Transcriptome Analysis of Stigmas of Vicia faba L. Flowers.

Pollination in angiosperms depends on complex communication between pollen grains and stigmas, classified as wet or dry, depending on the presence or absence of secretions at the stigma surface, respectively. In species with wet stigma, the cuticle is disrupted and the presence of exudates is indicative of their receptivity. Most stigma studies are focused on a few species and families, many of them with self-incompatibility systems. However, there is scarce knowledge about the stigma composition in Fabaceae, the third angiosperm family, whose stigmas have been classified as semidry. Here we report the first transcriptome profiling and DEGs of Vicia faba L. styles and stigmas from autofertile (flowers able to self-fertilize in the absence of manipulation, whose exudate is released spontaneously) and autosterile (flowers that need to be manipulated to break the cuticle and release the exudates to be receptive) inbred lines. From the 76,269 contigs obtained from the de novo assembly, only 45.1% of the sequences were annotated with at least one GO term. A total of 115,920, 75,489, and 70,801 annotations were assigned to Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) categories, respectively, and 5918 differentially expressed genes (DEGs) were identified between the autofertile and the autosterile lines. Among the most enriched metabolic pathways in the DEGs subset were those related with amino acid biosynthesis, terpenoid metabolism, or signal transduction. Some DEGs have been related with previous QTLs identified for autofertility traits, and their putative functions are discussed. The results derived from this work provide an important transcriptomic reference for style-stigma processes to aid our understanding of the molecular mechanisms involved in faba bean fertilization.

Study and QTL mapping of reproductive and morphological traits implicated in the autofertility of faba bean.

Autofertility describes the ability of faba bean flowers to self-fertilize thereby ensuring the productivity of this crop in the absence of pollinators or mechanical disturbance. In the legume crop faba bean (Vicia faba L.), lack of autofertility in a context of insufficient pollination can lead to a severe decrease in grain yield. Here we performed the first QTL analysis aimed at identifying the genomic regions controlling autofertility in this crop. We combined pod and seed setting scores from a recombinant inbred population (RIL) segregating for autofertility in different environments and years with measurements of morphological floral traits and pollen production and viability. This approach revealed 19 QTLs co-localizing in six genomic regions. Extensive co-localization was evident for various floral features whose QTLs clustered in chrs. I, II and V, while other QTLs in chrs. III, IV and VI revealed co-localization of flower characteristics and pod and seed set data. The percentage of phenotypic variation explained by the QTLs ranged from 8.9 for style length to 25.7 for stigma angle. In the three QTLs explaining the highest phenotypic variation (R 2 > 20), the marker alleles derived from the autofertile line Vf27. We further inspected positional candidates identified by these QTLs which represent a valuable resource for further validation. Our results advance the understanding of autofertility in faba bean and will aid the identification of responsible genes for genomic-assisted breeding in this crop.

Linkage mapping and QTL analysis of flowering time in faba bean.

Flowering time marks the transition from vegetative to reproductive growth and is key for optimal yield in any crop. The molecular mechanisms controlling this trait have been extensively studied in model plants such as Arabidopsis thaliana and rice. While knowledge on the molecular regulation of this trait is rapidly increasing in sequenced galegoid legume crops, understanding in faba bean remains limited. Here we exploited translational genomics from model legume crops to identify and fine map QTLs linked to flowering time in faba bean. Among the 31 candidate genes relevant for flowering control in A. thaliana and Cicer arietinum assayed, 25 could be mapped in a segregating faba bean RIL population. While most of the genes showed conserved synteny among related legume species, none of them co-localized with the 9 significant QTL regions identified. The FT gene, previously implicated in the control of flowering time in numerous members of the temperate legume clade, mapped close to the most relevant stable and conserved QTL in chromosome V. Interestingly, QTL analysis suggests an important role of epigenetic modifications in faba bean flowering control. The new QTLs and candidate genes assayed here provide a robust framework for further genetic studies and will contribute to the elucidation of the molecular mechanisms controlling this trait.

First approach to pod dehiscence in faba bean: genetic and histological analyses.

Pod dehiscence causes important yield losses in cultivated crops and therefore has been a key trait strongly selected against in crop domestication. In spite of the growing knowledge on the genetic basis of dehiscence in different crops, no information is available so far for faba bean. Here we conduct the first comprehensive study for faba bean pod dehiscence by combining, linkage mapping, comparative genomics, QTL analysis and histological examination of mature pods. Mapping of dehiscence-related genes revealed conservation of syntenic blocks among different legumes. Three QTLs were identified in faba bean chromosomes II, IV and VI, although none of them was stable across years. Histological analysis supports the convergent phenotypic evolution previously reported in cereals and related legume species but revealed a more complex pattern in faba bean. Contrary to common bean and soybean, the faba bean dehiscence zone appears to show functional equivalence to that described in crucifers. The lignified wall fiber layer, which is absent in the paucijuga primitive line Vf27, or less lignified and vacuolated in other dehiscent lines, appears to act as the major force triggering pod dehiscence in this species. While our findings, provide new insight into the mechanisms underlying faba bean dehiscence, full understanding of the molecular bases will require further studies combining precise phenotyping with genomic analysis.

Whole Plastome Sequencing Within Silene Section Psammophilae Reveals Mainland Hybridization and Divergence With the Balearic Island Populations.

Reconstructing the phylogenetic relationships within Caryophyllaceae tribe Sileneae has been obscured by hybridization and incomplete lineage sorting. Silene is the largest genus in the Caryophyllaceae, and unraveling its evolutionary history has been particularly challenging. In order to infer the phylogenetic relationships among the five species in Silene section Psammophilae, we have performed a genome skimming approach to acquire the complete plastid genome (cpDNA), nuclear ribosomal cistron (nrDNA), and partial mitochondrial genome (mtDNA). We have included 26 populations, representing the range of each species' distribution. This section includes five morphologically similar species endemic to the Iberian Peninsula and Balearic Islands (Ibiza and Formentera), yet some of them occupy distinct edaphic habitats (e.g. maritime sands, calcareous sandstones). In addition to phylogeographic analyses, genetic structuring using the chloroplast data set was inferred with Discriminant Analysis of Principal Components (DAPC), analyses of molecular variance (AMOVA), and a partial Mantel test. Reference-guided assembly of 50 bp single-end and 250 bp paired-end Illumina reads produced the nearly complete cpDNA genome (154 kbp), partial mtDNA genome (from 81 to 114 kbp), and the nrDNA cistron (6.4 kbp). Selected variable regions of the cpDNA and mtDNA assemblies were confirmed by Sanger sequencing. Phylogenetic analyses of the mainland populations reveal incongruence among the three genomes. None of the three data sets produced relationships consistent with taxonomy or geography. In contrast, Silene cambessedesii, present in the Balearic Islands, is the only species that forms a strongly supported monophyletic clade in the cpDNA genome and is strongly differentiated with respect to the remaining taxa of the Iberian Peninsula. These results contrast with those obtained for mainland populations. Across the entire analysis, only one well-supported mainland clade of Silene littorea and Silene stockenii emerges from the southern region of the Iberian Peninsula. DAPC and AMOVA results suggest the absence of genetic structure among mainland populations of Silene section Psammophilae, whereas partial Mantel test discarded spatial correlation of genetic differentiation. The widespread incongruence between morphology-based taxonomic boundaries and phylogeography suggests a history of interspecific hybridization, in which only a substantial geographic barrier, like isolation by the Mediterranean Sea, was sufficient to create and maintain species boundaries in Silene section Psammophilae.

Transcriptome and Biochemical Analysis of a Flower Color Polymorphism in Silene littorea (Caryophyllaceae).

Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a.

On flavonoid accumulation in different plant parts: variation patterns among individuals and populations in the shore campion (Silene littorea).

The presence of anthocyanins in flowers and fruits is frequently attributed to attracting pollinators and dispersers. In vegetative organs, anthocyanins and other non-pigmented flavonoids such as flavones and flavonols may serve protective functions against UV radiation, cold, heat, drought, salinity, pathogens, and herbivores; thus, these compounds are usually produced as a plastic response to such stressors. Although, the independent accumulation of anthocyanins in reproductive and vegetative tissues is commonly postulated due to differential regulation, the accumulation of flavonoids within and among populations has never been thoroughly compared. Here, we investigated the shore campion (Silene littorea, Caryophyllaceae) which exhibits variation in anthocyanin accumulation in its floral and vegetative tissues. We examined the in-situ accumulation of flavonoids in floral (petals and calyxes) and vegetative organs (leaves) from 18 populations representing the species' geographic distribution. Each organ exhibited considerable variability in the content of anthocyanins and other flavonoids both within and among populations. In all organs, anthocyanin and other flavonoids were correlated. At the plant level, the flavonoid content in petals, calyxes, and leaves was not correlated in most of the populations. However, at the population level, the mean amount of anthocyanins in all organs was positively correlated, which suggests that the variable environmental conditions of populations may play a role in anthocyanin accumulation. These results are unexpected because the anthocyanins are usually constitutive in petals, yet contingent to environmental conditions in calyxes and leaves. Anthocyanin variation in petals may influence pollinator attraction and subsequent plant reproduction, yet the amount of anthocyanins may be a direct response to environmental factors. In populations on the west coast, a general pattern of increasing accumulation of flavonoids toward southern latitudes was observed in calyxes and leaves. This pattern corresponds to a gradual increase of UV-B radiation and temperature, and a decrease of rainfall toward the south. However, populations along the southern coast exposed to similar climatic stressors showed highly variable flavonoid contents, implying that other factors may play a role in flavonoid accumulation.

Diversity of sexual systems within different lineages of the genus Silene.

Species and populations can be categorized by their sexual systems, depending on the spatial distribution of female and male reproductive structures within and among plants. Although a high diversity of sexual systems exists in Silene, their relative frequency at the genus and infrageneric level is unknown. Here, we carried out an extensive literature search for direct or indirect descriptions of sexual systems in Silene species. We found descriptions of sexual systems for 98 Silene species, where 63 and 35 correspond to the phylogenetically supported subgenera Silene and Behenantha, respectively. Hermaphroditism was the commonest sexual system (58.2 %), followed by dioecy (14.3 %), gynodioecy (13.3 %) and gynodioecy-gynomonoecy (i.e. hermaphroditic, female and gynomonoecious plants coexisting in the same population; 12.2 %). The presence of these sexual systems in both subgenera suggests their multiple origins. In 17 species, the description of sexual systems varied, and in most cases these differences corresponded to variations within or among populations. Interestingly, the poorly studied gynodioecy-gynomonoecy sexual system showed similar frequency to dioecy and gynodioecy in both subgenera. In addition, the incidence of gynodioecy-gynomonoecy was analysed in the species of section Psammophilae (Silene littorea, S. psammitis, S. adscendens and S. cambessedesii), in a survey of 26 populations across the distribution area of the species. The four species showed gynomonoecy-gynodioecy in most populations. Hermaphrodites were the most frequent morph, with a low number of females and gynomonoecious plants in all populations. The frequency of sexual morphs varied significantly among the studied populations but not among species. Female plants generally produced smaller numbers of flowers than hermaphroditic or gynomonoecious plants, and the percentages of female flowers per population were low. All these findings suggest that the gynodioecious-gynomonoecious sexual system in section Psammophilae is closer to hermaphroditism or gynomonoecy than gynodioecy.