Unique regulatory network of dragon fruit simultaneously mitigates the effect of vanadium pollutant and environmental factors.

Under changing climatic conditions, plants are simultaneously facing conflicting stresses in nature. Plants can sense different stresses, induce systematic ROS signals, and regulate transcriptomic, hormonal, and stomatal responses. We performed transcriptome analysis to reveal the integrative stress response regulatory mechanism underlying heavy metal stress alone or in combination with heat and drought conditions in pitaya (dragon fruit). A total of 70 genes were identified from 31,130 transcripts with conserved differential expression. Furthermore, weighted gene co-expression network analysis (WGCNA) identified trait-associated modules. By integrating information from three modules and protein-protein interaction (PPI) networks, we identified 10 interconnected genes associated with the multifaceted defense mechanism employed by pitaya against co-occurring stresses. To further confirm the reliability of the results, we performed a comparative analysis of 350 genes identified by three trait modules and 70 conserved genes exhibiting their dynamic expression under all treatments. Differential expression pattern of genes and comparative analysis, have proven instrumental in identifying ten putative structural genes. These ten genes were annotated as PLAT/LH2, CAT, MLP, HSP, PB1, PLA, NAC, HMA, and CER1 transcription factors involved in antioxidant activity, defense response, MAPK signaling, detoxification of metals and regulating the crosstalk between the complex pathways. Predictive analysis of putative candidate genes, potentially governing single, double, and multifactorial stress response, by several signaling systems and molecular patterns. These findings represent a valuable resource for pitaya breeding programs, offering the potential to develop resilient "super pitaya" plants.

Cicer super-pangenome provides insights into species evolution and agronomic trait loci for crop improvement in chickpea.

Chickpea (Cicer arietinum L.)-an important legume crop cultivated in arid and semiarid regions-has limited genetic diversity. Efforts are being undertaken to broaden its diversity by utilizing its wild relatives, which remain largely unexplored. Here, we present the Cicer super-pangenome based on the de novo genome assemblies of eight annual Cicer wild species. We identified 24,827 gene families, including 14,748 core, 2,958 softcore, 6,212 dispensable and 909 species-specific gene families. The dispensable genome was enriched for genes related to key agronomic traits. Structural variations between cultivated and wild genomes were used to construct a graph-based genome, revealing variations in genes affecting traits such as flowering time, vernalization and disease resistance. These variations will facilitate the transfer of valuable traits from wild Cicer species into elite chickpea varieties through marker-assisted selection or gene-editing. This study offers valuable insights into the genetic diversity and potential avenues for crop improvement in chickpea.

The genome of Haberlea rhodopensis provides insights into the mechanisms for tolerance to multiple extreme environments.

Haberlea rhodopensis, a resurrection species, is the only plant known to be able to survive multiple extreme environments, including desiccation, freezing temperatures, and long-term darkness. However, the molecular mechanisms underlying tolerance to these stresses are poorly studied. Here, we present a high-quality genome of Haberlea and found that ~ 23.55% of the 44,306 genes are orphan. Comparative genomics analysis identified 89 significantly expanded gene families, of which 25 were specific to Haberlea. Moreover, we demonstrated that Haberlea preserves its resurrection potential even in prolonged complete darkness. Transcriptome profiling of plants subjected to desiccation, darkness, and low temperatures revealed both common and specific footprints of these stresses, and their combinations. For example, PROTEIN PHOSPHATASE 2C (PP2C) genes were substantially induced in all stress combinations, while PHYTOCHROME INTERACTING FACTOR 1 (PIF1) and GROWTH RESPONSE FACTOR 4 (GRF4) were induced only in darkness. Additionally, 733 genes with unknown functions and three genes encoding transcription factors specific to Haberlea were specifically induced/repressed upon combination of stresses, rendering them attractive targets for future functional studies. The study provides a comprehensive understanding of the genomic architecture and reports details of the mechanisms of multi-stress tolerance of this resurrection species that will aid in developing strategies that allow crops to survive extreme and multiple abiotic stresses.

ScRNA-seq reveals dark- and light-induced differentially expressed gene atlases of seedling leaves in Arachis hypogaea L.

Although the regulatory mechanisms of dark and light-induced plant morphogenesis have been broadly investigated, the biological process in peanuts has not been systematically explored on single-cell resolution. Herein, 10 cell clusters were characterized using scRNA-seq-identified marker genes, based on 13 409 and 11 296 single cells from 1-week-old peanut seedling leaves grown under dark and light conditions. 6104 genes and 50 transcription factors (TFs) displayed significant expression patterns in distinct cell clusters, which provided gene resources for profiling dark/light-induced candidate genes. Further pseudo-time trajectory and cell cycle evidence supported that dark repressed the cell division and perturbed normal cell cycle, especially the PORA abundances correlated with 11 TFs highly enriched in mesophyll to restrict the chlorophyllide synthesis. Additionally, light repressed the epidermis cell developmental trajectory extending by inhibiting the growth hormone pathway, and 21 TFs probably contributed to the different genes transcriptional dynamic. Eventually, peanut AHL17 was identified from the profile of differentially expressed TFs, which encoded protein located in the nucleus promoted leaf epidermal cell enlargement when ectopically overexpressed in Arabidopsis through the regulatory phytohormone pathway. Overall, our study presents the different gene atlases in peanut etiolated and green seedlings, providing novel biological insights to elucidate light-induced leaf cell development at the single-cell level.

A genomic variation map provides insights into peanut diversity in China and associations with 28 agronomic traits.

Peanut (Arachis hypogaea L.) is an important allotetraploid oil and food legume crop. China is one of the world's largest peanut producers and consumers. However, genomic variations underlying the migration and divergence of peanuts in China remain unclear. Here we reported a genome-wide variation map based on the resequencing of 390 peanut accessions, suggesting that peanuts might have been introduced into southern and northern China separately, forming two cultivation centers. Selective sweep analysis highlights asymmetric selection between the two subgenomes during peanut improvement. A classical pedigree from South China offers a context for the examination of the impact of artificial selection on peanut genome. Genome-wide association studies identified 22,309 significant associations with 28 agronomic traits, including candidate genes for plant architecture and oil biosynthesis. Our findings shed light on peanut migration and diversity in China and provide valuable genomic resources for peanut improvement.

Celebrating Professor Rajeev K. Varshney's transformative research odyssey from genomics to the field on his induction as Fellow of the Royal Society.

Professor Rajeev K. Varshney's transformative impact on crop genomics, genetics, and agriculture is the result of his passion, dedication, and unyielding commitment to harnessing the potential of genomics to address the most pressing challenges faced by the global agricultural community. Starting from a small town in India and reaching the global stage, Professor Varshney's academic and professional trajectory has inspired many scientists active in research today. His ground-breaking work, especially his effort to list orphan tropical crops to genomic resource-rich entities, has been transformative. Beyond his scientific achievements, Professor Varshney is recognized by his colleagues as an exemplary mentor, fostering the growth of future researchers, building institutional capacity, and strengthening scientific capability. His focus on translational genomics and strengthening seed system in developing countries for the improvement of agriculture has made a tangible impact on farmers' lives. His skills have been best utilized in roles at leading research centres where he has applied his expertise to deliver a new vision for crop improvement. These efforts have now been recognized by the Royal Society with the award of the Fellowship (FRS). As we mark this significant milestone in his career, we not only celebrate Professor Varshney's accomplishments but also his wider contributions that continue to transform the agricultural landscape.

A Single-Nucleus Resolution Atlas of Transcriptome and Chromatin Accessibility for Peanut (Arachis Hypogaea L.) Leaves.

The peanut is an important worldwide cash-crop for edible oil and protein. However, the kinetic mechanisms that determine gene expression and chromatin accessibility during leaf development in peanut represented allotetraploid leguminous crops are poorly understood at single-cell resolution. Here, a single-nucleus atlas of peanut leaves is developed by simultaneously profiling the transcriptome and chromatin accessibility in the same individual-cell using fluorescence-activated sorted single-nuclei. In total, 5930 cells with 50 890 expressed genes are classified into 18 cell-clusters, and 5315 chromatin fragments are enriched with 26 083 target genes in the chromatin accessible landscape. The developmental trajectory analysis reveals the involvement of the ethylene-AP2 module in leaf cell differentiation, and cell-cycle analysis demonstrated that genome replication featured in distinct cell-types with circadian rhythms transcription factors (TFs). Furthermore, dual-omics illustrates that the fatty acid pathway modulates epidermal-guard cells differentiation and providescritical TFs interaction networks for understanding mesophyll development, and the cytokinin module (LHY/LOG) that regulates vascular growth. Additionally, an AT-hook protein AhAHL11 is identified that promotes leaf area expansion by modulating the auxin content increase. In summary, the simultaneous profiling of transcription and chromatin accessibility landscapes using snRNA/ATAC-seq provides novel biological insights into the dynamic processes of peanut leaf cell development at the cellular level.

Near-gapless genome assemblies of Williams 82 and Lee cultivars for accelerating global soybean research.

Complete, gapless telomere-to-telomere chromosome assemblies are a prerequisite for comprehensively investigating the architecture of complex regions, like centromeres or telomeres and removing uncertainties in the order, spacing, and orientation of genes. Using complementary genomics technologies and assembly algorithms, we developed highly contiguous, nearly gapless, genome assemblies for two economically important soybean [Glycine max (L.) Merr] cultivars (Williams 82 and Lee). The centromeres were distinctly annotated on all the chromosomes of both assemblies. We further found that the canonical telomeric repeats were present at the telomeres of all chromosomes of both Williams 82 and Lee genomes. A total of 10 chromosomes in Williams 82 and eight in Lee were entirely reconstructed in single contigs without any gap. Using the combination of ab initio prediction, protein homology, and transcriptome evidence, we identified 58,287 and 56,725 protein-coding genes in Williams 82 and Lee, respectively. The genome assemblies and annotations will serve as a valuable resource for studying soybean genomics and genetics and accelerating soybean improvement.

scRNA-seq Reveals the Mechanism of Fatty Acid Desaturase 2 Mutation to Repress Leaf Growth in Peanut (Arachis hypogaea L.).

Fatty Acid Desaturase 2 (FAD2) controls the conversion of oleic acids into linoleic acids. Mutations in FAD2 not only increase the high-oleic content, but also repress the leaf growth. However, the mechanism by which FAD2 regulates the growth pathway has not been elucidated in peanut leaves with single-cell resolution. In this study, we isolated fad2 mutant leaf protoplast cells to perform single-cell RNA sequencing. Approximately 24,988 individual cells with 10,249 expressed genes were classified into five major cell types. A comparative analysis of 3495 differentially expressed genes (DEGs) in distinct cell types demonstrated that fad2 inhibited the expression of the cytokinin synthesis gene LOG in vascular cells, thereby repressing leaf growth. Further, pseudo-time trajectory analysis indicated that fad2 repressed leaf cell differentiation, and cell-cycle evidence displayed that fad2 perturbed the normal cell cycle to induce the majority of cells to drop into the S phase. Additionally, important transcription factors were filtered from the DEG profiles that connected the network involved in high-oleic acid accumulation (WRKY6), activated the hormone pathway (WRKY23, ERF109), and potentially regulated leaf growth (ERF6, MYB102, WRKY30). Collectively, our study describes different gene atlases in high-oleic and normal peanut seedling leaves, providing novel biological insights to elucidate the molecular mechanism of the high-oleic peanut-associated agronomic trait at the single-cell level.

Single cell RNA-seq in phytohormone signaling: a promising future.

Phytohormone signaling regulates plant growth and development. Single cell RNA sequencing (scRNA-seq) provides unprecedented opportunities to decipher hormone-mediated spatiotemporal gene regulatory networks. In a recent study, Nolan et al. used time-series scRNA-seq to identify the cortex as a key site for brassinosteroid (BR)-mediated gene expression and revealed a signaling network during cell phase transition.

Transcriptome profiling reveals the expression and regulation of genes associated with Fusarium wilt resistance in chickpea (Cicer arietinum L.).

Fusarium wilt (FW) is one of the most significant biotic stresses limiting chickpea production worldwide. To dissect the molecular mechanism of FW resistance in chickpea, comparative transcriptome analyses of contrasting resistance sources of chickpea genotypes under control and Fusarium oxysporum f. sp. ciceris (Foc) inoculated conditions were performed. The high-throughput transcriptome sequencing generated about 1137 million sequencing reads from 24 samples representing two resistant genotypes, two susceptible genotypes, and two near-isogenic lines under control and stress conditions at two-time points (7th- and 12th-day post-inoculation). The analysis identified 5182 differentially expressed genes (DEGs) between different combinations of chickpea genotypes. Functional annotation of these genes indicated their involvement in various biological processes such as defense response, cell wall biogenesis, secondary metabolism, and disease resistance. A significant number (382) of transcription factor encoding genes exhibited differential expression patterns under stress. Further, a considerable number of the identified DEGs (287) co-localized with previously reported quantitative trait locus for FW resistance. Several resistance/susceptibility-related genes, such as SERINE/THREONINE PROTEIN KINASE, DIRIGENT, and MLO exhibiting contrasting expression patterns in resistant and susceptible genotypes upon Foc inoculation, were identified. The results presented in the study provide valuable insights into the transcriptional dynamics associated with FW stress response in chickpea and provide candidate genes for the development of disease-resistant chickpea cultivars.

Comprehensive Transcriptome Profiling Uncovers Molecular Mechanisms and Potential Candidate Genes Associated with Heat Stress Response in Chickpea.

Chickpea (Cicer arietinum L.) production is highly susceptible to heat stress (day/night temperatures above 32/20 °C). Identifying the molecular mechanisms and potential candidate genes underlying heat stress response is important for increasing chickpea productivity. Here, we used an RNA-seq approach to investigate the transcriptome dynamics of 48 samples which include the leaf and root tissues of six contrasting heat stress responsive chickpea genotypes at the vegetative and reproductive stages of plant development. A total of 14,544 unique, differentially expressed genes (DEGs) were identified across different combinations studied. These DEGs were mainly involved in metabolic processes, cell wall remodeling, calcium signaling, and photosynthesis. Pathway analysis revealed the enrichment of metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction, under heat stress conditions. Furthermore, heat-responsive genes encoding bHLH, ERF, WRKY, and MYB transcription factors were differentially regulated in response to heat stress, and candidate genes underlying the quantitative trait loci (QTLs) for heat tolerance component traits, which showed differential gene expression across tolerant and sensitive genotypes, were identified. Our study provides an important resource for dissecting the role of candidate genes associated with heat stress response and also paves the way for developing climate-resilient chickpea varieties for the future.

Chromosome-length genome assemblies of six legume species provide insights into genome organization, evolution, and agronomic traits for crop improvement.

INTRODUCTION: Legume crops are an important source of protein and oil for human health and in fixing atmospheric N2 for soil enrichment. With an objective to accelerate much-needed genetic analyses and breeding applications, draft genome assemblies were generated in several legume crops; many of them are not high quality because they are mainly based on short reads. However, the superior quality of genome assembly is crucial for a detailed understanding of genomic architecture, genome evolution, and crop improvement. OBJECTIVES: Present study was undertaken with an objective of developing improved chromosome-length genome assemblies in six different legumes followed by their systematic investigation to unravel different aspects of genome organization and legume evolution. METHODS: We employed in situ Hi-C data to improve the existing draft genomes and performed different evolutionary and comparative analyses using improved genome assemblies. RESULTS: We have developed chromosome-length genome assemblies in chickpea, pigeonpea, soybean, subterranean clover, and two wild progenitor species of cultivated groundnut (A. duranensis and A. ipaensis). A comprehensive comparative analysis of these genome assemblies offered improved insights into various evolutionary events that shaped the present-day legume species. We highlighted the expansion of gene families contributing to unique traits such as nodulation in legumes, gravitropism in groundnut, and oil biosynthesis in oilseed legume crops such as groundnut and soybean. As examples, we have demonstrated the utility of improved genome assemblies for enhancing the resolution of "QTL-hotspot" identification for drought tolerance in chickpea and marker-trait associations for agronomic traits in pigeonpea through genome-wide association study. Genomic resources developed in this study are publicly available through an online repository, 'Legumepedia'. CONCLUSION: This study reports chromosome-length genome assemblies of six legume species and demonstrates the utility of these assemblies in crop improvement. The genomic resources developed here will have significant role in accelerating genetic improvement applications of legume crops.

Genetic variation in CaTIFY4b contributes to drought adaptation in chickpea.

Chickpea production is vulnerable to drought stress. Identifying the genetic components underlying drought adaptation is crucial for enhancing chickpea productivity. Here, we present the fine mapping and characterization of 'QTL-hotspot', a genomic region controlling chickpea growth with positive consequences on crop production under drought. We report that a non-synonymous substitution in the transcription factor CaTIFY4b regulates seed weight and organ size in chickpea. Ectopic expression of CaTIFY4b in Medicago truncatula enhances root growth under water deficit. Our results suggest that allelic variation in 'QTL-hotspot' improves pre-anthesis water use, transpiration efficiency, root architecture and canopy development, enabling high-yield performance under terminal drought conditions. Gene expression analysis indicated that CaTIFY4b may regulate organ size under water deficit by modulating the expression of GRF-INTERACTING FACTOR1 (GIF1), a transcriptional co-activator of Growth-Regulating Factors. Taken together, our study offers new insights into the role of CaTIFY4b and on diverse physiological and molecular mechanisms underpinning chickpea growth and production under specific drought scenarios.

Analysis of Small RNA Sequencing Data in Plants.

Over the past decades, next-generation sequencing (NGS) has been employed extensively for investigating the regulatory mechanisms of small RNAs. Several bioinformatics tools are available for aiding biologists to extract meaningful information from enormous amounts of data generated by NGS platforms. This chapter describes a detailed methodology for analyzing small RNA sequencing data using different open source tools. We elaborate on various steps involved in analysis, from processing the raw sequencing reads to identifying miRNAs, their targets, and differential expression studies.

A chickpea genetic variation map based on the sequencing of 3,366 genomes.

Zero hunger and good health could be realized by 2030 through effective conservation, characterization and utilization of germplasm resources1. So far, few chickpea (Cicer arietinum) germplasm accessions have been characterized at the genome sequence level2. Here we present a detailed map of variation in 3,171 cultivated and 195 wild accessions to provide publicly available resources for chickpea genomics research and breeding. We constructed a chickpea pan-genome to describe genomic diversity across cultivated chickpea and its wild progenitor accessions. A divergence tree using genes present in around 80% of individuals in one species allowed us to estimate the divergence of Cicer over the last 21 million years. Our analysis found chromosomal segments and genes that show signatures of selection during domestication, migration and improvement. The chromosomal locations of deleterious mutations responsible for limited genetic diversity and decreased fitness were identified in elite germplasm. We identified superior haplotypes for improvement-related traits in landraces that can be introgressed into elite breeding lines through haplotype-based breeding, and found targets for purging deleterious alleles through genomics-assisted breeding and/or gene editing. Finally, we propose three crop breeding strategies based on genomic prediction to enhance crop productivity for 16 traits while avoiding the erosion of genetic diversity through optimal contribution selection (OCS)-based pre-breeding. The predicted performance for 100-seed weight, an important yield-related trait, increased by up to 23% and 12% with OCS- and haplotype-based genomic approaches, respectively.

Comprehensive analysis and identification of drought-responsive candidate NAC genes in three semi-arid tropics (SAT) legume crops.

BACKGROUND: Chickpea, pigeonpea, and groundnut are the primary legume crops of semi-arid tropics (SAT) and their global productivity is severely affected by drought stress. The plant-specific NAC (NAM - no apical meristem, ATAF - Arabidopsis transcription activation factor, and CUC - cup-shaped cotyledon) transcription factor family is known to be involved in majority of abiotic stresses, especially in the drought stress tolerance mechanism. Despite the knowledge available regarding NAC function, not much information is available on NAC genes in SAT legume crops. RESULTS: In this study, genome-wide NAC proteins - 72, 96, and 166 have been identified from the genomes of chickpea, pigeonpea, and groundnut, respectively, and later grouped into 10 clusters in chickpea and pigeonpea, while 12 clusters in groundnut. Phylogeny with well-known stress-responsive NACs in Arabidopsis thaliana, Oryza sativa (rice), Medicago truncatula, and Glycine max (soybean) enabled prediction of putative stress-responsive NACs in chickpea (22), pigeonpea (31), and groundnut (33). Transcriptome data revealed putative stress-responsive NACs at various developmental stages that showed differential expression patterns in the different tissues studied. Quantitative real-time PCR (qRT-PCR) was performed to validate the expression patterns of selected stress-responsive, Ca_NAC (Cicer arietinum - 14), Cc_NAC (Cajanus cajan - 15), and Ah_NAC (Arachis hypogaea - 14) genes using drought-stressed and well-watered root tissues from two contrasting drought-responsive genotypes of each of the three legumes. Based on expression analysis, Ca_06899, Ca_18090, Ca_22941, Ca_04337, Ca_04069, Ca_04233, Ca_12660, Ca_16379, Ca_16946, and Ca_21186; Cc_26125, Cc_43030, Cc_43785, Cc_43786, Cc_22429, and Cc_22430; Ah_ann1.G1V3KR.2, Ah_ann1.MI72XM.2, Ah_ann1.V0X4SV.1, Ah_ann1.FU1JML.2, and Ah_ann1.8AKD3R.1 were identified as potential drought stress-responsive candidate genes. CONCLUSION: As NAC genes are known to play role in several physiological and biological activities, a more comprehensive study on genome-wide identification and expression analyses of the NAC proteins have been carried out in chickpea, pigeonpea and groundnut. We have identified a total of 21 potential drought-responsive NAC genes in these legumes. These genes displayed correlation between gene expression, transcriptional regulation, and better tolerance against drought. The identified candidate genes, after validation, may serve as a useful resource for molecular breeding for drought tolerance in the SAT legume crops.