Drought response revealed by chromatin organization variation and transcriptional regulation in cotton.

BACKGROUND: Cotton is a major world cash crop and an important source of natural fiber, oil, and protein. Drought stress is becoming a restrictive factor affecting cotton production. To facilitate the development of drought-tolerant cotton varieties, it is necessary to study the molecular mechanism of drought stress response by exploring key drought-resistant genes and related regulatory factors. RESULTS: In this study, two cotton varieties, ZY007 (drought-sensitive) and ZY168 (drought-tolerant), showing obvious phenotypic differences under drought stress, were selected. A total of 25,898 drought-induced genes were identified, exhibiting significant enrichment in pathways related to plant stress responses. Under drought induction, At subgenome expression bias was observed at the whole-genome level, which may be due to stronger inhibition of Dt subgenome expression. A gene co-expression module that was significantly associated with drought resistance was identified. About 90% of topologically associating domain (TAD) boundaries were stable, and 6613 TAD variation events were identified between the two varieties under drought. We identified 92 genes in ZY007 and 98 in ZY168 related to chromatin 3D structural variation and induced by drought stress. These genes are closely linked to the cotton response to drought stress through canonical hormone-responsive pathways, modulation of kinase and phosphatase activities, facilitation of calcium ion transport, and other related molecular mechanisms. CONCLUSIONS: These results lay a foundation for elucidating the molecular mechanism of the cotton drought response and provide important regulatory locus and gene resources for the future molecular breeding of drought-resistant cotton varieties.

Pangenome analysis reveals transposon-driven genome evolution in cotton.

BACKGROUND: Transposable elements (TEs) have a profound influence on the trajectory of plant evolution, driving genome expansion and catalyzing phenotypic diversification. The pangenome, a comprehensive genetic pool encompassing all variations within a species, serves as an invaluable tool, unaffected by the confounding factors of intraspecific diversity. This allows for a more nuanced exploration of plant TE evolution. RESULTS: Here, we constructed a pangenome for diploid A-genome cotton using 344 accessions from representative geographical regions, including 223 from China as the main component. We found 511 Mb of non-reference sequences (NRSs) and revealed the presence of 5479 previously undiscovered protein-coding genes. Our comprehensive approach enabled us to decipher the genetic underpinnings of the distinct geographic distributions of cotton. Notably, we identified 3301 presence-absence variations (PAVs) that are closely tied to gene expression patterns within the pangenome, among which 2342 novel expression quantitative trait loci (eQTLs) were found residing in NRSs. Our investigation also unveiled contrasting patterns of transposon proliferation between diploid and tetraploid cotton, with long terminal repeat (LTR) retrotransposons exhibiting a synchronized surge in polyploids. Furthermore, the invasion of LTR retrotransposons from the A subgenome to the D subgenome triggered a substantial expansion of the latter following polyploidization. In addition, we found that TE insertions were responsible for the loss of 36.2% of species-specific genes, as well as the generation of entirely new species-specific genes. CONCLUSIONS: Our pangenome analyses provide new insights into cotton genomics and subgenome dynamics after polyploidization and demonstrate the power of pangenome approaches for elucidating transposon impacts and genome evolution.

A dominant negative mutation of GhMYB25-like alters cotton fiber initiation, reducing lint and fuzz.

Cotton (Gossypium hirsutum) fibers, vital natural textile materials, are single-cell trichomes that differentiate from the ovule epidermis. These fibers are categorized as lint (longer fibers useful for spinning) or fuzz (shorter, less useful fibers). Currently, developing cotton varieties with high lint yield but without fuzz remains challenging due to our limited knowledge of the molecular mechanisms underlying fiber initiation. This study presents the identification and characterization of a naturally occurring dominant negative mutation GhMYB25-like_AthapT, which results in a reduced lint and fuzzless phenotype. The GhMYB25-like_AthapT protein exerts its dominant negative effect by suppressing the activity of GhMYB25-like during lint and fuzz initiation. Intriguingly, the negative effect of GhMYB25-like_AthapT could be alleviated by high expression levels of GhMYB25-like. We also uncovered the role of GhMYB25-like in regulating the expression of key genes such as GhPDF2 (PROTODERMAL FACTOR 2), CYCD3; 1 (CYCLIN D3; 1) and PLD (Phospholipase D), establishing its significance as a pivotal transcription factor in fiber initiation. We identified other genes within this regulatory network, expanding our understanding of the determinants of fiber cell fate. These findings offer valuable insights for cotton breeding and contribute to our fundamental understanding of fiber development.

Synergistic interplay of redox homeostasis and polysaccharide synthesis promotes cotton fiber elongation.

Cell polarity is the foundation of cell development and tissue morphogenesis. The investigation of polarized growth provides opportunities to gain profound insights into morphogenesis and tissue functionality in organisms. Currently, there are still many mysteries surrounding the mechanisms that regulate polarized cell growth. Cotton fiber cells serve as an excellent model for studying polarized growth, and provide important clues for unraveling the molecular mechanisms, signaling pathways, and regulatory networks of polarized growth. In this study, we characterized two functional genes, GhMDHAR1AT/DT and GhDHAR2AT/DT with predominant expression during fiber elongation. Loss of function of both genes contributed to a significant increase in fiber length. Transcriptomic data revealed up-regulated expression of antioxidant genes in CRISPR mutant lines, along with delayed expression of secondary wall-related genes and temporally prolonged expression of primary wall-related genes. Experimental evidence demonstrated that the increase in GSH content and glutathione peroxidase (GPX) enzyme activity led to enhanced total antioxidant capacity (T-AOC), resulting in reduced H2O2 levels, which contributed to the extension of fiber elongation stage in CRISPR mutant lines. Moreover, the increased polysaccharide synthesis in CRISPR mutant lines was found to provide an abundant supply of raw materials for fiber cell wall elongation, suggesting that synergistic interplay between redox homeostasis and polysaccharide synthesis in fiber cells may facilitate cell wall remodeling and fiber elongation. This study provides valuable insights for deciphering the mechanisms of cell polarized growth and improving cotton fiber quality.

High-quality Gossypium hirsutum and Gossypium barbadense genome assemblies reveal the landscape and evolution of centromeres.

Centromere positioning and organization are crucial for genome evolution; however, research on centromere biology is largely influenced by the quality of available genome assemblies. Here, we combined Oxford Nanopore and Pacific Biosciences technologies to de novo assemble two high-quality reference genomes for Gossypium hirsutum (TM-1) and Gossypium barbadense (3-79). Compared with previously published reference genomes, our assemblies show substantial improvements, with the contig N50 improved by 4.6-fold and 5.6-fold, respectively, and thus represent the most complete cotton genomes to date. These high-quality reference genomes enable us to characterize 14 and 5 complete centromeric regions for G. hirsutum and G. barbadense, respectively. Our data revealed that the centromeres of allotetraploid cotton are occupied by members of the centromeric repeat for maize (CRM) and Tekay long terminal repeat families, and the CRM family reshapes the centromere structure of the At subgenome after polyploidization. These two intertwined families have driven the convergent evolution of centromeres between the two subgenomes, ensuring centromere function and genome stability. In addition, the repositioning and high sequence divergence of centromeres between G. hirsutum and G. barbadense have contributed to speciation and centromere diversity. This study sheds light on centromere evolution in a significant crop and provides an alternative approach for exploring the evolution of polyploid plants.

Evolutionary insights into the organization of chromatin structure and landscape of transcriptional regulation in plants.

Development of complex traits necessitates the functioning and coordination of intricate regulatory networks involving multiple genes. Understanding 3D chromatin structure can facilitate insight into the regulation of gene expression by regulatory elements. This potential, of visualizing the role of chromatin organization in the evolution and function of regulatory elements, remains largely unexplored. Here, we describe new perspectives that arise from the dual considerations of sequence variation of regulatory elements and chromatin structure, with a special focus on whole-genome doubling or polyploidy. We underscore the significance of hierarchical chromatin organization in gene regulation during evolution. In addition, we describe strategies for exploring chromatin organization in future investigations of regulatory evolution in plants, enabling insights into the evolutionary influence of regulatory elements on gene expression and, hence, phenotypes.

Regulatory controls of duplicated gene expression during fiber development in allotetraploid cotton.

Polyploidy complicates transcriptional regulation and increases phenotypic diversity in organisms. The dynamics of genetic regulation of gene expression between coresident subgenomes in polyploids remains to be understood. Here we document the genetic regulation of fiber development in allotetraploid cotton Gossypium hirsutum by sequencing 376 genomes and 2,215 time-series transcriptomes. We characterize 1,258 genes comprising 36 genetic modules that control staged fiber development and uncover genetic components governing their partitioned expression relative to subgenomic duplicated genes (homoeologs). Only about 30% of fiber quality-related homoeologs show phenotypically favorable allele aggregation in cultivars, highlighting the potential for subgenome additivity in fiber improvement. We envision a genome-enabled breeding strategy, with particular attention to 48 favorable alleles related to fiber phenotypes that have been subjected to purifying selection during domestication. Our work delineates the dynamics of gene regulation during fiber development and highlights the potential of subgenomic coordination underpinning phenotypes in polyploid plants.

Tcbf: a novel user-friendly tool for pan-3D genome analysis of topologically associating domain in eukaryotic organisms.

SUMMARY: TAD boundaries are essential for organizing the chromatin spatial structure and regulating gene expression in eukaryotes. However, for large-scale pan-3D genome research, identifying conserved and specific TAD boundaries across different species or individuals is computationally challenging. Here, we present Tcbf, a rapid and powerful Python/R tool that integrates gene synteny blocks and homologous sequences to automatically detect conserved and specific TAD boundaries among multiple species, which can efficiently analyze huge genome datasets, greatly reduce the computational burden and enable pan-3D genome research. AVAILABILITY AND IMPLEMENTATION: Tcbf is implemented by Python/R and is available at https://github.com/TcbfGroup/Tcbf under the MIT license.

Histone H3 lysine 27 trimethylation suppresses jasmonate biosynthesis and signaling to affect male fertility under high temperature in cotton.

High-temperature (HT) stress causes male sterility in crops, thus decreasing yields. To explore the possible contribution of histone modifications to male fertility under HT conditions, we defined the histone methylation landscape for the marks histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) by chromatin immunoprecipitation sequencing (ChIP-seq) in two differing upland cotton (Gossypium hirsutum) varieties. We observed a global disruption in H3K4me3 and H3K27me3 modifications, especially H3K27me3, in cotton anthers subjected to HT. HT affected the bivalent H3K4me3-H3K27me3 modification more than either monovalent modification. We determined that removal of H3K27me3 at the promoters of jasmonate-related genes increased their expression, maintaining male fertility under HT in the HT-tolerant variety at the anther dehiscence stage. Modulating jasmonate homeostasis or signaling resulted in an anther indehiscence phenotype under HT. Chemical suppression of H3K27me3 deposition increased jasmonic acid contents and maintained male fertility under HT. In summary, our study provides new insights into the regulation of male fertility by histone modifications under HT and suggests a potential strategy for improving cotton HT tolerance.

Gossypium mustelinum genome and an introgression population enrich interspecific genetics and breeding in cotton.

KEY MESSAGE: Genomic and genetic resources of G. mustelinum were effective for identifying genes for qualitative and quantitative traits. Gossypium mustelinum represents the earliest diverging evolutionary lineage of polyploid Gossypium, representing a rich gene pool for numerous desirable traits lost in cotton cultivars. Accurate information of the genomic features and the genetic architecture of objective traits are essential for the discovery and utilization of G. mustelinum genes. Here, we presented a chromosome-level genome assembly of G. mustelinum and developed an introgression population of the G. mustelinum in the background of G. hirsutum that contained 264 lines. We precisely delimited the boundaries of the 1,662 introgression segments with the help of G. mustelinum genome assembly, and 87% of crossover regions (COs) were less than 5 Kb. Genes for fuzzless and green fuzz were discovered, and a total of 14 stable QTLs were identified with 12 novel QTLs across four independent environments. A new fiber length QTL, qUHML/SFC-A11, was confined to a 177-Kb region, and GmOPB4 and GmGUAT11 were considered as the putative candidate genes as potential negative regulator for fiber length. We presented a genomic and genetic resource of G. mustelinum, which we demonstrated that it was efficient for identifying genes for qualitative and quantitative traits. Our study built a valuable foundation for cotton genetics and breeding.

Genetic interrogation of phenotypic plasticity informs genome-enabled breeding in cotton.

Phenotypic plasticity, or the ability to adapt to and thrive in changing climates and variable environments, is essential for developmental programs in plants. Despite its importance, the genetic underpinnings of phenotypic plasticity for key agronomic traits remain poorly understood in many crops. In this study, we aim to fill this gap by using genome-wide association studies to identify genetic variations associated with phenotypic plasticity in upland cotton (Gossypium hirsutum L.). We identified 73 additive quantitative trait loci (QTLs), 32 dominant QTLs, and 6799 epistatic QTLs associated with 20 traits. We also identified 117 additive QTLs, 28 dominant QTLs, and 4691 epistatic QTLs associated with phenotypic plasticity in 19 traits. Our findings reveal new genetic factors, including additive, dominant, and epistatic QTLs, that are linked to phenotypic plasticity and agronomic traits. Meanwhile, we find that the genetic factors controlling the mean phenotype and phenotypic plasticity are largely independent in upland cotton, indicating the potential for simultaneous improvement. Additionally, we envision a genomic design strategy by utilizing the identified QTLs to facilitate cotton breeding. Taken together, our study provides new insights into the genetic basis of phenotypic plasticity in cotton, which should be valuable for future breeding.

N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner.

N6 -methyladenosine (m6 A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post-transcriptional levels. However, the biological function and regulatory mechanism of m6 A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m6 A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6 A methylation in cotton response to drought stress. The results reveal that m6 A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6 A was deposited in the drought-resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m6 A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6 A levels, facilitating the mRNA decay of ABA signal-related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca2+ signal-related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus-induced gene silencing (VIGS) of two Ca2+ -related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m6 A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post-transcriptional modification involved in affecting drought response in cotton, by mediating m6 A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways.

A comprehensive overview of cotton genomics, biotechnology and molecular biological studies.

Cotton is an irreplaceable economic crop currently domesticated in the human world for its extremely elongated fiber cells specialized in seed epidermis, which makes it of high research and application value. To date, numerous research on cotton has navigated various aspects, from multi-genome assembly, genome editing, mechanism of fiber development, metabolite biosynthesis, and analysis to genetic breeding. Genomic and 3D genomic studies reveal the origin of cotton species and the spatiotemporal asymmetric chromatin structure in fibers. Mature multiple genome editing systems, such as CRISPR/Cas9, Cas12 (Cpf1) and cytidine base editing (CBE), have been widely used in the study of candidate genes affecting fiber development. Based on this, the cotton fiber cell development network has been preliminarily drawn. Among them, the MYB-bHLH-WDR (MBW) transcription factor complex and IAA and BR signaling pathway regulate the initiation; various plant hormones, including ethylene, mediated regulatory network and membrane protein overlap fine-regulate elongation. Multistage transcription factors targeting CesA 4, 7, and 8 specifically dominate the whole process of secondary cell wall thickening. And fluorescently labeled cytoskeletal proteins can observe real-time dynamic changes in fiber development. Furthermore, research on the synthesis of cotton secondary metabolite gossypol, resistance to diseases and insect pests, plant architecture regulation, and seed oil utilization are all conducive to finding more high-quality breeding-related genes and subsequently facilitating the cultivation of better cotton varieties. This review summarizes the paramount research achievements in cotton molecular biology over the last few decades from the above aspects, thereby enabling us to conduct a status review on the current studies of cotton and provide strong theoretical support for the future direction.

Genomic confirmation of Gossypium barbadense introgression into G. hirsutum and a subsequent MAGIC population.

Introgression of superior fiber traits from Pima cotton (Gossypium barbadense, GB) into high yield Upland cotton (G. hirsutum) has been a breeding objective for many years in a few breeding programs in the world. However, progress has been very slow due to introgression barriers resulting from whole genome hybridization between the two species. To minimize such barriers, chromosome substitution lines (CS-B) from Pima cotton 3-79 in an Upland cotton cultivar TM-1 were developed. A multiparent advanced generation inter-cross (MAGIC) population consisting of 180 recombinant inbred lines (RILs) was subsequently made using the 18 CS-B lines and three Upland cotton cultivars as parents. In this research, we sequenced the whole genomes of the 21 parents and 180 RILs to examine the G. barbadense introgression. Of the 18 CS-B lines, 11 contained the target GB chromosome or chromosome segment, two contained more than two GB chromosomes, and five did not have the expected introgression. Residual introgression in non-target chromosomes was prevalent in all CS-B lines. A clear structure existed in the MAGIC population and the 180 RILs were distributed into three groups, i.e., high, moderate, and low GB introgression. Large blocks of GB chromosome introgression were still present in some RILs after five cycles of random-mating, an indication of recombination suppression or other unknown reasons present in the population. Identity by descent analysis revealed that the MAGIC RILs contained less introgression than expected. This research presents an insight on understanding the complex problems of introgression between cotton species.

Genomic innovation and regulatory rewiring during evolution of the cotton genus Gossypium.

Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypium genus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.

Genome-wide association study and transcriptome analysis reveal key genes controlling fruit branch angle in cotton.

Fruit branch angle (FBA), a pivotal component of cotton plant architecture, is vital for field and mechanical harvesting. However, the molecular mechanism of FBA formation is poorly understood in cotton. To uncover the genetic basis for FBA formation in cotton, we performed a genome-wide association study (GWAS) of 163 cotton accessions with re-sequencing data. A total of 55 SNPs and 18 candidate genes were significantly associated with FBA trait. By combining GWAS and transcriptome analysis, four genes underlying FBA were identified. An FBA-associated candidate gene Ghi_A09G08736, which is homologous to SAUR46 in Arabidopsis thaliana, was detected in our study. In addition, transcriptomic evidence was provided to show that gravity and light were implicated in the FBA formation. This study provides new insights into the genetic architecture of FBA that informs architecture breeding in cotton.

Single-cell RNA-seq reveals fate determination control of an individual fibre cell initiation in cotton (Gossypium hirsutum).

Cotton fibre is a unicellular seed trichome, and lint fibre initials per seed as a factor determines fibre yield. However, the mechanisms controlling fibre initiation from ovule epidermis are not understood well enough. Here, with single-cell RNA sequencing (scRNA-seq), a total of 14 535 cells were identified from cotton ovule outer integument of Xu142_LF line at four developmental stages (1.5, 1, 0.5 days before anthesis and the day of anthesis). Three major cell types, fibre, non-fibre epidermis and outer pigment layer were identified and then verified by RNA in situ hybridization. A comparative analysis on scRNA-seq data between Xu142 and its fibreless mutant Xu142 fl further confirmed fibre cluster definition. The developmental trajectory of fibre cell was reconstructed, and fibre cell was identified differentiated at 1 day before anthesis. Gene regulatory networks at four stages revealed the spatiotemporal pattern of core transcription factors, and MYB25-like and HOX3 were demonstrated played key roles as commanders in fibre differentiation and tip-biased diffuse growth respectively. A model for early development of a single fibre cell was proposed here, which sheds light on further deciphering mechanism of plant trichome and the improvement of cotton fibre yield.

Genomic insights into the recent chromosome reduction of autopolyploid sugarcane Saccharum spontaneum.

Saccharum spontaneum is a founding Saccharum species and exhibits wide variation in ploidy levels. We have assembled a high-quality autopolyploid genome of S. spontaneum Np-X (2n = 4x = 40) into 40 pseudochromosomes across 10 homologous groups, that better elucidates recent chromosome reduction and polyploidization that occurred circa 1.5 million years ago (Mya). One paleo-duplicated chromosomal pair in Saccharum, NpChr5 and NpChr8, underwent fission followed by fusion accompanied by centromeric split around 0.80 Mya. We inferred that Np-X, with x = 10, most likely represents the ancestral karyotype, from which x = 9 and x = 8 evolved. Resequencing of 102 S. spontaneum accessions revealed that S. spontaneum originated in northern India from an x = 10 ancestor, which then radiated into four major groups across the Indian subcontinent, China, and Southeast Asia. Our study suggests new directions for accelerating sugarcane improvement and expands our knowledge of the evolution of autopolyploids.