crwn mutant seeds including seed abortion and reduced germination rate. Quadruple crwn null genotypes were rarely transmitted through gametophytes. Because defects in seeds often stem from abnormal endosperm development, we focused on crwn1 crwn2 (crwn1/2) endosperm. These mutant seeds exhibited enlarged chalazal endosperm cysts and increased expression of stress-related genes and the MADS-box transcription factor PHERES1 and its targets. Previously, it was shown that PHERES1 expression is regulated by H3K27me3 and that CRWN1 interacts with the PRC2 interactor PWO1. Thus, we tested whether crwn1/2 alters H3K27me3 patterns. We observed a mild loss of H3K27me3 at several hundred loci, which differed between endosperm and leaves. These data indicate that CRWNs are necessary to maintain the H3K27me3 landscape, with tissue-specific chromatin and transcriptional consequences.

A high-quality chromosome-scale genome assembly of blood orange, an important pigmented sweet orange variety

Blood orange (BO) is a rare red-fleshed sweet orange (SWO) with a high anthocyanin content and is associated with numerous health-related benefits. Here, we reported a high-quality chromosome-scale genome assembly for Neixiu (NX) BO, reaching 336.63 Mb in length with contig and scaffold N50 values of 30.6 Mb. Furthermore, 96% of the assembled sequences were successfully anchored to 9 pseudo-chromosomes. The genome assembly also revealed the presence of 37.87% transposon elements and 7.64% tandem repeats, and the annotation of 30,395 protein-coding genes. A high level of genome synteny was observed between BO and SWO, further supporting their genetic similarity. The speciation event that gave rise to the Citrus species predated the duplication event found within them. The genome-wide variation between NX and SWO was also compared. This first high-quality BO genome will serve as a fundamental basis for future studies on functional genomics and genome evolution.

Functional genomics reveals functions of terpene synthases for volatile terpene formation in peach

Functional genomics reveals functions of terpene synthases for volatile terpene formation in peach

Application of Developmental Regulators for Enhancing Plant Regeneration and Genetic Transformation.

Establishing plant regeneration systems and efficient genetic transformation techniques plays a crucial role in plant functional genomics research and the development of new crop varieties. The inefficient methods of transformation and regeneration of recalcitrant species and the genetic dependence of the transformation process remain major obstacles. With the advancement of plant meristematic tissues and somatic embryogenesis research, several key regulatory genes, collectively known as developmental regulators, have been identified. In the field of plant genetic transformation, the application of developmental regulators has recently garnered significant interest. These regulators play important roles in plant growth and development, and when applied in plant genetic transformation, they can effectively enhance the induction and regeneration capabilities of plant meristematic tissues, thus providing important opportunities for improving genetic transformation efficiency. This review focuses on the introduction of several commonly used developmental regulators. By gaining an in-depth understanding of and applying these developmental regulators, it is possible to further enhance the efficiency and success rate of plant genetic transformation, providing strong support for plant breeding and genetic engineering research.

Functional Genomics and Insights into the Pathogenesis and Treatment of Psoriasis

Psoriasis is a lifelong, systemic, immune mediated inflammatory skin condition, affecting 1–3% of the world’s population, with an impact on quality of life similar to diseases like cancer or diabetes. Genetics are the single largest risk factor in psoriasis, with Genome-Wide Association (GWAS) studies showing that many psoriasis risk genes lie along the IL-23/Th17 axis. Potential psoriasis risk genes determined through GWAS can be annotated and characterised using functional genomics, allowing the identification of novel drug targets and the repurposing of existing drugs. This review is focused on the IL-23/Th17 axis, providing an insight into key cell types, cytokines, and intracellular signaling pathways involved. This includes examination of currently available biological treatments, time to relapse post drug withdrawal, and rates of primary/secondary drug failure, showing the need for greater understanding of the underlying genetic mechanisms of psoriasis and how they can impact treatment. This could allow for patient stratification towards the treatment most likely to reduce the burden of disease for the longest period possible.

Unveiling the genetic blueprint of a desert scorpion: A chromosome-level genome of Hadrurus arizonensis provides the first reference for Parvorder Iurida.

Over 400 million years old, scorpions represent an ancient group of arachnids and one of the first animals to adapt to life on land. Presently, the lack of available genomes within scorpions hinders research on their evolution. This study leverages ultra-long nanopore sequencing and Pore-C to generate the first chromosome level assembly and annotation for the desert hairy scorpion, Hadrurus arizonensis. The assembled genome is 2.23 Gb in size with an N50 of 280 Mb. Pore-C scaffolding re-oriented 99.6% of bases into nine chromosomes and BUSCO identified 998 (98.6%) complete arthropod single copy orthologs. Repetitive elements represent 54.69% of the assembled bases, including 872,874 (29.39%) LINE elements. A total of 18,996 protein-coding genes and 75,256 transcripts were predicted, and extracted protein sequences yielded a BUSCO score of 97.2%. This is the first genome assembled and annotated within the family Hadruridae, representing a crucial resource for closing gaps in genomic knowledge of scorpions, resolving arachnid phylogeny, and advancing studies in comparative and functional genomics.

A novel and efficient Apple latent spherical virus-based gene silencing method for functional genomic studies in Chenopodium quinoa

Quinoa (Chenopodium quinoa Willd.), with its resilience in harsh environments and excellent nutritional value, has become crucial for global food security. Despite recent progress in genomic research, the inability to perform functional studies in quinoa due to the absence of transformation techniques remains a significant obstacle. In this work, we present the development of a novel Apple Latent Spherical Virus (ALSV)-mediated virus-induced gene silencing (VIGS) protocol that will allow to perform functional genomics studies in quinoa in a fast and simple way. The method was fine-tuned using ALSV plasmids that carry partial gene sequences of phytoene desaturase (PDS) from Nicotiana benthamiana and quinoa. The developed technique involves an initial inoculation in Nicotiana plants through agroinfiltration with Agrobacterium tumefaciens cultures carrying the different viral constructs. Viral extracts were prepared using local or systemic leaves, which were then used as inoculum to infect quinoa leaves through mechanical damage. The method was successfully tested in two contrasting quinoa varieties, although some differences were observed in infection phenotype and viral susceptibility. The effect of insertion sequence size in the viral vectors was also analyzed, resulting in differences in bleaching or chlorosis phenotype and impact on plant growth. The presence of the virus in inoculated plants was confirmed, and the reduction in PDS gene expression in silenced plants was verified. Because quinoa lacks stable transformation protocols, limiting heterologous expression assays, our ALSV-based VIGS protocol is very attractive for loss-of-function gene studies.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs

ABSTRACT Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

The full spectrum of SLC22 OCT1 mutations illuminates the bridge between drug transporter biophysics and pharmacogenomics

Mutations in transporters can impact an individual's response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.

Rapid production of abundant transgenic pomegranate (Punica granatum) hairy roots

Pomegranates (Punica granatum) are known for their high levels of health-beneficial compounds that belong to the hydrolyzable tannin (HT) and flavonoid families. However, a significant gap in our understanding exists of the biosynthetic and regulatory genes related to the accumulation of these compounds. To this end, the induction and transformation of hairy roots presents an exciting opportunity to decipher genetically the functions of candidate genes involved in the HT and flavonoid metabolism. This study aims at improving methods for rapid and abundant hairy root production from pomegranate explants by increasing seed germination rates and expediting the attainment of requisite biomass for multifaceted analyses. We found that treating seeds with sandpaper and concentrated sulfuric acid significantly enhanced their germination rates. Interestingly, no correlation between seed-coat texture and seed germination rate was observed among the 20 pomegranate accessions evaluated in this study. Notably, transferring hairy roots from agar plates to soil promoted rapid root biomass growth compared to maintaining them on agar plates. Root biomass as well as levels of gallic acid (a precursor for HT biosynthesis) and punicalagins (the major HTs in roots) across 15 pomegranate accessions were also analyzed. Three of these accessions exhibited higher seed germination percentages along with augmented biomass and elevated punicalagin levels. These pomegranate accessions emerge as promising germplasm for future genetic transformation and functional genomics studies.

Assessment of Soil Bacterial Community Compositions by Structural–Functional Genomics and Soil Edaphic Factors Largely Explain the Impact of Coal Mining

Assessment of Soil Bacterial Community Compositions by Structural–Functional Genomics and Soil Edaphic Factors Largely Explain the Impact of Coal Mining

Knowledge-guided learning methods for integrative analysis of multi-omics data

Integrative analysis of multi-omics data has the potential to yield valuable and comprehensive insights into the molecular mechanisms underlying complex diseases such as cancer and Alzheimer's disease. However, a number of analytical challenges complicate multi-omics data integration. For instance, -omics data are usually high-dimensional, and sample sizes in multi-omics studies tend to be modest. Furthermore, when genes in an important pathway have relatively weak signal, it can be difficult to detect them individually. There is a growing body of literature on knowledge-guided learning methods that can address these challenges by incorporating biological knowledge such as functional genomics and functional proteomics into multi-omics data analysis. These methods have been shown to outperform their counterparts that do not utilize biological knowledge in tasks including prediction, feature selection, clustering, and dimension reduction. In this review, we survey recently developed methods and applications of knowledge-guided multi-omics data integration methods and discuss future research directions.