Genome resequencing reveals the evolutionary history of garlic reproduction traits.

The propagation of cultivated garlic relies on vegetative cloves, thus flowers become non-essential for reproduction in this species, driving the evolution of reproductive feature-derived traits. To obtain insights into the evolutionary alteration of reproductive traits in the clonally propagated garlic, the evolutionary histories of two main reproduction-related traits, bolting and flower differentiation, were explored by genome analyses using 134 accessions displaying wide diversity in these two traits. Resequencing identified 272.8 million variations in the garlic genome, 198.0 million of which represent novel variants. Population analysis identified five garlic groups that have evolved into two clades. Gene expression, single-cell transcriptome sequencing, and genome-wide trait association analyses have identified numerous candidates that correlate with reproductive transition and flower development, some of which display distinct selection signatures. Selective forces acting on the B-box zinc finger protein-encoding Asa2G00291.1, the global transcription factor group E protein-encoding Asa5G01527.1, and VERNALIZATION INSENSITIVE 3-like Asa3G03399.1 appear to be representative of the evolution of garlic bolting. Plenty of novel genomic variations and trait-related candidates represent valuable resources for biological studies of garlic. Numerous selective signatures from genes associated with the two chosen reproductive traits provide important insights into the evolutionary history of reproduction in this clonally propagated crop.

Ongoing Evolution in the Genus Crocus: Diversity of Flowering Strategies on the Way to Hysteranthy.

Species of the genus Crocus are found over a wide range of climatic areas. In natural habitats, these geophytes diverge in the flowering strategies. This variability was assessed by analyzing the flowering traits of the Spanish collection of wild crocuses, preserved in the Bank of Plant Germplasm of Cuenca. Plants of the seven Spanish species were analyzed both in their natural environments (58 native populations) and in common garden experiments (112 accessions). Differences among species observed in the native habitats were maintained under uniform environmental conditions, suggesting a genetic basis for flowering mechanisms. Two eco-morphological types, autumn- and spring-flowering species, share similar patterns of floral induction and differentiation period in summer. The optimal temperature for this process was 23 °C for both types. Unlike Irano-Turanian crocuses, spring-flowering Spanish species do not require low winter temperatures for flower elongation. Hysteranthous crocuses flower in autumn prior to leaf elongation. We conclude that the variability in flowering traits in crocuses is related to the genetic and environmental regulation of flower primordia differentiation and elongation prior to emergence above the soil surface. The elucidation of the physiological differences between eco-morphological types of crocuses: synanthous with cold requirements and synanthous and hysteranthous without cold requirements, unlocks a new approach to the flowering evolution of geophytes in Mediterranean regions. Crocus species can serve both as a new model in the study of the molecular basis of hysteranthy and for the purposes of developing the molecular markers for desirable flowering traits.

Insights into the maternal pathway for Cucumber green mottle mosaic virus infection of cucurbit seeds.

Cucumber green mottle mosaic virus (CGMMV), genus Tobamovirus, is a major pathogen of cucurbits that primarily affects cucumber, melon, and watermelon crops. The aim of this study was to reveal the contribution of CGMMV-infected female flowers to disease spread. Using a fluorescent in situ hybridization (FISH) technique, we show that ovaries and ovules of CGMMV-infected cucumber and melon plants showed a CGMMV-specific fluorescence signal prior to and following anthesis. The fluorescence signal was prominent but sporadic. Ripe fruits of infected melon plants showed strong signals in the funiculus, the seed stalk, which connects the developing seed to the interior ovary wall. Importantly, in seeds, a strong fluorescence signal was observed in the perisperm-endosperm (PE) envelope, which underlies the seed coat and surrounds the embryo. Interestingly, the fluorescence signal was not uniformly distributed in the PE envelope but was localized to a specific envelope layer. These results have important epidemiological implications for CGMMV management and commercial seed production, particularly regarding the improvement of seed disinfection methods that will contribute to limit the global distribution of the virus.

Architecture and Florogenesis in Female Cannabis sativa Plants.

The inflorescence is the main product of medical cannabis. Hundreds of specialized metabolites with potential bioactivity are produced and accumulated in the glandular trichomes that are highly abundant mainly on female inflorescences. Understanding the morphophysiological and genetic mechanisms governing flower and inflorescence development is therefore of high scientific and practical importance. However, in-depth investigations of cannabis florogenesis are limited. Cannabis producers and researchers consider long photoperiod to be "non-inductive" or "vegetative," but under these growth conditions, the development of solitary flowers and bracts in shoot internodes clearly indicates that the plant cannot be defined as vegetative or non-inductive in the classical sense. Most probably, induction of solitary flowers is age-dependent and controlled by internal signals, but not by photoperiod. Short photoperiod induces intense branching, which results in the development of a compound raceme. Each inflorescence consists of condensed branchlets with the same phytomer structure as that of the larger phytomers developed under long day. Each phytomer consists of reduced leaves, bracts, one or two solitary flowers, and an axillary shoot (or inflorescence). Therefore, the effect of short photoperiod on cannabis florogenesis is not flower induction, but rather a dramatic change in shoot apex architecture to form a compound racemose inflorescence structure. An understanding of the morphophysiological characteristics of cannabis inflorescence will lay the foundation for biotechnological and physiological applications to modify architecture and to maximize plant productivity and uniformity in medical Cannabis.

Garlic (Allium sativum L.) fertility: transcriptome and proteome analyses provide insight into flower and pollen development.

Commercial cultivars of garlic, a popular condiment, are sterile, making genetic studies and breeding of this plant challenging. However, recent fertility restoration has enabled advanced physiological and genetic research and hybridization in this important crop. Morphophysiological studies, combined with transcriptome and proteome analyses and quantitative PCR validation, enabled the identification of genes and specific processes involved in gametogenesis in fertile and male-sterile garlic genotypes. Both genotypes exhibit normal meiosis at early stages of anther development, but in the male-sterile plants, tapetal hypertrophy after microspore release leads to pollen degeneration. Transcriptome analysis and global gene-expression profiling showed that >16,000 genes are differentially expressed in the fertile vs. male-sterile developing flowers. Proteome analysis and quantitative comparison of 2D-gel protein maps revealed 36 significantly different protein spots, 9 of which were present only in the male-sterile genotype. Bioinformatic and quantitative PCR validation of 10 candidate genes exhibited significant expression differences between male-sterile and fertile flowers. A comparison of morphophysiological and molecular traits of fertile and male-sterile garlic flowers suggests that respiratory restrictions and/or non-regulated programmed cell death of the tapetum can lead to energy deficiency and consequent pollen abortion. Potential molecular markers for male fertility and sterility in garlic are proposed.

Storage temperature controls the timing of garlic bulb formation via shoot apical meristem termination.

MAIN CONCLUSION: Timing of bulb formation and floral stem induction in garlic is controlled by preplanting storage temperature and shoot apical meristem termination, probably via FLOWERING LOCUS T (FT) genes. Garlic is planted in the winter, undergoes a vegetative stage, then forms bulbs in response to increasing temperature and lengthening photoperiod. Herein, the storage conditions for propagation bulbs are shown to potentially affect future vegetative-stage length and timing of bulb formation. Storage temperatures of 2 or 33 °C inhibited internal bud growth. Levels of endogenous abscisic acid (ABA) and its inactive isomer trans-ABA were significantly higher in the internal bud of cloves stored at 33 vs. 2 °C, and exogenous ABA treatment before planting confirmed its inhibitory effect on foliage leaf development. Bulb formation started 30 and 60 days after planting of cloves stored at 2 and 33 °C, respectively. Warm storage temperature induced the formation of multiple leaves and cloves after planting. Plants from cloves stored at warm temperature developed a floral stem, whereas those from cold storage did not. Allium sativum FLOWERING LOCUS T1 (AsFT1) was upregulated 2.5- and 4.5-fold in the internal bud and storage leaf, respectively, after 90 and 150 days of cold vs. warm storage. Expression of AsFT4, expected to be antagonist to AsFT1, was 2- to 3-fold lower in the internal bud from cold storage. Expression of AsFT2, associated with floral termination, was 2- to 3- and 10- to 12-fold higher for cold vs. warm storage temperatures, in the internal bud and storage leaf, respectively. Early bulb formation, induced by cold storage, is suggested to inhibit normal foliage leaf development and transition of the shoot apical meristem to reproductive meristem, through regulation of FT genes.

Integrated transcriptome catalogue and organ-specific profiling of gene expression in fertile garlic (Allium sativum L.).

BACKGROUND: Garlic is cultivated and consumed worldwide as a popular condiment and green vegetable with medicinal and neutraceutical properties. Garlic cultivars do not produce seeds, and therefore, this plant has not been the subject of either classical breeding or genetic studies. However, recent achievements in fertility restoration in a number of genotypes have led to flowering and seed production, thus enabling genetic studies and breeding in garlic. RESULTS: A transcriptome catalogue of fertile garlic was produced from multiplexed gene libraries, using RNA collected from various plant organs, including inflorescences and flowers. Over 32 million 250-bp paired-end reads were assembled into an extensive transcriptome of 240,000 contigs. An abundant transcriptome assembled separately from 102,000 highly expressed contigs was annotated and analyzed for gene ontology and metabolic pathways. Organ-specific analysis showed significant variation of gene expression between plant organs, with the highest number of specific reads in inflorescences and flowers. Analysis of the enriched biological processes and molecular functions revealed characteristic patterns for stress response, flower development and photosynthetic activity. Orthologues of key flowering genes were differentially expressed, not only in reproductive tissues, but also in leaves and bulbs, suggesting their role in flower-signal transduction and the bulbing process. More than 100 variants and isoforms of enzymes involved in organosulfur metabolism were differentially expressed and had organ-specific patterns. In addition to plant genes, viral RNA of at least four garlic viruses was detected, mostly in the roots and cloves, whereas only 1-4% of the reads were found in the foliage leaves. CONCLUSIONS: The de novo transcriptome of fertile garlic represents a new resource for research and breeding of this important crop, as well as for the development of effective molecular markers for useful traits, including fertility and seed production, resistance to pests and neutraceutical characteristics.

Effects of different temperature regimes on flower development, microsporogenesis and fertility in bolting garlic (Allium sativum).

Garlic (Allium sativum L.) cultivars do not develop fertile flowers and seeds. Therefore, garlic production and improvement depend exclusively on vegetative propagation. Recent advances in garlic research have enabled fertility restoration and the discovery of fertile and male-sterile genotypes; however, the environmental regulation of the reproductive process is still not clear. Garlic seeds are successfully produced in the Mediterrenean region, where the photoperiod is relatively short, whereas spring and summer temperatures are high. We hypothesise that, in bolting garlic, various stages of florogenesis are differentially regulated by temperature and that high temperatures might obstruct pollen production. The effects of eight combinations of controlled growth temperatures on fertile and male-sterile garlic clones were studied. In both genotypes, a gradual temperature increase before and during anthesis favoured intact flower development. Surprisingly, continuous exposure to moderate temperatures during the entire growth period resulted in poor flowering, anther abortion and reduced pollen production. In the male-sterile genotype, no growth regime improved pollen production, which is controlled by genetic mechanisms. In the male-fertile genotype, gradual temperature increase supported pollen production but a sharp transition to high temperatures resulted in rapid flower senescence and pollen abortion, thus supporting our research hypothesis. In both fertile and male-sterile plants, the most vulnerable phase of microsporogenesis is the unicellular microspore stage. Tapetal malformation is the major cause for malnutrition of the microspores, with consequent production of nonviable pollen grains.

Turned on by heat: differential expression of FT and LFY-like genes in Narcissus tazetta during floral transition.

In Narcissus tazetta, a monocotyledonous bulbous geophyte, floral initiation and differentiation occur within the bulb during the quiescent period in summer, when ambient temperatures are relatively high and the bulb is located underground with no foliage or roots. In many plant species, FLOWERING LOCUS T (FT) and its homologues are considered powerful promoters of flowering. The Narcissus FT gene homologue (NtFT) was isolated, and organ-specific expression patterns of NtFT during the annual cycle and reproductive development under different temperature regimes were analysed using quantitative reverse transcription-PCR (qRT-PCR) and RNA in situ hybridization. During floral induction, NtFT was not expressed in bulb scales, roots, or foliage leaves, but it was detected inside the bulb in the apical meristem and leaf primordia. The expression of another key flowering gene, NLF, the LEAFY homologue in N. tazetta, was also observed only in meristem and leaf primordia within the bulbs; however, its expression did not coincide with that of NtFT during meristem transition to reproductive stage. Under high temperatures (25-30 °C) in the dark, NtFT expression occurred simultaneously with floral induction timing, indicating that floral induction is affected by high temperatures but not by photoperiod or vernalization. Monitoring the apical meristem of Narcissus in February-August of two growing seasons under ambient and controlled storage conditions showed that transition to flowering is temperature dependent and varies between years. Lack of NtFT and NLF expression in foliage leaves suggests that flower initiation control in Narcissus differs from that in common model plants.

Male gametogenesis and sterility in garlic (Allium sativum L.): barriers on the way to fertilization and seed production.

Commercial cultivars of garlic (Allium sativum) do not produce flowers and seed; hence, information on microgametogenesis and genetic knowledge of this important crop is unavailable. Recently, physiological studies enabled flowering and fertility restoration in garlic bolting genotypes by environmental manipulations, thus broadening of the genetic variation and facilitating genetic studies. The present report provides first detailed description of the development of male gametophytes in 11 garlic genotypes varying in their fertility traits. Morphological and anatomical studies revealed completely fertile genotypes, as well as variation in anther and pollen development and disruption of the male organs and gametes at different developmental stages. Three types of plant sterility were observed, including complete sterility, male sterility and environmentally induced male sterility. The ITS1 and ITS2 regions of rRNA of the studied genotypes proved to be strongly conservative and thus did not correspond with the phenotypic expression of fertility or sterility in garlic. On the other hand, two-dimensional protein separation maps revealed significant differences between fertile and sterile genotypes, as well as between developmental stages of microsporogenesis. Further research is needed to investigate the internal mechanisms and environmental component of garlic sterility, as well as the possible molecular markers of these traits.

Flower development in garlic: the ups and downs of gaLFY expression.

The lack of sexual processes prohibits genetic studies and conventional breeding in commercial cultivars of garlic. Recent restoration of garlic flowering ability by environmental manipulations has opened new avenues for physiological and genetic studies. The LEAFY homologue gaLFY has been shown to be involved in the floral development, while two alternatively spliced gaLFY transcripts are expressed in flowering genotypes. In the present work, quantitative real-time PCR and two techniques of RNA in situ hybridization were employed to analyze spatiotemporal expression patterns of the gaLFY during consequent stages of the garlic reproductive process. Temporal accumulation of gaLFY is strongly associated with reproductive organs, significantly increased during florogenesis and gametogenesis, and is down-regulated in the vegetative meristems and topsets in the inflorescence. The two alternative transcripts of the gene show different expression patterns: a high level of the long gaLFY transcript coincided only with floral transition, while further up-regulation of this gene in the reproductive organs is associated mainly with the short gaLFY transcript. It is concluded that gaLFY is involved at different stages of the sexual reproduction of garlic. These new insights broaden our basic understanding of flower biology of garlic and help to establish conventional and molecular breeding systems for this important crop.

Unlocking variability: inherent variation and developmental traits of garlic plants originated from sexual reproduction.

Recent collections of fertile garlic (Allium sativum) accessions from Central Asia allow a detailed study of seedling developments and the evaluation of inherent variations. We hereby provide a comprehensive account of the ontogenesis of a population of garlic seedlings and their vegetative and reproductive traits. A nucleotide binding site profiling marker technology was applied to provide conclusive evidence for the cross-pollination nature of garlic, and to compare the levels of polymorphism between progeny derived from a single mother clone fertilized by several pollinators. The seedlings' population demonstrates a large variation in vegetative and reproductive characters, including bulbing ability, bulb color and size, clove number, and response to environmental conditions, similar to that of the genepool of vegetatively propagated garlic clones. In addition, a large variation in flowering and seed production ability was recorded. The understanding of garlic physiology, the availability of the large variability unleashed by sexual reproduction, and the possible utilization of sexual hybridization opens the way for genetic studies and breeding work.

Reproductive development and phenotypic differences in garlic are associated with expression and splicing of LEAFY homologue gaLFY.

Modern garlic (Allium sativum L.) cultivars are sterile and propagated only vegetatively. The recent discovery of fertile genotypes in Central Asia and the restoration of flowering and fertility by environmental manipulations open the way for in-depth florogenetic, genetic, and molecular research in garlic. In the present work, two bolting garlic accessions were employed: #3026, developing normal flowers and seeds, and #2509, in which flowers abort at the early stages of development. Morphological studies showed transition of the apical meristems from the vegetative to the reproductive stage and inflorescence initiation in both genotypes. Low temperatures promote transition of the apex and stem elongation, but have no effect on the phenotypic expression of the inflorescence development. The initial stages of reproductive development in non-flowering #2509 plants were followed by abortion of floral primordia at the differentiation stage. A search for genes involved in the control of flowering in garlic resulted in identification of the garlic LEAFY/FLO homologue, gaLFY. Further comparative analyses of gene expression revealed two gaLFY transcripts, differing in 64 nucleotides, with clear splicing borders. The short variant transcript was identified in both genotypes throughout all development stages, whereas the long variant appears in the flowering genotype #3026 only during reproductive development. The phenotypic differences in garlic, with regard to flowering, may be associated with the efficacy of the splicing process.

Seasonal adaptations of the tuberous roots of Ranunculus asiaticus to desiccation and resurrection by changes in cell structure and protein content.

The annual developmental cycle of tuberous roots of Ranunculus asiaticus was studied with respect to structure and content of their cells, to understand how these roots are adapted to desiccation, high temperature and rehydration. Light microscopy, histochemical analysis, and protein analyses by SDS-PAGE were employed at eight stages of annual root development. During growth and maturation of the roots, cortical cells increased in size and their cell walls accumulated pectin materials in a distinct layer to the inside of the primary walls, with pits between adjoining cells. The number of starch granules and protein bodies also increased within the cells. Several discrete proteins accumulated. Following quiescence and rehydration of the roots there was a loss of starch and proteins from the cells, and cell walls decreased in thickness. The resurrection geophyte R. asiaticus possesses desiccation-tolerant annual roots. They store carbon and nitrogen reserves within their cells, and pectin within the walls to support growth of the plant following summer quiescence and rehydration.