The maize single-nucleus transcriptome comprehensively describes signaling networks governing movement and development of grass stomata.

The unique morphology of grass stomata enables rapid responses to environmental changes. Deciphering the basis for these responses is critical for improving food security. We have developed a planta platform of single-nucleus RNA-sequencing by combined fluorescence-activated nuclei flow sorting, and used it to identify cell types in mature and developing stomata from 33,098 nuclei of the maize epidermis-enriched tissues. Guard cells (GCs) and subsidiary cells (SCs) displayed differential expression of genes, besides those encoding transporters, involved in the abscisic acid, CO2, Ca2+, starch metabolism, and blue light signaling pathways, implicating coordinated signal integration in speedy stomatal responses, and of genes affecting cell wall plasticity, implying a more sophisticated relationship between GCs and SCs in stomatal development and dumbbell-shaped guard cell formation. The trajectory of stomatal development identified in young tissues, and by comparison to the bulk RNA-seq data of the MUTE defective mutant in stomatal development, confirmed known features, and shed light on key participants in stomatal development. Our study provides a valuable, comprehensive, and fundamental foundation for further insights into grass stomatal function.

Introgressing the Aegilops tauschii genome into wheat as a basis for cereal improvement.

Increasing crop production is necessary to feed the world's expanding population, and crop breeders often utilize genetic variations to improve crop yield and quality. However, the narrow diversity of the wheat D genome seriously restricts its selective breeding. A practical solution is to exploit the genomic variations of Aegilops tauschii via introgression. Here, we established a rapid introgression platform for transferring the overall genetic variations of A. tauschii to elite wheats, thereby enriching the wheat germplasm pool. To accelerate the process, we assembled four new reference genomes, resequenced 278 accessions of A. tauschii and constructed the variation landscape of this wheat progenitor species. Genome comparisons highlighted diverse functional genes or novel haplotypes with potential applications in wheat improvement. We constructed the core germplasm of A. tauschii, including 85 accessions covering more than 99% of the species' overall genetic variations. This was crossed with elite wheat cultivars to generate an A. tauschii-wheat synthetic octoploid wheat (A-WSOW) pool. Laboratory and field analysis with two examples of the introgression lines confirmed its great potential for wheat breeding. Our high-quality reference genomes, genomic variation landscape of A. tauschii and the A-WSOW pool provide valuable resources to facilitate gene discovery and breeding in wheat.

Genome-wide characterization of the WAK gene family and expression analysis under plant hormone treatment in cotton.

BACKGROUND: Wall-associated kinases (WAK), one of the receptor-like kinases (RLK), function directly in the connection and communication between the plant cell wall and the cytoplasm. WAK genes are highly conserved and have been identified in plants, such as rice, but there is little research on the WAK gene family in cotton. RESULTS: In the present study, we identified 29 GhWAK genes in Gossypium hirsutum. Phylogenetic analysis showed that cotton WAK proteins can be divided into five clades. The results of synteny and Ka/Ks analysis showed that the GhWAK genes mainly originated from whole genome duplication (WGD) and were then mainly under purifying selection. Transcriptome data and real-time PCR showed that 97% of GhWAK genes highly expressed in cotton fibers and ovules. ß-glucuronidase (GUS) staining assays showed that GhWAK5 and GhWAK16 expressed in Arabidopsis leaf trichomes. Fourteen GhWAK genes were found to possess putative gibberellin (GA) response elements in the promoter regions, 13 of which were significantly induced by GA treatment. Ten GhWAK genes contained auxin (IAA) response elements and the expression level of nine GhWAKs significantly increased under auxin treatment. CONCLUSIONS: We provide a preliminary analysis of the WAK gene family in G. hirsutum, which sheds light on the potantial roles of GhWAK genes in cotton fiber cell development. Our data also provides a useful resource for future studies on the functional roles of GhWAK genes.

GhARF16-1 modulates leaf development by transcriptionally regulating the GhKNOX2-1 gene in cotton.

The leaf is a crucial organ evolved with remarkable morphological diversity to maximize plant photosynthesis. The leaf shape is a key trait that affects photosynthesis, flowering rates, disease resistance and yield. Although many genes regulating leaf development have been identified in the past years, the precise regulatory architecture underlying the generation of diverse leaf shapes remains to be elucidated. We used cotton as a reference model to probe the genetic framework underlying divergent leaf forms. Comparative transcriptome analysis revealed that the GhARF16-1 and GhKNOX2-1 genes might be potential regulators of leaf shape. We functionally characterized the auxin-responsive factor ARF16-1 acting upstream of GhKNOX2-1 to determine leaf morphology in cotton. The transcription of GhARF16-1 was significantly higher in lobed-leaved cotton than in smooth-leaved cotton. Furthermore, the overexpression of GhARF16-1 led to the up-regulation of GhKNOX2-1 and resulted in more and deeper serrations in cotton leaves, similar to the leaf shape of cotton plants overexpressing GhKNOX2-1. We found that GhARF16-1 specifically bound to the promoter of GhKNOX2-1 to induce its expression. The heterologous expression of GhARF16-1 and GhKNOX2-1 in Arabidopsis led to lobed and curly leaves, and a genetic analysis revealed that GhKNOX2-1 is epistatic to GhARF16-1 in Arabidopsis, suggesting that the GhARF16-1 and GhKNOX2-1 interaction paradigm also functions to regulate leaf shape in Arabidopsis. To our knowledge, our results uncover a novel mechanism by which auxin, through the key component ARF16-1 and its downstream-activated gene KNOX2-1, determines leaf morphology in eudicots.

The plasma-membrane polyamine transporter PUT3 is regulated by the Na+ /H+ antiporter SOS1 and protein kinase SOS2.

In Arabidopsis, the plasma membrane transporter PUT3 is important to maintain the cellular homeostasis of polyamines and plays a role in stabilizing mRNAs of some heat-inducible genes. The plasma membrane Na+ /H+ transporter SOS1 and the protein kinase SOS2 are two salt-tolerance determinants crucial for maintaining intracellular Na+ and K+ homeostasis. Here, we report that PUT3 genetically and physically interacts with SOS1 and SOS2, and these interactions modulate PUT3 transport activity. Overexpression of PUT3 (PUT3OE) results in hypersensitivity of the transgenic plants to polyamine and paraquat. The hypersensitivity of PUT3OE is inhibited by the sos1 and sos2 mutations, which indicates that SOS1 and SOS2 are required for PUT3 transport activity. A protein interaction assay revealed that PUT3 physically interacts with SOS1 and SOS2 in yeast and plant cells. SOS2 phosphorylates PUT3 both in vitro and in vivo. SOS1 and SOS2 synergistically activate the polyamine transport activity of PUT3, and PUT3 also modulates SOS1 activity by activating SOS2 in yeast cells. Overall, our findings suggest that both plasma-membrane proteins PUT3 and SOS1 could form a complex with the protein kinase SOS2 in response to stress conditions and modulate the transport activity of each other through protein interactions and phosphorylation.

Genome-wide comparative analysis of RNA-binding Glycine-rich protein family genes between Gossypium arboreum and Gossypium raimondii.

RB-GRP (RNA-binding Glycine-rich protein gene) family belongs to the fourth subfamily of the GRP (Glycine-rich protein gene) superfamily, which plays a great role in plant growth and development, as well as in abiotic stresses response, while it has not been identified in cotton. Here, we identified 37 and 32 RB-GRPs from two cotton species (Gossypium arboreum and Gossypium raimondii, respectively), which were divided into four distinct subfamilies based on the presence of additional motifs and the arrangement of the glycine repeats. The distribution of RB-GRPs was nonrandom and uneven among the chromosomes both in two cotton species. The expansion of RB-GRP gene family between two cultivars was mainly attributed to segmental and tandem duplication events indicated by synteny analysis, and the tandem duplicated genes were mapped into homologous collinear blocks, indicated that they shared a common ancestral gene in both species. Furthermore, most RB-GRPs in two cotton species undergone stronger negative selective pressure by evolutionary analysis of RB-GRP orthologous genes. Meanwhile, RB-GRPs participated in different abiotic stresses (Abscisic acid, salt and Polyethylene glycol) responses and tissues at different developmental stages between two cotton species were showed by gene expression analysis. This research would provide insight into the evolution and function of the RB-GRPs in Gossypium species.

The genome of broomcorn millet.

Broomcorn millet (Panicum miliaceum L.) is the most water-efficient cereal and one of the earliest domesticated plants. Here we report its high-quality, chromosome-scale genome assembly using a combination of short-read sequencing, single-molecule real-time sequencing, Hi-C, and a high-density genetic map. Phylogenetic analyses reveal two sets of homologous chromosomes that may have merged ~5.6 million years ago, both of which exhibit strong synteny with other grass species. Broomcorn millet contains 55,930 protein-coding genes and 339 microRNA genes. We find Paniceae-specific expansion in several subfamilies of the BTB (broad complex/tramtrack/bric-a-brac) subunit of ubiquitin E3 ligases, suggesting enhanced regulation of protein dynamics may have contributed to the evolution of broomcorn millet. In addition, we identify the coexistence of all three C4 subtypes of carbon fixation candidate genes. The genome sequence is a valuable resource for breeders and will provide the foundation for studying the exceptional stress tolerance as well as C4 biology.

Mutations in a subfamily of abscisic acid receptor genes promote rice growth and productivity.

Abscisic acid (ABA) is a key phytohormone that controls plant growth and stress responses. It is sensed by the pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory components of the ABA receptor (RCAR) family of proteins. Here, we utilized CRISPR/Cas9 technology to edit group I (PYL1-PYL6 and PYL12) and group II (PYL7-PYL11 and PYL13) PYL genes in rice. Characterization of the combinatorial mutants suggested that genes in group I have more important roles in stomatal movement, seed dormancy, and growth regulation than those in group II. Among all of the single pyl mutants, only pyl1 and pyl12 exhibited significant defects in seed dormancy. Interestingly, high-order group I mutants, but not any group II mutants, displayed enhanced growth. Among group I mutants, pyl1/4/6 exhibited the best growth and improved grain productivity in natural paddy field conditions, while maintaining nearly normal seed dormancy. Our results suggest that a subfamily of rice PYLs has evolved to have particularly important roles in regulating plant growth and reveal a genetic strategy to improve rice productivity.

A high-quality genome assembly of quinoa provides insights into the molecular basis of salt bladder-based salinity tolerance and the exceptional nutritional value.

Chenopodium quinoa is a halophytic pseudocereal crop that is being cultivated in an ever-growing number of countries. Because quinoa is highly resistant to multiple abiotic stresses and its seed has a better nutritional value than any other major cereals, it is regarded as a future crop to ensure global food security. We generated a high-quality genome draft using an inbred line of the quinoa cultivar Real. The quinoa genome experienced one recent genome duplication about 4.3 million years ago, likely reflecting the genome fusion of two Chenopodium parents, in addition to the γ paleohexaploidization reported for most eudicots. The genome is highly repetitive (64.5% repeat content) and contains 54 438 protein-coding genes and 192 microRNA genes, with more than 99.3% having orthologous genes from glycophylic species. Stress tolerance in quinoa is associated with the expansion of genes involved in ion and nutrient transport, ABA homeostasis and signaling, and enhanced basal-level ABA responses. Epidermal salt bladder cells exhibit similar characteristics as trichomes, with a significantly higher expression of genes related to energy import and ABA biosynthesis compared with the leaf lamina. The quinoa genome sequence provides insights into its exceptional nutritional value and the evolution of halophytes, enabling the identification of genes involved in salinity tolerance, and providing the basis for molecular breeding in quinoa.

Genome-wide identification, phylogeny, and expression analysis of pectin methylesterases reveal their major role in cotton fiber development.

BACKGROUND: Pectin methylesterase (PME, EC 3.1.1.11) is a hydrolytic enzyme that utilizes pectin as substrates, and plays a significant role in regulating pectin reconstruction thereby regulating plant growth. Pectin is one of the important components of the plant cell wall, which forms the main structural material of cotton fiber. In this research, cotton genome information was used to identify PMEs. RESULTS: We identified 80 (GaPME01-GaPME80) PME genes from diploid G. arboreum (A genome), 78 (GrPME01-GrPME78) PME genes from G. raimondii (D genome), and 135 (GhPME001-GhPME135) PME genes from tetraploid cotton G. hirsutum (AD genome). We further analyzed their gene structure, conserved domain, gene expression, and systematic evolution to lay the foundation for deeper research on the function of PMEs. Phylogenetic data indicated that members from the same species demonstrated relatively high sequence identities and genetic similarities. Analysis of gene structures showed that most of the PMEs genes had 2-3 exons, with a few having a variable number of exons from 4 to 6. There are nearly no differences in the gene structure of PMEs among the three (two diploid and one tetraploid) cotton species. Selective pressure analysis showed that the Ka/Ks value for each of the three cotton species PME families was less than one. CONCLUSION: Conserved domain analysis showed that PMEs members had a relatively conserved C-terminal pectinesterase domain (PME) while the N-terminus was less conserved. Moreover, some of the family members contained a pectin methylesterase inhibitor (PMEI) domain. The Ka/Ks ratios suggested that the duplicated PMEs underwent purifying selection after the duplication events. This study provided an important basis for further research on the functions of cotton PMEs. Results from qRT-PCR indicated that the expression level of different PMEs at various fiber developmental stages was different. Moreover, some of the PMEs showed fiber predominant expression in secondary wall thickening indicating tissue-specific expression patterns.

Fine mapping and candidate gene analysis of the dominant glandless gene Gl 2 (e) in cotton (Gossypium spp.).

KEY MESSAGE: Dominant glandless gene Gl 2 (e) was fine-mapped to a 15 kb region containing one candidate gene encoding an MYC transcription factor, sequence and expression level of the gene were analyzed. Cottonseed product is an excellent source of oil and protein. However, this nutrition source is greatly limited in utilization by the toxic gossypol in pigment glands. It is reported that the Gl 2 (e) gene could effectively inhibit the formation of the pigment glands. Here, three F2 populations were constructed using two pairs of near isogenic lines (NILs), which differ nearly only by the gland trait, for fine mapping of Gl 2 (e) . DNA markers were identified from recently developed cotton genome sequence. The Gl 2 (e) gene was located within a 15-kb genomic interval between two markers CS2 and CS4 on chromosome 12. Only one gene was identified in the genomic interval as the candidate for Gl 2 (e) which encodes a family member of MYC transcription factor with 475-amino acids. Unexpectedly, the results of expression analysis indicated that the MYC gene expresses in glanded lines while almost does not express in glandless lines. These results suggest that the MYC gene probably serves as a vital positive regulator in the organogenesis pathway of pigment gland, and low expression of this gene will not launch the downstream pathway of pigment gland formation. This is the first pigment gland-related gene identification in cotton and will facilitate the research on glandless trait, cotton MYC proteins and low-gossypol cotton breeding.

Transcriptome analysis reveals long noncoding RNAs involved in fiber development in cotton (Gossypium arboreum).

Long noncoding RNAs (lncRNAs) play important roles in various biological regulatory processes in yeast, mammals, and plants. However, no systematic identification of lncRNAs has been reported in Gossypium arboreum. In this study, the strand-specific RNA sequencing (ssRNA-seq) of samples from cotton fibers and leaves was performed, and lncRNAs involved in fiber initiation and elongation processes were systematically identified and analyzed. We identified 5,996 lncRNAs, of which 3,510 and 2,486 can be classified as long intergenic noncoding RNAs (lincRNAs) and natural antisense transcripts (lncNAT), respectively. LincRNAs and lncNATs are similar in many aspects, but have some differences in exon number, exon length, and transcript length. Expression analysis revealed that 51.9% of lincRNAs and 54.5% of lncNATs transcripts were preferentially expressed at one stage of fiber development, and were significantly highly expressed than protein-coding transcripts (21.7%). During the fiber and rapid elongation stages, rapid and dynamic changes in lncRNAs may contribute to fiber development in cotton. This work describes a set of lncRNAs that are involved in fiber development. The characterization and expression analysis of lncRNAs will facilitate future studies on their roles in fiber development in cotton.

Comprehensive analysis of NAC transcription factors in diploid Gossypium: sequence conservation and expression analysis uncover their roles during fiber development.

Determining how function evolves following gene duplication is necessary for understanding gene expansion. Transcription factors (TFs) are a class of proteins that regulate gene expression by binding to specific cis-acting elements in the promoters of target genes, subsequently activating or repressing their transcription. In the present study, we systematically examined the functional diversification of the NAC transcription factor (NAC-TFs) family by analyzing their chromosomal location, structure, phylogeny, and expression pattern in Gossypium raimondii (Gr) and G. arboreum (Ga). The 145 and 141 NAC genes identified in the Gr and Ga genomes, respectively, were annotated and divided into 18 subfamilies, which showed distinct divergence in gene structure and expression patterns during fiber development. In addition, when the functional parameters were examined, clear divergence was observed within tandem clusters, which suggested that subfunctionalization had occurred among duplicate genes. The expression patterns of homologous gene pairs also changed, suggestive of the diversification of gene function during the evolution of diploid cotton. These findings provide insights into the mechanisms underlying the functional differentiation of duplicated NAC-TFs genes in two diploid cotton species.

[Recent progress in gene mapping through high-throughput sequencing technology and forward genetic approaches].

Traditional gene mapping using forward genetic approaches is conducted primarily through construction of a genetic linkage map, the process of which is tedious and time-consuming, and often results in low accuracy of mapping and large mapping intervals. With the rapid development of high-throughput sequencing technology and decreasing cost of sequencing, a variety of simple and quick methods of gene mapping through sequencing have been developed, including direct sequencing of the mutant genome, sequencing of selective mutant DNA pooling, genetic map construction through sequencing of individuals in population, as well as sequencing of transcriptome and partial genome. These methods can be used to identify mutations at the nucleotide level and has been applied in complex genetic background. Recent reports have shown that sequencing mapping could be even done without the reference of genome sequence, hybridization, and genetic linkage information, which made it possible to perform forward genetic study in many non-model species. In this review, we summarized these new technologies and their application in gene mapping.

Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution.

Gossypium hirsutum has proven difficult to sequence owing to its complex allotetraploid (AtDt) genome. Here we produce a draft genome using 181-fold paired-end sequences assisted by fivefold BAC-to-BAC sequences and a high-resolution genetic map. In our assembly 88.5% of the 2,173-Mb scaffolds, which cover 89.6%∼96.7% of the AtDt genome, are anchored and oriented to 26 pseudochromosomes. Comparison of this G. hirsutum AtDt genome with the already sequenced diploid Gossypium arboreum (AA) and Gossypium raimondii (DD) genomes revealed conserved gene order. Repeated sequences account for 67.2% of the AtDt genome, and transposable elements (TEs) originating from Dt seem more active than from At. Reduction in the AtDt genome size occurred after allopolyploidization. The A or At genome may have undergone positive selection for fiber traits. Concerted evolution of different regulatory mechanisms for Cellulose synthase (CesA) and 1-Aminocyclopropane-1-carboxylic acid oxidase1 and 3 (ACO1,3) may be important for enhanced fiber production in G. hirsutum.

Development of chromosome-specific markers with high polymorphism for allotetraploid cotton based on genome-wide characterization of simple sequence repeats in diploid cottons (Gossypium arboreum L. and Gossypium raimondii Ulbrich).

BACKGROUND: Tetraploid cotton contains two sets of homologous chromosomes, the At- and Dt-subgenomes. Consequently, many markers in cotton were mapped to multiple positions during linkage genetic map construction, posing a challenge to anchoring linkage groups and mapping economically-important genes to particular chromosomes. Chromosome-specific markers could solve this problem. Recently, the genomes of two diploid species were sequenced whose progenitors were putative contributors of the At- and Dt-subgenomes to tetraploid cotton. These sequences provide a powerful tool for developing chromosome-specific markers given the high level of synteny among tetraploid and diploid cotton genomes. In this study, simple sequence repeats (SSRs) on each chromosome in the two diploid genomes were characterized. Chromosome-specific SSRs were developed by comparative analysis and proved to distinguish chromosomes. RESULTS: A total of 200,744 and 142,409 SSRs were detected on the 13 chromosomes of Gossypium arboreum L. and Gossypium raimondii Ulbrich, respectively. Chromosome-specific SSRs were obtained by comparing SSR flanking sequences from each chromosome with those from the other 25 chromosomes. The average was 7,996 per chromosome. To confirm their chromosome specificity, these SSRs were used to distinguish two homologous chromosomes in tetraploid cotton through linkage group construction. The chromosome-specific SSRs and previously-reported chromosome markers were grouped together, and no marker mapped to another homologous chromosome, proving that the chromosome-specific SSRs were unique and could distinguish homologous chromosomes in tetraploid cotton. Because longer dinucleotide AT-rich repeats were the most polymorphic in previous reports, the SSRs on each chromosome were sorted by motif type and repeat length for convenient selection. The primer sequences of all chromosome-specific SSRs were also made publicly available. CONCLUSION: Chromosome-specific SSRs are efficient tools for chromosome identification by anchoring linkage groups to particular chromosomes during genetic mapping and are especially useful in mapping of qualitative-trait genes or quantitative trait loci with just a few markers. The SSRs reported here will facilitate a number of genetic and genomic studies in cotton, including construction of high-density genetic maps, positional gene cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among Gossypium species.

Genome sequence of the cultivated cotton Gossypium arboreum.

The complex allotetraploid nature of the cotton genome (AADD; 2n = 52) makes genetic, genomic and functional analyses extremely challenging. Here we sequenced and assembled the Gossypium arboreum (AA; 2n = 26) genome, a putative contributor of the A subgenome. A total of 193.6 Gb of clean sequence covering the genome by 112.6-fold was obtained by paired-end sequencing. We further anchored and oriented 90.4% of the assembly on 13 pseudochromosomes and found that 68.5% of the genome is occupied by repetitive DNA sequences. We predicted 41,330 protein-coding genes in G. arboreum. Two whole-genome duplications were shared by G. arboreum and Gossypium raimondii before speciation. Insertions of long terminal repeats in the past 5 million years are responsible for the twofold difference in the sizes of these genomes. Comparative transcriptome studies showed the key role of the nucleotide binding site (NBS)-encoding gene family in resistance to Verticillium dahliae and the involvement of ethylene in the development of cotton fiber cells.

Analyses of the NAC transcription factor gene family in Gossypium raimondii Ulbr.: chromosomal location, structure, phylogeny, and expression patterns.

NAC domain proteins are plant-specific transcription factors known to play diverse roles in various plant developmental processes. In the present study, we performed the first comprehensive study of the NAC gene family in Gossypium raimondii Ulbr., incorporating phylogenetic, chromosomal location, gene structure, conserved motif, and expression profiling analyses. We identified 145 NAC transcription factor (NAC-TF) genes that were phylogenetically clustered into 18 distinct subfamilies. Of these, 127 NAC-TF genes were distributed across the 13 chromosomes, 80 (55%) were preferentially retained duplicates located in both duplicated regions and six were located in triplicated chromosomal regions. The majority of NAC-TF genes showed temporal-, spatial-, and tissue-specific expression patterns based on transcriptomic and qRT-PCR analyses. However, the expression patterns of several duplicate genes were partially redundant, suggesting the occurrence of sub-functionalization during their evolution. Based on their genomic organization, we concluded that genomic duplications contributed significantly to the expansion of the NAC-TF gene family in G. raimondii. Comprehensive analysis of their expression profiles could provide novel insights into the functional divergence among members of the NAC gene family in G. raimondii.