Chromosome-level genomes of three key Allium crops and their trait evolution.

Allium crop breeding remains severely hindered due to the lack of high-quality reference genomes. Here we report high-quality chromosome-level genome assemblies for three key Allium crops (Welsh onion, garlic and onion), which are 11.17 Gb, 15.52 Gb and 15.78 Gb in size with the highest recorded contig N50 of 507.27 Mb, 109.82 Mb and 81.66 Mb, respectively. Beyond revealing the genome evolutionary process of Allium species, our pathogen infection experiments and comparative metabolomic and genomic analyses showed that genes encoding enzymes involved in the metabolic pathway of Allium-specific flavor compounds may have evolved from an ancient uncharacterized plant defense system widely existing in many plant lineages but extensively boosted in alliums. Using in situ hybridization and spatial RNA sequencing, we obtained an overview of cell-type categorization and gene expression changes associated with spongy mesophyll cell expansion during onion bulb formation, thus indicating the functional roles of bulb formation genes.

Comparative Transcriptomics of Multi-Stress Responses in Pachycladon cheesemanii and Arabidopsis thaliana.

The environment is seldom optimal for plant growth and changes in abiotic and biotic signals, including temperature, water availability, radiation and pests, induce plant responses to optimise survival. The New Zealand native plant species and close relative to Arabidopsis thaliana, Pachycladon cheesemanii, grows under environmental conditions that are unsustainable for many plant species. Here, we compare the responses of both species to different stressors (low temperature, salt and UV-B radiation) to help understand how P. cheesemanii can grow in such harsh environments. The stress transcriptomes were determined and comparative transcriptome and network analyses discovered similar and unique responses within species, and between the two plant species. A number of widely studied plant stress processes were highly conserved in A. thaliana and P. cheesemanii. However, in response to cold stress, Gene Ontology terms related to glycosinolate metabolism were only enriched in P. cheesemanii. Salt stress was associated with alteration of the cuticle and proline biosynthesis in A. thaliana and P. cheesemanii, respectively. Anthocyanin production may be a more important strategy to contribute to the UV-B radiation tolerance in P. cheesemanii. These results allowed us to define broad stress response pathways in A. thaliana and P. cheesemanii and suggested that regulation of glycosinolate, proline and anthocyanin metabolism are strategies that help mitigate environmental stress.

Identification of the genes at S and Z reveals the molecular basis and evolution of grass self-incompatibility.

Self-incompatibility (SI) is a feature of many flowering plants, whereby self-pollen is recognized and rejected by the stigma. In grasses (Poaceae), the genes controlling this phenomenon have not been fully elucidated. Grasses have a unique two-locus system, in which two independent genetic loci (S and Z) control self-recognition. S and Z are thought to have arisen from an ancient duplication, common to all grasses. With new chromosome-scale genome data, we examined the genes present at S- and Z-loci, firstly in ryegrass (Lolium perenne), and subsequently in ~20 other grass species. We found that two DUF247 genes and a short unstructured protein (SP/ZP) were present at both S- and Z- in all SI species, while in self-compatible species these genes were often lost or mutated. Expression data suggested that DUF247 genes acted as the male components and SP/ZP were the female components. Consistent with their role in distinguishing self- from non-self, all genes were hypervariable, although key secondary structure features were conserved, including the predicted N-terminal cleavage site of SP/ZP. The evolutionary history of these genes was probed, revealing that specificity groups at the Z-locus arose before the advent of various grass subfamilies/species, while specificity groups at the S-locus arose after the split of Panicoideae, Chloridoideae, Oryzoideae and Pooideae. Finally, we propose a model explaining how the proteins encoded at the S and Z loci might function to specify self-incompatibility.

Insight into the regulatory networks underlying the high lipid perennial ryegrass growth under different irradiances.

Under favourable conditions, perennial ryegrass (Lolium perenne) engineered to accumulated high lipid (HL) carbon sink in their leaves was previously shown to also enhance photosynthesis and growth. The greater aboveground biomass was found to be diminished in a dense canopy compared to spaced pots. Besides, the underlying genetic regulatory network linking between leaf lipid sinks and these physiological changes remains unknown. In this study, we demonstrated that the growth advantage was not displayed in HL Lolium grown in spaced pots under low lights. Under standard lights, analysis of differentiating transcripts in HL Lolium reveals that the plants had elevated transcripts involved in lipid metabolism, light capturing, photosynthesis, and sugar signalling while reduced expression of genes participating in sugar biosynthesis and transportation. The plants also had altered several transcripts involved in mitochondrial oxidative respiration and redox potential. Many of the above upregulated or downregulated transcript levels were found to be complemented by growing the plants under low light. Overall, this study emphasizes the importance of carbon and energy homeostatic regulatory mechanisms to overall productivity of the HL Lolium through photosynthesis, most of which are significantly impacted by low irradiances.

The 5'-3' mRNA Decay Pathway Modulates the Plant Circadian Network in Arabidopsis.

Circadian rhythms enable organisms to anticipate and adjust their physiology to periodic environmental changes. These rhythms are controlled by biological clocks that consist of a set of clock genes that regulate each other's expression. Circadian oscillations in messenger RNA (mRNA) levels require the regulation of mRNA production and degradation. While transcription factors controlling clock function have been well characterized from cyanobacteria to humans, the role of factors controlling mRNA decay is largely unknown. Here, we show that mutations in SM-LIKE PROTEIN 1 (LSM1) and exoribonucleases 4 (XRN4), components of the 5'-3' mRNA decay pathway, alter clock function in Arabidopsis. We found that lsm1 and xrn4 mutants display long-period phenotypes for clock gene expression. In xrn4, these circadian defects were associated with changes in circadian phases of expression, but not overall mRNA levels, of several core-clock genes. We then used noninvasive transcriptome-wide mRNA stability analysis to identify genes and pathways regulated by XRN4. Among genes affected in the xrn4 mutant at the transcriptional and posttranscriptional level, we found an enrichment in genes involved in auxin, ethylene and drought recovery. Large effects were not observed for canonical core-clock genes, although the mRNAs of several auxiliary clock genes that control the pace of the clock were stabilized in xrn4 mutants. Our results establish that the 5'-3' mRNA decay pathway constitutes a novel posttranscriptional regulatory layer of the circadian gene network, which probably acts through a combination of small effects on mRNA stability of several auxiliary and some core-clock genes.

A Point Mutation in Phytochromobilin synthase Alters the Circadian Clock and Photoperiodic Flowering of Medicago truncatula.

Plants use seasonal cues to initiate flowering at an appropriate time of year to ensure optimal reproductive success. The circadian clock integrates these daily and seasonal cues with internal cues to initiate flowering. The molecular pathways that control the sensitivity of flowering to photoperiods (daylengths) are well described in the model plant Arabidopsis. However, much less is known for crop species, such as legumes. Here, we performed a flowering time screen of a TILLING population of Medicago truncatula and found a line with late-flowering and altered light-sensing phenotypes. Using RNA sequencing, we identified a nonsense mutation in the Phytochromobilin synthase (MtPΦBS) gene, which encodes an enzyme that carries out the final step in the biosynthesis of the chromophore required for phytochrome (phy) activity. The analysis of the circadian clock in the MtpΦbs mutant revealed a shorter circadian period, which was shared with the MtphyA mutant. The MtpΦbs and MtphyA mutants showed downregulation of the FT floral regulators MtFTa1 and MtFTb1/b2 and a change in phase for morning and night core clock genes. Our findings show that phyA is necessary to synchronize the circadian clock and integration of light signalling to precisely control the timing of flowering.

A novel TFL1 gene induces flowering in the mast seeding alpine snow tussock, Chionochloa pallens (Poaceae).

Masting, the synchronous, highly variable flowering across years by a population of perennial plants, has been reported to be precipitated by various factors including nitrogen levels, drought conditions, and spring and summer temperatures. However, the molecular mechanism leading to the initiation of flowering in masting plants in particular years remains largely unknown, despite the potential impact of climate change on masting phenology. We studied genes controlling flowering in the alpine snow tussock Chionochloa pallens (Poaceae), a strongly masting perennial grass. We used a range of in situ and manipulated plants to obtain leaf samples from tillers (shoots) which subsequently remained vegetative or flowered. Here, we show that a novel orthologue of TERMINAL FLOWER 1 (TFL1; normally a repressor of flowering in other species) promotes the induction of flowering in C. pallens (hence Anti-TFL1), a conclusion supported by structural, functional and expression analyses. Global transcriptomic analysis indicated differential expression of CpTPS1, CpGA20ox1, CpREF6 and CpHDA6, emphasizing the role of endogenous cues and epigenetic regulation in terms of responsiveness of plants to initiate flowering. Our molecular-based study provides insights into the cellular mechanism of flowering in masting plants and will supplement ecological and statistical models to predict how masting will respond to global climate change.

Identification and Characterization of Perennial Ryegrass (Lolium perenne) Vernalization Genes.

Perennial ryegrass (Lolium perenne) is a temperate grass species commonly used as pasture for livestock. Flowering (heading) of ryegrass impacts metabolizable energy content and seed yield, therefore this trait is important for both farmers and seed producers. In related grass species, the VRN genes (VRN1-3) have been largely implicated in the determination of vernalization response and are responsible for much of the intra-species variation in this trait. Many other important flowering-time regulators have been cataloged in the model grass Brachypodium distachyon; however, in several cases, such as VRN2, their ryegrass homologs have not been well-characterized. Here, ryegrass homologs of important flowering time genes from B. distachyon were identified through available synteny data and sequence similarity. Phylogenetic analysis of VRN3/FT-like and VRN2-like genes was performed to elucidate these families further. The expression patterns of these genes were assessed during vernalization. This confirmed the key roles played by LpVRN1 and LpFT3 in the promotion of flowering. Furthermore, two orthologs of VRN2 identified here, as well as an ortholog of CO9, were expressed prior to vernalization, and were repressed in flowering plants, suggesting a role in floral repression. Significant variability in expression of these flowering pathway genes in diverse genotypes was detected and may underlie variation in flowering time and vernalization response.

Molecular control of the floral transition in the mast seeding plant Celmisia lyallii (Asteraceae).

Mast flowering (or masting) is synchronous, highly variable flowering among years in populations of perennial plants. Despite having widespread consequences for seed consumers, endangered fauna and human health, masting is hard to predict. While observational studies show links to various weather patterns in different plant species, the mechanism(s) underpinning the regulation of masting is still not fully explained. We studied floral induction in Celmisia lyallii (Asteraceae), a mast flowering herbaceous alpine perennial, comparing gene expression in flowering and nonflowering plants. We performed translocation experiments to induce the floral transition in C. lyallii plants followed by both global and targeted expression analysis of flowering-pathway genes. Differential expression analysis showed elevated expression of ClSOC1 and ClmiR172 (promoters of flowering) in leaves of plants that subsequently flowered, in contrast to elevated expression of ClAFT and ClTOE1 (repressors of flowering) in leaves of plants that did not flower. The warm summer conditions that promoted flowering led to differential regulation of age and hormonal pathway genes, including ClmiR172 and ClGA20ox2, known to repress the expression of floral repressors and permit flowering. Upregulated expression of epigenetic modifiers of floral promoters also suggests that plants may maintain a novel "summer memory" across years to induce flowering. These results provide a basic mechanistic understanding of floral induction in masting plants and evidence of their ability to imprint various environmental cues to synchronize flowering, allowing us to better predict masting events under climate change.

PHOSPHATIDYLETHANOLAMINE-BINDING PROTEINS: the conductors of dual reproduction in plants with vegetative storage organs.

Geophytes, the plants that form vegetative storage organs, are characterized by a dual reproduction system, in which vegetative and sexual propagation are tightly regulated to ensure fitness in harsh climatic conditions. Recent findings highlight the role of the PEBP (PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN) gene family in geophytes as major players in the molecular cascades underlying both types of reproduction. In this review, we briefly explain the life cycle and reproduction strategies of different geophytes and what is known about the physiological aspects related to these processes. Subsequently, an in-depth overview is provided of the molecular and genetic pathways driving these processes. In the evolution of plants, the PEBP gene family has expanded, followed by neo- and subfunctionalization. Careful characterization revealed that differential expression and differential protein complex formation provide the members of this gene family with unique functions, enabling them to mediate the crosstalk between the two reproductive events in geophytes in response to environmental and endogenous cues. Taking all these studies into account, we propose to regard the PEBPs as conductors of geophyte reproductive development.

RNAi-mediated repression of dormancy-related genes results in evergrowing apple trees.

DORMANCY-ASSOCIATED MADS-box (DAM) and SHORT VEGETATIVE PHASE (SVP) genes have been implicated in the regulation of winter dormancy in perennials. Ectopic expression of apple (Malus × domestica Borkh. 'Royal Gala') DAM and SVP genes delays budbreak and constrains lateral shoot outgrowth. In this study, we used RNA interference (RNAi) to simultaneously target all apple DAM and SVP genes in order to study their role and mode of action in the regulation of bud dormancy, budbreak and flowering. A synthetic construct carrying a hairpin fragment assembled from sequences specific to coding regions of three DAM and two SVP genes was used to generate transgenic lines. Reduced expression of DAM/SVP genes resulted in delayed leaf senescence and abscission in autumn, failure to enter bud dormancy in winter and continual growth of new leaves regardless of the season for over 3 years. Precocious flowering but normal flower morphology, fertility and fruit development were observed. The non-dormant phenotype was associated with modified phytohormone composition. The content of gibberellins (GAs) and jasmonates (JAs) was significantly increased in terminal buds of RNAi lines compared with wildtype plants, accompanied by elevated expression of the key GA biosynthesis pathway gene GIBBERELLIN 20 OXIDASE-2 (MdGA20ox-2) along with the FLOWERING LOCUS T gene MdFT2. The key mediator of plasmodesmatal closure, MdCALLOSE SYNTHASE 1 (MdCALS1), was repressed in RNAi lines. This study provides functional evidence for the role of DAM/SVP genes in vegetative phenology of apple and paves the way for production of low-chill varieties suitable for growth in warming climates.

Genome draft of the Arabidopsis relative Pachycladon cheesemanii reveals novel strategies to tolerate New Zealand's high ultraviolet B radiation environment.

BACKGROUND: Pachycladon cheesemanii is a close relative of Arabidopsis thaliana and is an allotetraploid perennial herb which is widespread in the South Island of New Zealand. It grows at altitudes of up to 1000 m where it is subject to relatively high levels of ultraviolet (UV)-B radiation. To gain first insights into how Pachycladon copes with UV-B stress, we sequenced its genome and compared the UV-B tolerance of two Pachycladon accessions with those of two A. thaliana accessions from different altitudes. RESULTS: A high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologs. Synteny analysis with genomes from other species of the Brassicaceae family found a close phylogenetic relationship of P. cheesemanii with Boechera stricta from Brassicaceae lineage I. While UV-B radiation caused a greater growth reduction in the A. thaliana accessions than in the P. cheesemanii accessions, growth was not reduced in one P. cheesemanii accession. The homologues of A. thaliana UV-B radiation response genes were duplicated in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii appears to differ from that in A. thaliana. CONCLUSION: Although the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies allowing the plant to tolerate relatively high UV-B radiation.

Identification of flowering-time genes in mast flowering plants using De Novo transcriptomic analysis.

Mast flowering is synchronised highly variable flowering by a population of perennial plants over a wide geographical area. High seeding years are seen as a threat to native and endangered species due to high predator density caused by the abundance of seed. An understanding of the molecular pathways that influence masting behaviour in plants could provide better prediction of a forthcoming masting season and enable conservation strategies to be deployed. The goal of this study was to identify candidate flowering genes that might be involved in regulating mast flowering. To achieve this, high-throughput large-scale RNA-sequencing was performed on two masting plant species, Celmisia lyallii (Asteraceae), and Chionochloa pallens (Poaceae) to develop a reference transcriptome for functional and molecular analysis. An average total of 33 million 150 base-paired reads, for both species, were assembled using the Trinity pipeline, resulting in 151,803 and 348,649 transcripts respectively for C. lyallii and C. pallens. For both species, about 56% of the unigenes were annotated with gene descriptions to known proteins followed by Gene Ontology analysis, categorising them on the basis of putative biological processes, molecular function, and cellular localization. A total of 543 transcripts from C. lyallii and 470 transcripts from C. pallens were also mapped to unique flowering-time proteins identified in Arabidopsis thaliana, suggesting the conservation of the flowering network in these wild alpine plants growing in natural field conditions. Expression analysis of several selected homologous flowering-pathway genes showed seasonal and photoperiodic variations. These genes can further be analysed to understand why seasonal cues, such as the increasing photoperiod in spring, that triggers the annual flowering of most plants, are insufficient to always trigger flowering in masting plants and to uncover the molecular basis of how additional cues (such as temperature during the previous growing seasons) then determines flowering in mast years.

Molecular Characterisation of a Supergene Conditioning Super-High Vitamin C in Kiwifruit Hybrids.

During analysis of kiwifruit derived from hybrids between the high vitamin C (ascorbic acid; AsA) species Actinidia eriantha and A. chinensis, we observed bimodal segregation of fruit AsA concentration suggesting major gene segregation. To test this hypothesis, we performed whole-genome sequencing on pools of hybrid genotypes with either high or low AsA fruit. Pool-GWAS (genome-wide association study) revealed a single Quantitative Trait Locus (QTL) spanning more than 5 Mbp on chromosome 26, which we denote as qAsA26.1. A co-dominant PCR marker was used to validate this association in four diploid (A. chinensis × A. eriantha) × A. chinensis backcross families, showing that the A. eriantha allele at this locus increases fruit AsA levels by 250 mg/100 g fresh weight. Inspection of genome composition and recombination in other A. chinensis genetic maps confirmed that the qAsA26.1 region bears hallmarks of suppressed recombination. The molecular fingerprint of this locus was examined in leaves of backcross validation families by RNA sequencing (RNASEQ). This confirmed strong allelic expression bias across this region as well as differential expression of transcripts on other chromosomes. This evidence suggests that the region harbouring qAsA26.1 constitutes a supergene, which may condition multiple pleiotropic effects on metabolism.

Histone modification and activation by SOC1-like and drought stress-related transcription factors may regulate AcSVP2 expression during kiwifruit winter dormancy.

The SHORT VEGETATIVE PHASE (SVP)-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes have been shown to regulate winter dormancy in woody perennials. In kiwifruit, AcSVP2 affects the duration of dormancy in cultivars that require high chill for dormancy release. In this study, we used a low-chill kiwifruit Actinidia chinensis 'Hort16A' to further study the function and regulation of AcSVP2. Overexpression of AcSVP2 in transgenic A. chinensis delayed budbreak in spring. A reduction in the active trimethylation histone marks of the histone H3K4 and acetylation of histone H3 contributed to the reduction of AcSVP2 expression towards dormancy release, while the inactive histone marks of trimethylation of the histone H3K27 and H3K9 in AcSVP2 locus did not show significant enrichment at the end of winter dormancy. Analysis of expression in shoot buds showed that AcSVP2 transcript was elevated in dormant buds during winter months and declined prior to budbreak, which was coordinated with expression of some of kiwifruit SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1)-like genes. Screening of 101 transcription factors in an assay with a 2.3 kb promoter region of AcSVP2 found that kiwifruit SOC1-like genes are able to activate the AcSVP2 promoter. We further identified additional transcription factors associated with drought/osmotic stress and dormancy which may regulate AcSVP2 expression.

Comparative transcriptome analysis of the wild-type model apomict Hieracium praealtum and its loss of parthenogenesis (lop) mutant.

BACKGROUND: Asexual seed formation (apomixis) has been observed in diverse plant families but is rare in crop plants. The generation of apomictic crops would revolutionize agriculture, as clonal seed production provides a low cost and efficient way to produce hybrid seed. Hieracium (Asteraceae) is a model system for studying the molecular components of gametophytic apomixis (asexual seed reproduction). RESULTS: In this study, a reference transcriptome was produced from apomictic Hieracium undergoing the key apomictic events of apomeiosis, parthenogenesis and autonomous endosperm development. In addition, transcriptome sequences from pre-pollination and post-pollination stages were generated from a loss of parthenogenesis (lop) mutant accession that exhibits loss of parthenogenesis and autonomous endosperm development. The transcriptome is composed of 147,632 contigs, 50% of which were annotated with orthologous genes and their probable function. The transcriptome was used to identify transcripts differentially expressed during apomictic and pollination dependent (lop) seed development. Gene Ontology enrichment analysis of differentially expressed transcripts showed that an important difference between apomictic and pollination dependent seed development was the expression of genes relating to epigenetic gene regulation. Genes that mark key developmental stages, i.e. aposporous embryo sac development and seed development, were also identified through their enhanced expression at those stages. CONCLUSION: The production of a comprehensive floral reference transcriptome for Hieracium provides a valuable resource for research into the molecular basis of apomixis and the identification of the genes underlying the LOP locus.

Functional Genomics and Flowering Time in Medicago truncatula: An Overview.

Flowering time is an important trait that influences adaptation and yield in many crop legumes. Both the inherent earliness of flowering and the degree to which it is responsive to environmental factors determine both the eco-geographic range across which crops can be successfully grown and the seasonal cycles most suitable for production. This chapter will provide a brief review of studies investigating the genetic control of flowering time in Medicago truncatula.

Medicago truncatula SOC1 Genes Are Up-regulated by Environmental Cues That Promote Flowering.

Like Arabidopsis thaliana, the flowering of the legume Medicago truncatula is promoted by long day (LD) photoperiod and vernalization. However, there are differences in the molecular mechanisms involved, with orthologs of two key Arabidopsis thaliana regulators, FLOWERING LOCUS C (FLC) and CONSTANS (CO), being absent or not having a role in flowering time function in Medicago. In Arabidopsis, the MADS-box transcription factor gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (AtSOC1), plays a key role in integrating the photoperiodic and vernalization pathways. In this study, we set out to investigate whether the Medicago SOC1 genes play a role in regulating flowering time. Three Medicago SOC1 genes were identified and characterized (MtSOC1a-MtSOC1c). All three MtSOC1 genes, when heterologously expressed, were able to promote earlier flowering of the late-flowering Arabidopsis soc1-2 mutant. The three MtSOC1 genes have different patterns of expression. However, consistent with a potential role in flowering time regulation, all three MtSOC1 genes are expressed in the shoot apex and are up-regulated in the shoot apex of plants in response to LD photoperiods and vernalization. The up-regulation of MtSOC1 genes was reduced in Medicago fta1-1 mutants, indicating that they are downstream of MtFTa1. Insertion mutant alleles of Medicago soc1b do not flower late, suggestive of functional redundancy among Medicago SOC1 genes in promoting flowering.

Complete Genome Sequence of a New Zealand Isolate of the Bovine Pathogen Streptococcus uberis.

Streptococcus uberis forms part of the native microbiota of cattle and is able to opportunistically infect the mammary gland; as such, it is a leading cause of bovine mastitis globally. Here, we report the complete genome sequence of S. uberis NZ01, isolated in New Zealand from a cow with a clinical case of bovine mastitis.