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.

The chromosome-scale reference genome of mirid bugs (Adelphocoris suturalis) genome provides insights into omnivory, insecticide resistance, and survival adaptation.

BACKGROUND: Adelphocoris suturalis (Hemiptera: Miridae) is a notorious agricultural pest, which causes serious economic losses to a diverse range of agricultural crops around the world. The poor understanding of its genomic characteristics has seriously hindered the establishment of sustainable and environment-friendly agricultural pest management through biotechnology and biological insecticides. RESULTS: Here, we report a chromosome-level assembled genome of A. suturalis by integrating Illumina short reads, PacBio, 10x Chromium, and Hi-C mapping technologies. The resulting 1.29 Gb assembly contains twelve chromosomal pseudomolecules with an N50 of 1.4 and 120.6 Mb for the contigs and scaffolds, respectively, and carries 20,010 protein-coding genes. The considerable size of the A. suturalis genome is predominantly attributed to a high amount of retrotransposons, especially long interspersed nuclear elements (LINEs). Transcriptomic and phylogenetic analyses suggest that A. suturalis-specific candidate effectors, and expansion and expression of gene families associated with omnivory, insecticide resistance and reproductive characteristics, such as digestion, detoxification, chemosensory receptors and long-distance migration likely contribute to its strong environmental adaptability and ability to damage crops. Additionally, 19 highly credible effector candidates were identified and transiently overexpressed in Nicotiana benthamiana for functional assays and potential targeting for insect resistance genetic engineering. CONCLUSIONS: The high-quality genome of A. suturalis provides an important genomic landscape for further investigations into the mechanisms of omnivory, insecticide resistance and survival adaptation, and for the development of integrated management strategies.

Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem.

BACKGROUND: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. RESULTS: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. CONCLUSIONS: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Genome and haplotype provide insights into the population differentiation and breeding improvement of Gossypium barbadense.

INTRODUCTION: Sea-island cotton (Gossypium barbadense, Gb) is one of the major sources of high-grade natural fiber. Besides the common annual Gb cotton, perennial Gb cotton is also cultivated, but studies on perennial Gb cotton are rare. OBJECTIVES: We aimed to make a systematic analysis of perennial sea-island cotton and lay a foundation for its utilization in breeding, and try to identify the representative structural variations (SVs) in sea-island cotton, and to reveal the population differentiation and adaptive improvement of sea-island cotton. METHODS: Through genome assembly of one perennial Gb cotton accession (named Gb_M210936) and comparative genome analysis, variations during Gb cotton domestication were identified by comparing Gb_M210936 with annual Gb accession 3-79 and with wild allotetraploid cotton G. darwinii. Six perennial Gb accessions combining with the resequenced 1,129 cotton accessions were used to conduct population and genetic analysis. Large haplotype blocks (haploblocks), generated from interspecific introgressions and intraspecific inversions, were identified and were used to analyze their effects on population differentiation and agronomic traits of sea-island cotton. RESULTS: One reference genome of perennial sea-island cotton was assembled. Representative SVs in sea-island cotton were identified, and 31 SVs were found to be associated with agronomic traits. Perennial Gb cotton had a closer kinship with the wild-to-landrace continuum Gb cotton from south America where Gb cotton is originally domesticated. Haploblocks were associated with agronomic traits improvement of sea-island cotton, promoted sea-island cotton differentiation into three subgroups, were suffered from breeding selection, and may drive Gb cotton to be adapted to central Asian. CONCLUSION: Our study made up the lack of perennial Gb cotton genome, and clarified that exotic introgressions improved the traits of sea-island cotton, promoted the population differentiation, and drove sea-island cotton adaptive to central Asia, which will provide new insights for the genetic breeding improvement of sea-island cottons.

Variation in mitogenome structural conformation in wild and cultivated lineages of sorghum corresponds with domestication history and plastome evolution.

BACKGROUND: Mitochondria are organelles within eukaryotic cells that are central to the metabolic processes of cellular respiration and ATP production. However, the evolution of mitochondrial genomes (mitogenomes) in plants is virtually unknown compared to animal mitogenomes or plant plastids, due to complex structural variation and long stretches of repetitive DNA making accurate genome assembly more challenging. Comparing the structural and sequence differences of organellar genomes within and between sorghum species is an essential step in understanding evolutionary processes such as organellar sequence transfer to the nuclear genome as well as improving agronomic traits in sorghum related to cellular metabolism. RESULTS: Here, we assembled seven sorghum mitochondrial and plastid genomes and resolved reticulated mitogenome structures with multilinked relationships that could be grouped into three structural conformations that differ in the content of repeats and genes by contig. The grouping of these mitogenome structural types reflects the two domestication events for sorghum in east and west Africa. CONCLUSIONS: We report seven mitogenomes of sorghum from different cultivars and wild sources. The assembly method used here will be helpful in resolving complex genomic structures in other plant species. Our findings give new insights into the structure of sorghum mitogenomes that provides an important foundation for future research into the improvement of sorghum traits related to cellular respiration, cytonuclear incompatibly, and disease resistance.

Dual Domestication, Diversity, and Differential Introgression in Old World Cotton Diploids.

Domestication in the cotton genus is remarkable in that it has occurred independently four different times at two different ploidy levels. Relatively little is known about genome evolution and domestication in the cultivated diploid species Gossypium herbaceum and Gossypium arboreum, due to the absence of wild representatives for the latter species, their ancient domestication, and their joint history of human-mediated dispersal and interspecific gene flow. Using in-depth resequencing of a broad sampling from both species, we provide support for their independent domestication, as opposed to a progenitor-derivative relationship, showing that diversity (mean π = 6 × 10-3) within species is similar, and that divergence between species is modest (FST = 0.413). Individual accessions were homozygous for ancestral single-nucleotide polymorphisms at over half of variable sites, while fixed, derived sites were at modest frequencies. Notably, two chromosomes with a paucity of fixed, derived sites (i.e., chromosomes 7 and 10) were also strongly implicated as having experienced high levels of introgression. Collectively, these data demonstrate variable permeability to introgression among chromosomes, which we propose is due to divergent selection under domestication and/or the phenomenon of F2 breakdown in interspecific crosses. Our analyses provide insight into the evolutionary forces that shape diversity and divergence in the diploid cultivated species and establish a foundation for understanding the contribution of introgression and/or strong parallel selection to the extensive morphological similarities shared between species.

Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant.

Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes' function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1-10 bp in length. The main editing sites are located at positions 11-17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.

Identification and Functional Analysis of lncRNA by CRISPR/Cas9 During the Cotton Response to Sap-Sucking Insect Infestation.

Sap-sucking insects cause severe damage to cotton production. Long non-coding RNAs (lncRNAs) play vital regulatory roles in various development processes and stress response, however, the function of lncRNAs during sap-sucking insect infection in cotton is largely unknown. In this study, the transcriptome profiles between resistant (HR) and susceptible (ZS) cotton cultivars under whitefly infestation at different time points (0, 4, 12, 24, and 48 h) were compared. A total of 6,651 lncRNAs transcript and 606 differentially expressed lncRNAs were identified from the RNA-seq data. A co-expression network indicated that lncA07 and lncD09 were potential hub genes that play a regulatory role in cotton defense against aphid infestation. Furthermore, CRISPR/Cas9 knock-out mutant of lncD09 and lncA07 showed a decrease of jasmonic acid (JA) content, which potentially lead to increased susceptibility toward insect infestation. Differentially expressed genes between wild type and lncRNA knock-out plants are enriched in modulating development and resistance to stimulus. Additionally, some candidate genes such as Ghir_A01G022270, Ghir_D04G014430, and Ghir_A01G022270 are involved in the regulation of the JA-mediated signaling pathway. This result provides a novel insight of the lncRNA role in the cotton defense system against pests.

The Gossypium anomalum genome as a resource for cotton improvement and evolutionary analysis of hybrid incompatibility.

Cotton is an important crop that has been the beneficiary of multiple genome sequencing efforts, including diverse representatives of wild species for germplasm development. Gossypium anomalum is a wild African diploid species that harbors stress-resistance and fiber-related traits with potential application to modern breeding efforts. In addition, this species is a natural source of cytoplasmic male sterility and a resource for understanding hybrid lethality in the genus. Here, we report a high-quality de novo genome assembly for G. anomalum and characterize this genome relative to existing genome sequences in cotton. In addition, we use the synthetic allopolyploids 2(A2D1) and 2(A2D3) to discover regions in the G. anomalum genome potentially involved in hybrid lethality, a possibility enabled by introgression of regions homologous to the D3 (Gossypium davidsonii) lethality loci into the synthetic 2(A2D3) allopolyploid.

Two gap-free reference genomes and a global view of the centromere architecture in rice.

Rice (Oryza sativa), a major staple throughout the world and a model system for plant genomics and breeding, was the first crop genome sequenced almost two decades ago. However, reference genomes for all higher organisms to date contain gaps and missing sequences. Here, we report the assembly and analysis of gap-free reference genome sequences for two elite O. sativa xian/indica rice varieties, Zhenshan 97 and Minghui 63, which are being used as a model system for studying heterosis and yield. Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres. We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures. In addition, the similarity of CentO repeats in the same chromosome is higher than across chromosomes, supporting a model of local expansion and homogenization. Both genomes have over 395 non-TE genes located in centromere regions, of which ∼41% are actively transcribed. Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes. The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.

Chloroplast genomes in Populus (Salicaceae): comparisons from an intensively sampled genus reveal dynamic patterns of evolution.

The chloroplast is one of two organelles containing a separate genome that codes for essential and distinct cellular functions such as photosynthesis. Given the importance of chloroplasts in plant metabolism, the genomic architecture and gene content have been strongly conserved through long periods of time and as such are useful molecular tools for evolutionary inferences. At present, complete chloroplast genomes from over 4000 species have been deposited into publicly accessible databases. Despite the large number of complete chloroplast genomes, comprehensive analyses regarding genome architecture and gene content have not been conducted for many lineages with complete species sampling. In this study, we employed the genus Populus to assess how more comprehensively sampled chloroplast genome analyses can be used in understanding chloroplast evolution in a broadly studied lineage of angiosperms. We conducted comparative analyses across Populus in order to elucidate variation in key genome features such as genome size, gene number, gene content, repeat type and number, SSR (Simple Sequence Repeat) abundance, and boundary positioning between the four main units of the genome. We found that some genome annotations were variable across the genus owing in part from errors in assembly or data checking and from this provided corrected annotations. We also employed complete chloroplast genomes for phylogenetic analyses including the dating of divergence times throughout the genus. Lastly, we utilized re-sequencing data to describe the variations of pan-chloroplast genomes at the population level for P. euphratica. The analyses used in this paper provide a blueprint for the types of analyses that can be conducted with publicly available chloroplast genomes as well as methods for building upon existing datasets to improve evolutionary inference.

Parallel and Intertwining Threads of Domestication in Allopolyploid Cotton.

The two cultivated allopolyploid cottons, Gossypium hirsutum and Gossypium barbadense, represent a remarkable example of parallel independent domestication, both involving dramatic morphological transformations under selection from wild perennial plants to annualized row crops. Deep resequencing of 643 newly sampled accessions spanning the wild-to-domesticated continuum of both species, and their allopolyploid relatives, are combined with existing data to resolve species relationships and elucidate multiple aspects of their parallel domestication. It is confirmed that wild G. hirsutum and G. barbadense were initially domesticated in the Yucatan Peninsula and NW South America, respectively, and subsequently spread under domestication over 4000-8000 years to encompass most of the American tropics. A robust phylogenomic analysis of infraspecific relationships in each species is presented, quantify genetic diversity in both, and describe genetic bottlenecks associated with domestication and subsequent diffusion. As these species became sympatric over the last several millennia, pervasive genome-wide bidirectional introgression occurred, often with striking asymmetries involving the two co-resident genomes of these allopolyploids. Diversity scans revealed genomic regions and genes unknowingly targeted during domestication and additional subgenomic asymmetries. These analyses provide a comprehensive depiction of the origin, divergence, and adaptation of cotton, and serve as a rich resource for cotton improvement.

Orchestration of plant development and defense by indirect crosstalk of salicylic acid and brassinosteorid signaling via transcription factor GhTINY2.

Salicylic acid (SA) and brassinosteroids (BRs) are well known to regulate diverse processes of plant development and stress responses, but the mechanisms by which these phytohormones mediate the growth and defense trade-off are largely unclear. In addition, little is known about the roles of DEHYDRATION RESPONSIVE ELEMENT BINDING transcription factors, especially in biotic stress and plant growth. Here, we identified a cotton (Gossypium hirsutum) APETALA2/ETHYLENE RESPONSIVE FACTOR gene GhTINY2 that is strongly induced by Verticillium dahliae. Overexpression of GhTINY2 in cotton and Arabidopsis enhanced tolerance to V. dahliae, while knockdown of expression increased the susceptibility of cotton to the pathogen. GhTINY2 was found to promote SA accumulation and SA signaling transduction by directly activating expression of WRKY51. Moreover, GhTINY2-overexpressing cotton and Arabidopsis showed retardation of growth, increased sensitivity to inhibitors of BR biosynthesis, down-regulation of several BR-induced genes, and up-regulation of BR-repressed genes, while GhTINY2-RNAi cotton showed the opposite effects. We further determined that GhTINY2 negatively regulates BR signaling by interacting with BRASSINAZOLE-RESISTANT 1 (BZR1) and restraining its transcriptional activation of the expression of INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19). These findings indicate that GhTINY2 fine-tunes the trade-off between immunity and growth via indirect crosstalk between WRKY51-mediated SA biosynthesis and BZR1-IAA19-regulated BR signaling.

Cotton pan-genome retrieves the lost sequences and genes during domestication and selection.

BACKGROUND: Millennia of directional human selection has reshaped the genomic architecture of cultivated cotton relative to wild counterparts, but we have limited understanding of the selective retention and fractionation of genomic components. RESULTS: We construct a comprehensive genomic variome based on 1961 cottons and identify 456 Mb and 357 Mb of sequence with domestication and improvement selection signals and 162 loci, 84 of which are novel, including 47 loci associated with 16 agronomic traits. Using pan-genome analyses, we identify 32,569 and 8851 non-reference genes lost from Gossypium hirsutum and Gossypium barbadense reference genomes respectively, of which 38.2% (39,278) and 14.2% (11,359) of genes exhibit presence/absence variation (PAV). We document the landscape of PAV selection accompanied by asymmetric gene gain and loss and identify 124 PAVs linked to favorable fiber quality and yield loci. CONCLUSIONS: This variation repertoire points to genomic divergence during cotton domestication and improvement, which informs the characterization of favorable gene alleles for improved breeding practice using a pan-genome-based approach.

The Gossypium stocksii genome as a novel resource for cotton improvement.

Cotton is an important textile crop whose gains in production over the last century have been challenged by various diseases. Because many modern cultivars are susceptible to several pests and pathogens, breeding efforts have included attempts to introgress wild, naturally resistant germplasm into elite lines. Gossypium stocksii is a wild cotton species native to Africa, which is part of a clade of vastly understudied species. Most of what is known about this species comes from pest resistance surveys and/or breeding efforts, which suggests that G. stocksii could be a valuable reservoir of natural pest resistance. Here, we present a high-quality de novo genome sequence for G. stocksii. We compare the G. stocksii genome with resequencing data from a closely related, understudied species (Gossypium somalense) to generate insight into the relatedness of these cotton species. Finally, we discuss the utility of the G. stocksii genome for understanding pest resistance in cotton, particularly resistance to cotton leaf curl virus.

An enhanced photosynthesis and carbohydrate metabolic capability contributes to heterosis of the cotton (Gossypium hirsutum) hybrid 'Huaza Mian H318', as revealed by genome-wide gene expression analysis.

BACKGROUND: Heterosis has been exploited for decades in different crops due to resulting in dramatic increases in yield, but relatively little molecular evidence on this topic was reported in cotton. RESULTS: The elite cotton hybrid variety 'Huaza Mian H318' (H318) and its parental lines were used to explore the source of its yield heterosis. A four-year investigation of yield-related traits showed that the boll number of H318 showed higher stability than that of its two parents, both in suitable and unsuitable climate years. In addition, the hybrid H318 grew faster and showed higher fresh and dry weights than its parental lines at the seedling stage. Transcriptome analysis of seedlings identified 17,308 differentially expressed genes (DEGs) between H318 and its parental lines, and 3490 extremely changed DEGs were screened out for later analysis. Most DEGs (3472/3490) were gathered between H318 and its paternal line (4-5), and only 64 DEGs were found between H318 and its maternal line (B0011), which implied that H318 displays more similar transcriptional patterns to its maternal parent at the seedling stage. GO and KEGG analyses showed that these DEGs were highly enriched in photosynthesis, lipid metabolic, carbohydrate metabolic and oxidation-reduction processes, and the expression level of these DEGs was significantly higher in H318 relative to its parental lines, which implied that photosynthesis, metabolism and stress resistances were enhanced in H318. CONCLUSION: The enhanced photosynthesis, lipid and carbohydrate metabolic capabilities contribute to the heterosis of H318 at the seedling stage, and establishes a material foundation for subsequent higher boll-setting rates in complex field environments.

The Gossypium longicalyx Genome as a Resource for Cotton Breeding and Evolution.

Cotton is an important crop that has made significant gains in production over the last century. Emerging pests such as the reniform nematode have threatened cotton production. The rare African diploid species Gossypium longicalyx is a wild species that has been used as an important source of reniform nematode immunity. While mapping and breeding efforts have made some strides in transferring this immunity to the cultivated polyploid species, the complexities of interploidal transfer combined with substantial linkage drag have inhibited progress in this area. Moreover, this species shares its most recent common ancestor with the cultivated A-genome diploid cottons, thereby providing insight into the evolution of long, spinnable fiber. Here we report a newly generated de novo genome assembly of G. longicalyx This high-quality genome leveraged a combination of PacBio long-read technology, Hi-C chromatin conformation capture, and BioNano optical mapping to achieve a chromosome level assembly. The utility of the G. longicalyx genome for understanding reniform immunity and fiber evolution is discussed.

Genetic Analysis of the Transition from Wild to Domesticated Cotton (Gossypium hirsutum L.).

The evolution and domestication of cotton is of great interest from both economic and evolutionary standpoints. Although many genetic and genomic resources have been generated for cotton, the genetic underpinnings of the transition from wild to domesticated cotton remain poorly known. Here we generated an intraspecific QTL mapping population specifically targeting domesticated cotton phenotypes. We used 466 F2 individuals derived from an intraspecific cross between the wild Gossypium hirsutum var. yucatanense (TX2094) and the elite cultivar G. hirsutum cv. Acala Maxxa, in two environments, to identify 120 QTL associated with phenotypic changes under domestication. While the number of QTL recovered in each subpopulation was similar, only 22 QTL were considered coincident (i.e., shared) between the two locations, eight of which shared peak markers. Although approximately half of QTL were located in the A-subgenome, many key fiber QTL were detected in the D-subgenome, which was derived from a species with unspinnable fiber. We found that many QTL are environment-specific, with few shared between the two environments, indicating that QTL associated with G. hirsutum domestication are genomically clustered but environmentally labile. Possible candidate genes were recovered and are discussed in the context of the phenotype. We conclude that the evolutionary forces that shape intraspecific divergence and domestication in cotton are complex, and that phenotypic transformations likely involved multiple interacting and environmentally responsive factors.