Examination of S-Locus Regulated Differential Expression in Primula vulgaris Floral Development.
ABSTRACT: Recent findings on the molecular basis of heteromorphic self-incompatibility in Primula have shown that the controlling self-incompatibility (S)-locus is not allelic, but is instead a small hemizygous region of only a few genes in the thrum genotype. How these genes alter the development of floral morphology and the specificity of self-incompatibility is still not completely clear. In order to start to identify genes regulated by the S-locus and elucidate the large-scale biological processes affected, we used RNA-seq data from floral buds of heteromorphic P. vulgaris pin (long style, short anthers) and thrum (short style, long anthers) morphs at early and late developmental time points. Differential expression between the two morphs was assessed at both time points and Gene Ontology term analyses of these gene sets were conducted. Our findings suggest that the S-locus regulates a large number of genes outside its physical bounds and likely sets up a cascade of expression changes. Additionally, we found evidence to suggest that there may be a timing difference in pollen development between the morphs, with pin pollen development proceeding earlier than thrum pollen development. This finding provides insight into how morphological differences in pollen between the morphs may be established, but intriguingly, could also be related to the self-incompatibility phenotype.
Project description:Heteromorphic self-incompatibility (SI) is an important system for preventing inbreeding in the genus Primula. However, investigations into the molecular mechanisms of Primula SI are lacking. To explore the mechanisms of SI in Primula maximowiczii, the pollen germination and fruiting rates of self- and cross-pollinations between pin and thrum morphs were investigated, and transcriptomics analyses of the pistils after pollination were performed to assess gene expression patterns in pin and thrum SI. The results indicated that P. maximowiczii exhibits strong SI and that the mechanisms of pollen tube inhibition differ between pin and thrum morphs. While self-pollen tubes of the pin morph were able to occasionally, though rarely, enter the style, those of the thrum morph were never observed to enter the style. The transcriptomics analysis of the pistils revealed 1311 and 1048 differentially expressed genes (DEGs) that were identified by comparing pin self-pollination (PS) vs. pin cross-pollination (PT) and thrum self-pollination (TS) vs. thrum cross-pollination (TP). Notably, about 90% of these DEGs exhibited different expression patterns in the two comparisons. Moreover, pin and thrum DEGs were associated with different Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways following enrichment analyses. Based on our results, the molecular mechanisms underlying the pin and thrum SI in P. maximowiczii appear to be distinct. Furthermore, the genes involved in the SI processes are commonly associated with carbohydrate metabolism and environmental adaptation. These results provide new insight into the molecular mechanisms of Primula SI.
Project description:Common buckwheat (Fagopyrum esculentum) is a heteromorphic self-incompatible (SI) species with two types of floral architecture: thrum (short style) and pin (long style). The floral morphology and intra-morph incompatibility are controlled by a single genetic locus, S. However, the molecular mechanisms underlying the heteromorphic self-incompatibility of common buckwheat remain unclear. To identify these mechanisms, we performed proteomic, quantitative reverse-transcription PCR, and linkage analyses. Comparison of protein profiles between the long and short styles revealed a protein unique to the short style. Amino-acid sequencing revealed that it was a truncated form of polygalacturonase (PG); we designated the gene encoding this protein FePG1. Phylogenetic analysis classified FePG1 into the same clade as PGs that function in pollen development and floral morphology. FePG1 expression was significantly higher in short styles than in long styles. It was expressed in flowers of a short-homostyle line but not in flowers of a long-homostyle line. Linkage analysis indicated that FePG1 was not linked to the S locus; it could be a factor downstream of this locus. Our finding of a gene putatively working under the regulation of the S locus provides useful information for elucidation of the mechanism of heteromorphic self-incompatibility.
Project description:Primula vulgaris (primrose) exhibits heterostyly: plants produce self-incompatible pin- or thrum-form flowers, with anthers and stigma at reciprocal heights. Darwin concluded that this arrangement promotes insect-mediated cross-pollination; later studies revealed control by a cluster of genes, or supergene, known as the S (Style length) locus. The P. vulgaris S locus is absent from pin plants and hemizygous in thrum plants (thrum-specific); mutation of S locus genes produces self-fertile homostyle flowers with anthers and stigma at equal heights. Here, we present a 411?Mb?P. vulgaris genome assembly of a homozygous inbred long homostyle, representing ~87% of the genome. We annotate over 24,000?P. vulgaris genes, and reveal more genes up-regulated in thrum than pin flowers. We show reduced genomic read coverage across the S locus in other Primula species, including P. veris, where we define the conserved structure and expression of the S locus genes in thrum. Further analysis reveals the S locus has elevated repeat content (64%) compared to the wider genome (37%). Our studies suggest conservation of S locus genetic architecture in Primula, and provide a platform for identification and evolutionary analysis of the S locus and downstream targets that regulate heterostyly in diverse heterostylous species.
Project description:Distyly is a widespread floral polymorphism characterized by the flowers within a population showing reciprocal placement of the anthers and stigma. Darwin hypothesizes that distyly evolves to promote precise pollen transfer between morphs. Primula chungensis exhibits two types of anther heights, and these two types of anthers show pollen of two different size classes. To understand whether the stigma could capture more pollen grains from the anthers of the pollen donor as the separation between the stigma of pollen receiver and the anther of pollen donor decreased, the present research assessed the source of the pollen load in a series of open-pollinated flowers with continuous variation of style lengths. Individuals with continuous variation of style length were tagged, and the selected flowers in the tagged plants were emasculated the day before dehiscence. The stigma of the emasculated flowers was fixed in fuchsin gel at the end of blooming. We assessed the pollen sources on each stigma by taking photos under a microscope and measured the diameter of each conspecific pollen grain with ImageJ. We found that a shorter distance from the stigmas to the anthers of a pollen donor gave the flower a higher capacity to receive pollen from those anthers. Our result provides a new evidence that distyly could promote the pollen transfer between morphs, which is consistent with Darwin's hypothesis of disassortative pollination. An alternative hypothesis for the evolution of distyly (e.g. selfing avoidance) might also be true, but less likely, because self-incompatibility would greatly avoid self-fertilization for many distylous species.
Project description:Heterostyly employs distinct hermaphroditic floral morphs to enforce outbreeding. Morphs differ structurally in stigma/anther positioning, promoting cross-pollination, and physiologically blocking self-fertilization. Heterostyly is controlled by a self-incompatibility (S)-locus of a small number of linked S-genes specific to short-styled morph genomes. Turnera possesses three S-genes, namely TsBAHD (controlling pistil characters), TsYUC6, and TsSPH1 (controlling stamen characters). Here, we compare pistil and stamen transcriptomes of floral morphs of T. subulata to investigate hypothesized S-gene function(s) and whether hormonal differences might contribute to physiological incompatibility. We then use network analyses to identify genetic networks underpinning heterostyly. We found a depletion of brassinosteroid-regulated genes in short styled (S)-morph pistils, consistent with hypothesized brassinosteroid-inactivating activity of TsBAHD. In S-morph anthers, auxin-regulated genes were enriched, consistent with hypothesized auxin biosynthesis activity of TsYUC6. Evidence was found for auxin elevation and brassinosteroid reduction in both pistils and stamens of S- relative to long styled (L)-morph flowers, consistent with reciprocal hormonal differences contributing to physiological incompatibility. Additional hormone pathways were also affected, however, suggesting S-gene activities intersect with a signaling hub. Interestingly, distinct S-genes controlling pistil length, from three species with independently evolved heterostyly, potentially intersect with phytochrome interacting factor (PIF) network hubs which mediate red/far-red light signaling. We propose that modification of the activities of PIF hubs by the S-locus could be a common theme in the evolution of heterostyly.
Project description:The different forms of flowers in a species have attracted the attention of many evolutionary biologists, including Charles Darwin. In Fagopyrum esculentum (common buckwheat), the occurrence of dimorphic flowers, namely short-styled and long-styled flowers, is associated with a type of self-incompatibility (SI) called heteromorphic SI. The floral morphology and intra-morph incompatibility are both determined by a single genetic locus named the S-locus. Plants with short-styled flowers are heterozygous (S/s) and plants with long-styled flowers are homozygous recessive (s/s) at the S-locus. Despite recent progress in our understanding of the molecular basis of flower development and plant SI systems, the molecular mechanisms underlying heteromorphic SI remain unresolved. By examining differentially expressed genes from the styles of the two floral morphs, we identified a gene that is expressed only in short-styled plants. The novel gene identified was completely linked to the S-locus in a linkage analysis of 1,373 plants and had homology to EARLY FLOWERING 3. We named this gene S-LOCUS EARLY FLOWERING 3 (S-ELF3). In an ion-beam-induced mutant that harbored a deletion in the genomic region spanning S-ELF3, a phenotype shift from short-styled flowers to long-styled flowers was observed. Furthermore, S-ELF3 was present in the genome of short-styled plants and absent from that of long-styled plants both in world-wide landraces of buckwheat and in two distantly related Fagopyrum species that exhibit heteromorphic SI. Moreover, independent disruptions of S-ELF3 were detected in a recently emerged self-compatible Fagopyrum species and a self-compatible line of buckwheat. The nonessential role of S-ELF3 in the survival of individuals and the prolonged evolutionary presence only in the genomes of short-styled plants exhibiting heteromorphic SI suggests that S-ELF3 is a suitable candidate gene for the control of the short-styled phenotype of buckwheat plants.
Project description:BACKGROUND AND AIMS:Heterostyly is a floral polymorphism characterized by the reciprocal position of stamens and stigmas in different flower morphs in a population. This reciprocal herkogamy is usually associated with an incompatibility system that prevents selfing and intra-morph fertilization, termed a heteromorphic incompatibility system. In different evolutionary models explaining heterostyly, it has been alternately argued that heteromorphic incompatibility either preceded or followed the evolution of reciprocal herkogamy. In some models, reciprocal herkogamy and incompatibility have been hypothesized to be linked together during the evolution of the heterostylous system. METHODS:We examine the incompatibility systems in species with different stylar polymorphisms from the genera Lithodora and Glandora (Boraginaceae). We then test whether evolution towards reciprocal herkogamy is associated with the acquisition of incompatibility. To this end, a phylogeny of these genera and related species is reconstructed and the morphological and reproductive changes that occurred during the course of evolution are assessed. KEY RESULTS:Both self-compatibility and self-incompatibility are found within the studied genera, along with different degrees of intra-morph compatibility. We report for the first time extensive variability among members of the genus Glandora and related species in terms of the presence or absence of intraspecies polymorphism and heteromorphic incompatibility. Overall, our results do not support a tight link between floral polymorphism and incompatibility systems. CONCLUSIONS:The independent evolution of stylar polymorphism and incompatibility appears to have occurred in this group of plants. This refutes the canonical view that there is strong linkage between these reproductive traits.
Project description:Floral morphology determines the pattern of pollen transfer within and between individuals. In hermaphroditic species, the spatial arrangement of sexual organs influences the rate of self-pollination as well as the placement of pollen in different areas of the pollinator's body. Studying the evolutionary modification of floral morphology in closely related species offers an opportunity to investigate the causes and consequences of floral variation. Here, we investigate the recurrent modification of flower morphology in three closely related pairs of taxa in Solanum section Androceras (Solanaceae), a group characterized by the presence of two morphologically distinct types of anthers in the same flower (heteranthery). We use morphometric analyses of plants grown in a common garden to characterize and compare the changes in floral morphology observed in parallel evolutionary transitions from relatively larger to smaller flowers. Our results indicate that the transition to smaller flowers is associated with a reduction in the spatial separation of anthers and stigma, changes in the allometric relationships among floral traits, shifts in pollen allocation to the two anther morphs and reduced pollen : ovule ratios. We suggest that floral modification in this group reflects parallel evolution towards increased self-fertilization and discuss potential selective scenarios that may favour this recurrent shift in floral morphology and function.
Project description:Tristyly is a genetic polymorphism in which populations are comprised of three floral morphs (mating types) differing reciprocally in sex-organ height. Intermorph (disassortative) mating governed by a trimorphic incompatibility system should result in 1:1:1 morph ratios at equilibrium, but both deterministic and stochastic processes can cause skewed morph ratios in tristylous populations. Here, we investigate mechanisms causing morph-ratio bias in Pontederia parviflora, an emergent aquatic native to tropical America. We compared reproductive traits among morphs and surveyed 71 populations to determine patterns of morph-ratio bias. We then used simulation models of morph-frequency dynamics to test the hypothesis that morph-specific differences in pollen production and their influence on male fertility can explain patterns of morph-ratio bias. Ninety-seven percent of populations that we sampled were tristylous, but with a significant excess of the short-styled morph and a deficiency of the long-styled morph. Atypically for a tristylous species, mid-level anthers of the short-styled morph produced over twice as much pollen compared with the corresponding anthers of the long-styled morph. Our computer models incorporating this difference in male fertility resulted in morph ratios not significantly different from the average frequencies from our survey suggesting that the short-styled morph is more successful than the long-styled morph in siring ovules of the mid-styled morph. We propose that the difference in male fertility between morphs may be a non-adaptive consequence of a developmental constraint caused by the architecture of tristyly in Pontederiaceae.
Project description:Heteromorphic flower development in Primula is controlled by the S locus. The S locus genes, which control anther position, pistil length and pollen size in pin and thrum flowers, have not yet been characterized. We have integrated S-linked genes, marker sequences and mutant phenotypes to create a map of the P. vulgaris S locus region that will facilitate the identification of key S locus genes. We have generated, sequenced and annotated BAC sequences spanning the S locus, and identified its chromosomal location. We have employed a combination of classical genetics and three-point crosses with molecular genetic analysis of recombinants to generate the map. We have characterized this region by Illumina sequencing and bioinformatic analysis, together with chromosome in situ hybridization. We present an integrated genetic and physical map across the P. vulgaris S locus flanked by phenotypic and DNA sequence markers. BAC contigs encompass a 1.5-Mb genomic region with 1 Mb of sequence containing 82 S-linked genes anchored to overlapping BACs. The S locus is located close to the centromere of the largest metacentric chromosome pair. These data will facilitate the identification of the genes that orchestrate heterostyly in Primula and enable evolutionary analyses of the S locus.