olyploidy or whole-genome duplication provides genomic opportunities for evolutionary innovations in many animal groups and all flowering plants 1-5 , including most important crops such as wheat, cotton and canola or oilseed rape 6-8. The common occurrence of polyploidy may suggest its advantage and potential for selection and adaptation 2,3,9 , through rapid genetic and genomic changes as observed in newly formed Brassica napus 10 , Tragopogon miscellus 11 and polyploid wheat 12 , and/or largely epigenetic modifications as in Arabidopsis and cotton polyploids 5,13. Cotton is a powerful model for revealing genomic insights into polyploidy 3 , providing a phylogenetically defined framework of polyploidization (~1.5 million years ago (Ma)) 14 , followed by natural diversification and crop domestication 15. The evolutionary history of the polyploid cotton clade is longer than that of some other allopolyploids, such as hexaploid wheat (~8,000 years) 12 , tetraploid canola (~7,500 years) 16 and tetraploid Tragopogon (~150 years) 11. Polyploidization between an A-genome African species (Gossypium arboreum (Ga)-like) and a D-genome American species (G. raimondii (Gr)-like) in the New World created a new allotetraploid or amphidiploid (AD-genome) cotton clade (Fig. 1a) 14 , which has diversified into five polyploid lineages, G. hirsutum (Gh) (AD) 1 , G. barbadense (Gb) (AD) 2 , G. tomentosum (Gt) (AD) 3 , G. mustelinum (Gm) (AD) 4 and G. darwinii (Gd) (AD) 5. G. ekmanianum and G. stephensii are recently characterized and closely related to Gh 17. Gh and Gb were separately domesticated from perennial shrubs to become annualized Upland and Pima cottons 15. To date, global cotton production provides income for ~100 million families across ~150 countries, with an annual economic impact of ~US$500 billion worldwide 6. However, cotton supply is reduced due to aridification, climate change and pest emergence. Future improvements in cotton and sustainability will involve use of the genomic resources and gene-editing tools becoming available in many crops 9,18,19. Cotton genomes have been sequenced for the D-genome (Gr) 20 and A-genome (Ga) 21 diploids and two cultivated tetraploids 22-26. These analyses have shown structural, genetic and gene expression variation related to fiber traits and stress responses in cultivated

Background: Polyploidy provides new genetic material that facilitates evolutionary novelty, species adaptation, and crop domestication. Polyploidy often leads to an increase in cell or organism size, which may affect transcript abundance or transcriptome size, but the relationship between polyploidy and transcriptome changes remains poorly understood. Plant cells often undergo endoreduplication, confounding the polyploid effect. Results: To mitigate these effects, we select female gametic cells that are developmentally stable and void of endoreduplication. Using single-cell RNA sequencing (scRNA-seq) in Arabidopsis thaliana tetraploid lines and isogenic diploids, we show that transcriptome abundance doubles in the egg cell and increases approximately 1.6-fold in the central cell, consistent with cell size changes. In the central cell of tetraploid plants, DEMETER (DME) is upregulated, which can activate PRC2 family members FIS2 and MEA, and may suppress the expression of other genes. Upregulation of cell size regulators in tetraploids, including TOR and OSR2, may increase the size of reproductive cells. In diploids, the order of transcriptome abundance is central cell, synergid cell, and egg cell, consistent with their cell size variation. Remarkably, we uncover new sets of female gametophytic cell-specific transcripts with predicted biological roles; the most abundant transcripts encode families of cysteine-rich peptides, implying roles in cell-cell recognition during double fertilization. Conclusions: Transcriptome in single cells doubles in tetraploid plants compared to diploid, while the degree of change and relationship to the cell size depends on cell types. These scRNA-seq resources are free of cross-contamination and are uniquely valuable for advancing plant hybridization, reproductive biology, and polyploid genomics.

Heterosis is widely applied in agriculture; however, the underlying molecular mechanisms for superior performance are not well understood. Ethylene biosynthesis and signaling genes are shown to be down-regulated in interspecific hybrids. Ethylene is a plant hormone that promotes fruit ripening and maturation but inhibits hypocotyl elongation. Here we report that application of exogenous ethylene could eliminate biomass vigor in F1 hybrids, suggesting a negative role of ethylene in heterosis. Ethylene biosynthesis is mediated by the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthase (ACS). Down-regulation of genes led to the decrease of ethylene production, which was associated with the high-vigor F1 hybrids, but not with the low-vigor ones. At the mechanistic level, expression of genes was down-regulated diurnally and indirectly by () during the day and directly by () at night. Consistent with the negative role of ethylene in plant growth, biomass vigor was higher in the mutants than in wild-type plants, while increasing endogenous ethylene production in the hybridizing parents reduced growth vigor in the hybrids. Thus, integrating circadian rhythms and light signaling into ethylene production is another regulatory module of complex biological networks, leading to biomass heterosis in plants.

Like those of many agricultural crops, the cultivated cotton is an allotetraploid and has a large genome (~2.5 gigabase pairs). The two sub genomes, A and D, are highly similar but unequally sized and repeat-rich, which pose significant challenges for accurate genome reconstruction using standard approaches. Here we report the development of BAC libraries, sub genome specific physical maps, and a new-generation sequencing approach that will lead to a reference-grade genome assembly for Upland cotton. Three BAC libraries were constructed, fingerprinted, and integrated with BAC-end sequences (BES) to produce a de novo whole-genome physical map. The BAC map was partitioned by sub genomes through alignment to the diploid progenitor D-genome reference sequence with densely spaced BES anchor points and computational filtering. The physical maps were validated with FISH and genetic mapping of SNP markers derived from BES. Two pairs of homeologous chromosomes, A11/D11 and A12/D12, were used to assess multiplex sequencing approaches for completeness and scalability. The results represent the first sub genome anchored physical maps of Upland cotton, and a new-generation approach to the whole-genome sequencing, which will lead to the reference-grade assembly of allopolyploid cotton and serve as a general strategy for sequencing other polyploid species.

Background: Circadian rhythms modulate growth and development in all organisms through interlocking transcriptional-translational feedback loops. The transcriptional loop involves chromatin modifications of central circadian oscillators in mammals and plants. However, the molecular basis for rhythmic epigenetic modifications and circadian regulation is poorly understood. Results: Here we report a feedback relationship between diurnal regulation of circadian clock genes and histone modifications in Arabidopsis. On one hand, the circadian oscillators CCA1 and LHY regulate diurnal expression of genes coding for the eraser (JMJ14) directly and writer (SDG2) indirectly for H3K4me3 modification, leading to rhythmic H3K4me3 changes in target genes. On the other hand, expression of circadian oscillator genes including CCA1 and LHY is associated with H3K4me3 levels and decreased in the sdg2 mutant but increased in the jmj14 mutant. At the genome-wide level, diurnal rhythms of H3K4me3 and another histone mark H3K9ac are associated with diurnal regulation of 20-30% of the expressed genes. While the majority (86%) of H3K4me3 and H3K9ac target genes overlap, only 13% of morning-phased and 22% of evening-phased genes had both H3K4me3 and H3K9ac peaks, suggesting specific roles of different histone modifications in diurnal gene expression. Conclusions: Circadian clock genes promote diurnal regulation of SDG2 and JMJ14 expression, which in turn regulate rhythmic histone modification dynamics for the clock and its output genes. This reciprocal regulatory module between chromatin modifiers and circadian clock oscillators orchestrates diurnal gene expression that governs plant growth and development.

Seed size is related to plant evolution and crop yield and is affected by genetic mutations, imprinting, and genome dosage. Imprinting is a widespread epigenetic phenomenon in mammals and flowering plants. ETHYLENE INSENSITIVE2 (EIN2) encodes a membrane protein that links the ethylene perception to transcriptional regulation. Interestingly, during seed development EIN2 is maternally-expressed in Arabidopsis and maize, but the role of EIN2 in seed development is unknown. Here we show that EIN2 is expressed specifically in the endosperm, and the maternal-specific EIN2 expression affects temporal regulation of endosperm cellularization. As a result, seed size increases in the genetic cross using the ein2 mutant as the maternal parent or in the ein2 mutant. The maternal-specific expression of EIN2 in the endosperm is controlled by DNA methylation but not by H3K27me3 or by ethylene and several ethylene pathway genes tested. RNA-seq analysis in the endosperm isolated by laser-capture microdissection show upregulation of many endosperm-expressed genes such as AGAMOUS-LIKEs (AGLs) in the ein2 mutant or when the maternal EIN2 allele is not expressed. EIN2 does not interact with DNA and may act through ETHYLENE INSENSITIVE3 (EIN3), a DNA binding protein present in sporophytic tissues, to activate target genes like AGLs, which in turn mediate temporal regulation of endosperm cellularization and seed size. These results provide mechanistic insights into endosperm and maternal-specific expression of EIN2 on endosperm cellularization and seed development, which could help improve seed production in plants and crops.

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Background Cotton fibers provide a powerful model for studying cell differentiation and elongation. Each cotton fiber is a singular and elongated cell derived from epidermal-layer cells of a cotton seed. Efforts to understand this dramatic developmental shift have been impeded by the difficulty of separation between fiber and epidermal cells. Results Here we employed laser-capture microdissection (LCM) to separate these cell types. RNA-seq analysis revealed transitional differences between fiber and epidermal-layer cells at 0 or 2 days post anthesis. Specifically, down-regulation of putative cell cycle genes was coupled with upregulation of ribosome biosynthesis and translation-related genes, which may suggest their respective roles in fiber cell initiation. Indeed, the amount of fibers in cultured ovules was increased by cell cycle progression inhibitor, Roscovitine, and decreased by ribosome biosynthesis inhibitor, Rbin-1. Moreover, subfunctionalization of homoeologs was pervasive in fiber and epidermal cells, with expression bias towards 10% more D than A homoeologs of cell cycle related genes and 40–50% more D than A homoeologs of ribosomal protein subunit genes. Key cell cycle regulators were predicted to be epialleles in allotetraploid cotton. MYB-transcription factor genes displayed expression divergence between fibers and ovules. Notably, many phytohormone-related genes were upregulated in ovules and down-regulated in fibers, suggesting spatial-temporal effects on fiber cell development. Conclusions Fiber cell initiation is accompanied by cell cycle arrest coupled with active ribosome biosynthesis, spatial-temporal regulation of phytohormones and MYB transcription factors, and homoeolog expression bias of cell cycle and ribosome biosynthesis genes. These valuable genomic resources and molecular insights will help develop breeding and biotechnological tools to improve cotton fiber production.