Search

The development of new varieties of horticultural crops benefits from the integration of conventional and molecular marker-assisted breeding schemes in order to combine phenotyping and genotyping information. In this study, a selected panel of 16 microsatellite markers were used in different steps of a breeding programme of lettuce ( Lactuca sativa L., 2 n = 18). Molecular markers were first used to genotype 71 putative parental lines and to plan 89 controlled crosses designed to maximise recombination potentials. The resulting 871 progeny plants were then molecularly screened, and their marker allele profiles were compared with the profiles expected based on the parental lines. The average cross-pollination success rate was 68 ± 33%, so 602 F1 hybrids were completely identified. Unexpected genotypes were detected in 5% of cases, consistent with this species' spontaneous out-pollination rate. Finally, in a later step of the breeding programme, 47 different F3 progenies, selected by phenotyping for a number of morphological descriptors, were characterised in terms of their observed homozygosity and within-population genetic uniformity and stability. Ten of these populations had a median homozygosity above 90% and a median genetic similarity above 95% and are, therefore, particularly suitable for pre-commercial trials. In conclusion, this study shows the synergistic effects and advantages of conventional and molecular methods of selection applied in different steps of a breeding programme aimed at developing new varieties of lettuce.

Pollination by nectarivorous birds is predicted to result in different patterns of pollen dispersal and plant mating compared to pollination by insects. We tested the prediction that paternal genetic diversity, outcrossing rate and realized pollen dispersal will be reduced when the primary pollinator group is excluded from bird-pollinated plants. Pollinator exclusion experiments in conjunction with paternity analysis of progeny were applied to Eucalyptus caesia Benth. (Myrtaceae), a predominantly honeyeater-pollinated tree that is visited by native insects and the introduced Apis mellifera (Apidae). Microsatellite genotyping at 14 loci of all adult E. caesia at two populations (n = 580 and 315), followed by paternity analysis of 705 progeny, revealed contrasting results between populations. Honeyeater exclusion did not significantly impact pollen dispersal or plant mating at Mount Caroline. In contrast, at the Chiddarcooping site, the exclusion of honeyeaters led to lower outcrossing rates, a threefold reduction in the average number of sires per fruit, a decrease in intermediate-distance mating and an increase in near-neighbour mating. The results from Chiddarcooping suggest that bird pollination may increase paternal genetic diversity, potentially leading to higher fitness of progeny and favouring the evolution of this strategy. However, further experimentation involving additional trees and study sites is required to test this hypothesis. Alternatively, insects may be effective pollinators in some populations of bird-adapted plants, but ineffective in others., (© 2019 John Wiley & Sons Ltd.)

Studies on foraging partitioning in pollinators can provide critical information to the understanding of food-web niche and pollination functions, thus aiding conservation. Metabarcoding based on PCR amplification and high-throughput sequencing has seen increasing applications in characterizing pollen loads carried by pollinators. However, amplification bias across taxa could lead to unpredictable artefacts in estimation of pollen compositions. We examined the efficacy of a genome-skimming method based on direct shotgun sequencing in quantifying mixed pollen, using mock samples (five and 14 mocks of flower and bee pollen, respectively). The results demonstrated a high level of repeatability and accuracy in identifying pollen from mixtures of varied species ratios. All pollen species were detected in all mocks, and pollen frequencies estimated from the number of sequence reads of each species were significantly correlated with pollen count proportions (linear model, R 2  = 86.7%, p = 2.2e-16). For >97% of the mixed taxa, pollen proportion could be quantified by sequencing to the correct order of magnitude, even for species which constituted only 0.2% of the total pollen. In addition, DNA extracted from pollen grains equivalent to those collected from a single honeybee corbicula was sufficient for genome-skimming. We conclude that genome-skimming is a feasible approach to identifying and quantifying mixed pollen samples. By providing reliable and sensitive taxon identification and relative abundance, this method is expected to improve our understanding in studies that involve plant-pollinator interactions, such as pollen preference in corbiculate bees, pollen diet analyses and identification of landscape pollen resource use from beehives., (© 2019 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F 2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F 2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

Arlequin (Alq) is a gain-of-function mutant whose most relevant feature is that sepals are able to become fruit-like organs due to the ectopic expression of the ALQ-TAGL1 gene. The role of this gene in tomato fruit ripening was previously demonstrated. To discover new functional roles for ALQ-TAGL1, and most particularly its involvement in the fruit set process, a detailed characterization of Alq yield-related traits was performed. Under standard conditions, the Alq mutant showed a much higher fruit set rate than the wild type. A significant percentage of Alq fruits were seedless. The results showed that pollination-independent fruit set in Alq is due to early transition from flower to fruit. Analysis of endogenous hormones in Alq suggests that increased content of cytokinins and decreased level of abscisic acid may account for precocious fruit set. Comparative expression analysis showed relevant changes of several genes involved in cell division, gibberellin metabolism, and the auxin signalling pathway. Since pollination-independent fruit set may be a very useful strategy for maintaining fruit production under adverse conditions, fruit set and yield in Alq plants under moderate salinity were assessed. Interestingly, Alq mutant plants showed a high yield under saline conditions, similar to that of Alq and the wild type under unstressed conditions., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Taxonomic delimitations are challenging because of the convergent and variable nature of phenotypic traits. This is evident in species-rich lineages, where the ancestral and derived states and their gains and losses are difficult to assess. Phylogenetic comparative methods help to evaluate the convergent evolution of a given morphological character, thus enabling the discovery of traits useful for classifications. In this study, we investigate the evolution of selected traits to test for their suitability for generic delimitations in the clade Lepanthes, one of the Neotropical species-richest groups. We evaluated every generic name proposed in the Lepanthes clade producing densely sampled phylogenies with Maximum Parsimony, Maximum Likelihood, and Bayesian approaches. Using Ancestral State Reconstructions, we then assessed 18 phenotypic characters that have been traditionally employed to diagnose genera. We propose the recognition of 14 genera based on solid morphological delimitations. Among the characters assessed, we identified 16 plesiomorphies, 12 homoplastic characters, and seven synapomorphies, the latter of which are reproductive features mostly related to the pollination by pseudocopulation and possibly correlated with rapid diversifications in Lepanthes. Furthermore, the ancestral states of some reproductive characters suggest that these traits are associated with pollination mechanisms alike promoting homoplasy. Our methodological approach enables the discovery of useful traits for generic delimitations in the Lepanthes clade and offers various other testable hypotheses on trait evolution for future research on Pleurothallidinae orchids because the phenotypic variation of some characters evaluated here also occurs in other diverse genera.

Gnetum possesses morphologically bisexual but functionally unisexual reproductive structures that exude sugary pollination drops to attract insects. Previous studies have revealed that the arborescent species ( G. gnemon L.) and the lianoid species ( G. luofuense C.Y.Cheng) possess different pollination syndromes. This study compared the proteome in the pollination drops of these two species using label-free quantitative techniques. The transcriptomes of fertile reproductive units (FRUs) and sterile reproductive units (SRUs) for each species were furthermore compared using Illumina Hiseq sequencing, and integrated proteomic and transcriptomic analyses were subsequently performed. Our results show that the differentially expressed proteins between FRUs and SRUs were involved in carbohydrate metabolism, the biosynthesis of amino acids and ovule defense. In addition, the differentially expressed genes between the FRUs and SRUs (e.g., MADS-box genes) were engaged in reproductive development and the formation of pollination drops. The integrated protein-transcript analyses revealed that FRUs and their exudates were relatively conservative while the SRUs and their exudates were more diverse, probably functioning as pollinator attractants. The evolution of reproductive organs appears to be synchronized with changes in the pollination drop proteome of Gnetum , suggesting that insect-pollinated adaptations are not restricted to angiosperms but also occur in gymnosperms., Competing Interests: The authors declare no conflicts of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Gene flow via pollen movement affects genetic variation in plant populations and is an important consideration in plant domestication. Fontainea picrosperma is a subcanopy rainforest tree that is of commercial interest because it is the source of tigilanol tiglate, a natural product used for the treatment of solid tumors. We identify patterns of pollen-mediated gene flow within natural populations of F. picrosperma and estimate genetic parameters and genetic structure between adult and juvenile groups using microsatellite markers. Our results show pollination events occur over much shorter distances than reported for tropical canopy species. At least 63% of seeds are sired by male trees located within 30 m of the mother. On average, 27% of the local male population contributed to successful reproduction of F. picrosperma with most fathers siring a single seed, however, the contributions to reproduction were uneven. Larger male trees with more flowers had greater reproductive success than those with less flowers (P < 0.05). There were comparatively low levels of genetic variation across the species (H E  = 0.405 for adult trees and 0.379 for juveniles) and we found no loss of genetic diversity between adult and juvenile trees. Short distance pollen flow and low genetic diversity is theoretically a prelude to genetic impoverishment, however F. picrosperma has persisted through multiple significant climatic oscillations. Nevertheless, the remaining low genetic diversity is of concern for domestication programs which require maximal genetic diversity to facilitate efficient selective breeding and genetic improvement of this commercially significant species.

This micromorphological, chemical and ultrastructural study is a continuation of research conducted on the section Lepidorhiza. The Bulbophyllum echinolabium flowers comprised features that characterize a sapromyophilous syndrome, having large, glistening parts that emit an intense scent of rotten meat. The secretory activity was described in the hypochile (nectary in longitudinal groove and in the prickles) and the epichile (putative osmophore). The ultrastructural studies revealed a dense cytoplasm in the epidermis and subepidermal tissue with large nuclei and numerous mitochondria, the profiles of SER and RER, and dictyosomes. Large amounts of heterogeneous residues of secreted material (possibly phenolic) were present on the cuticle surface, similar to the unusual prominent periplasmic space with flocculent secretory material. The chemical analysis (GC/MS) of the scent profile of lips comprised carbohydrates and their derivatives (29.55% of all compounds), amino acids (1.66%), lipids (8.04%) and other organic compounds (60.73%). A great number of identified compounds are Diptera attractants (mainly Milichiidae, Tephritidae, Drosophilidae, Muscidae, Sarcophagidae, Tachinidae). The examination of visual and olfactory features indicates correlation between colour of flowers and the type of olfactory mimicry, where a dark colour labellum emits strong smell of rotten waste.

Covariation among traits can modify the evolutionary trajectory of complex structures. This process is thought to operate at a microevolutionary scale, but its long-term effects remain controversial because trait covariation can itself evolve. Flower morphology, and particularly floral trait (co)variation, has been envisioned as the product of pollinator-mediated selection. Available evidence suggests that major changes in pollinator assemblages may affect the joint expression of floral traits and their phenotypic integration. We expect species within a monophyletic lineage sharing the same pollinator type will show not only similarity in trait means but also similar phenotypic variance-covariance structures. Here, we tested this expectation using eighteen Salvia species pollinated either by bees or by hummingbirds. Our findings indicated a nonsignificant multivariate phylogenetic signal and a decoupling between means and variance-covariance phenotypic matrices of floral traits during the evolution to hummingbird pollination. Mean trait value analyses revealed significant differences between bee- and hummingbird-pollinated Salvia species although fewer differences were detected in the covariance structure between groups. Variance-covariance matrices were much more similar among bee- than hummingbird-pollinated species. This pattern is consistent with the expectation that, unlike hummingbirds, bees physically manipulate the flower, presumably exerting stronger selection pressures favouring morphological convergence among species. Overall, we conclude that the evolution of hummingbird pollination proceeded through different independent transitions. Thus, although the evolution of hummingbird pollination led to a new phenotypic optimum, the process involved the diversification of the covariance structure., (© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.)

For seed-bearing plants, the basis of seed and fruit formation is pollination. The normal progression of pollination is through advances in continuous signal exchange and material transfer, which occur mainly in female reproductive organs; thus, the molecular mechanism of development in female reproductive organs is vital for understanding the principle of pollination. However, molecular biology studies on the development of female cones related to pollination are rare and unclear in gymnosperms, especially in Cupressaceae. In this study, Platycladus orientalis, a monotypic genus within Cupressaceae, was chosen to examine female cone transcriptomes at pre-pollination and pollination stages by Illumina paired-end sequencing technology to de novo sequence six libraries with 3 biological replicates. These libraries were used to construct a P. orientalis transcriptome database containing 71,669 unigenes (4,963 upregulated unigenes and 11,747 downregulated unigenes at the pollination stage) for subsequent analysis. Based on the annotations and expression levels, the functions of differentially expressed unigenes and enriched pathways between the developmental processes of female cones were analysed to detail the preliminary development and pollination mechanism of the female cone. Targeted investigations were specifically performed to determine the elementary mechanism of secretion and functioning of the pollination drop, a vital ovule secretion at the pollination stage. Ultimately, the expression of 15 unigenes selected between two stages were further assessed and confirmed using qRT-PCR, which demonstrated reliable data and significant differences in the expression profiles of key genes. As one of the largest available transcriptomic resources of this species, the database is constructed to prospectively adapt to the physiological and genomic data of woody plants. This work provided the first transcriptome profile of P. orientalis female cones at different developmental stages, and will promote the illumination of the pollination mechanism of P. orientalis, and will serve as the basis for in-depth genomic study in the Cupressaceae family. This initiative will arouse the interest and attention of scholars and pave the way for future studies.

Key Message: Elite wheat pollinators are critical for successful hybrid breeding. We identified Rht-B1 and Ppd-D1 loci affecting multiple pollinator traits and therefore represent major targets for improving hybrid seed production. Hybrid breeding has a great potential to significantly boost wheat yields. Ideal male pollinators would be taller in stature, contain many spikelets well-spaced along the spike and exhibit high extrusion of large anthers. Most importantly, flowering time would match with that of the female parent. Available genetic resources for developing an elite wheat pollinator are limited, and the genetic basis for many of these traits is largely unknown. Here, we report on the genetic analysis of pollinator traits using biparental mapping populations. We identified two anther extrusion QTLs of medium effect, one on chromosome 1BL and the other on 4BS coinciding with the semi-dwarfing Rht-B1 locus. The effect of Rht-B1 alleles on anther extrusion is genotype dependent, while tall plant Rht-B1a allele is consistently associated with large anthers. Multiple QTLs were identified at the Ppd-D1 locus for anther length, spikelet number and spike length, with the photoperiod-sensitive Ppd-D1b allele associated with favourable pollinator traits in the populations studied. We also demonstrated that homeoloci, Rht-D1 and Ppd-B1, influence anther length among other traits. These results suggest that combinations of Rht-B1 and Ppd-D1 alleles control multiple pollinator traits and should be major targets of hybrid wheat breeding programs.

The pollination effectiveness of a flower visitor has traditionally been measured as the product of a quantity component that depends on the frequency of interaction and a quality component that measures the per-visit effects on plant reproduction. We propose that this could be complemented with a genetic component informing about each pollinator's contribution to the genetic diversity and composition of the plant progeny. We measured the quantity and quality components of effectiveness of most pollinator functional groups of the generalist herb Erysimum mediohispanicum. We used 10 microsatellite markers to calculate the genetic component as the diversity of sires among siblings and included it into the calculation of the pollination effectiveness. Functional groups varied in the quantity and quality components, which were shown to be decoupled. Functional groups also differed in the genetic component. This component changed the estimates of pollination effectiveness, increasing the differences between some functional groups and modifying the pollination effectiveness landscape. We demonstrate that including the genetic component in the calculation of the pollination effectiveness may allow a more complete quantification of the contribution of each pollinator to the reproductive success of a plant, providing information on its mating patterns and long-term fitness., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The natural regeneration of tree species depends on seed and pollen dispersal. To assess whether limited dispersal could be critical for the sustainability of selective logging practices, we performed parentage analyses in two Central African legume canopy species displaying contrasted floral and fruit traits: Distemonanthus benthamianus and Erythrophleum suaveolens. We also developed new tools linking forward dispersal kernels with backward migration rates to better characterize long-distance dispersal. Much longer pollen dispersal in D. benthamianus (mean distance d p  = 700 m, m p  = 52% immigration rate in 6 km 2 plot, s = 7% selfing rate) than in E. suaveolens (d p  = 294 m, m p  = 22% in 2 km 2 plot, s = 20%) might reflect different insect pollinators. At a local scale, secondary seed dispersal by vertebrates led to larger seed dispersal distances in the barochorous E. suaveolens (d s  = 175 m) than in the wind-dispersed D. benthamianus (d s  = 71 m). Yet, seed dispersal appeared much more fat-tailed in the latter species (15%-25% seeds dispersing >500 m), putatively due to storm winds (papery pods). The reproductive success was correlated to trunk diameter in E. suaveolens and crown dominance in D. benthamianus. Contrary to D. benthamianus, E. suaveolens underwent significant assortative mating, increasing further the already high inbreeding of its juveniles due to selfing, which seems offset by strong inbreeding depression. To achieve sustainable exploitation, seed and pollen dispersal distances did not appear limiting, but the natural regeneration of E. suaveolens might become insufficient if all trees above the minimum legal cutting diameter were exploited. This highlights the importance of assessing the diameter structure of reproductive trees for logged species., (© 2019 John Wiley & Sons Ltd.)

Species interactions are known to be key in driving patterns of biodiversity across the globe. Plant-plant interactions through heterospecific pollen (HP) transfer by their shared pollinators is common and has consequences for plant reproductive success and floral evolution, and thus has the potential to influence global patterns of biodiversity and plant community assembly. The literature on HP transfer is growing and it is therefore timely to review patterns and causes of among-species variation in HP receipt at a global scale, thus uncovering its potential contribution to global patterns of biodiversity. Here we analyzed published data on 245 species distributed across five continents to evaluate latitudinal and altitudinal patterns of HP receipt. We further analyzed the role of floral symmetry and evolutionary history in mediating patterns of HP receipt. Latitude and elevation affected the likelihood and intensity of HP receipt indicating that HP transfer increases in species-rich communities and in areas with high abundance of vertebrate pollinators. Floral symmetry and evolutionary history determined HP load size across plant communities worldwide. Overall, our results suggest that HP receipt may have the potential to contribute to global geographic patterns of plant diversity by imposing strong selective pressures in species-rich areas across the globe.

Ribes diacanthum Pall. (Grossulariaceae), a species with dioecious, unixsexual flowers, has great economic and medicinal value and is widespread in northeastern China. After the initiation of intact floral organs, male flowers develop an abnormal stigma, and female flowers develop fading stamens incapable of pollination. To explore the genes governing dioecious unisexual floral development in R. diacanthum, we used high-throughput sequencing to obtain transcriptome data for male and female inflorescences and analyzed expression patterns of candidate genes at various developmental stages of male and female flowers. The combined transcriptomic data were successfully assembled into 72,791 transcripts (N50 = 1467) and 48,600 unigenes (N50 = 1378); 62% of the unigenes were annotated by NR, Swissprot, KEGG, GO and COG database based on orthology. Analysis of the differentially expressed genes (DEGs) showed that 2785 annotated genes were differentially expressed, and significantly more genes were male-biased than were female-biased in expression in the inflorescences. Both male and female flowers were found to be complete hermaphroditic flowers during early floral development; sex determination was a late event. Several MADS-box genes such as comp53946&#95;c0 (putative AGL11) might be directly correlated with the establishment of sexual dimorphism. The sex-specific transcripts and genes identified may regulate coordinated events during floral development and be involved in the molecular regulation of dioecious, unisexual floral development in R. diacanthum. The transcriptome from the male and the female inflorescences will provide a valuable reference for further functional research on the development of dioecious, unisexual flowers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Multiple-allele-inherited male sterility (MAMS) is important in Chinese cabbage (Brassica rapa L. ssp. pekinensis) breeding, but the molecular mechanisms leading to male sterility are poorly understood. In this study, we cloned a novel gene, BrSKS13, that is differentially expressed in fertile and sterile flower buds of Chinese cabbage. BrSKS13 is most similar to Arabidopsis thaliana AT3G13400 (SKS13) and encodes a predicted 61.87 kDa protein with three cupredoxin superfamily conserved domains in the multicopper oxidase family. Semi-quantitative reverse-transcription PCR (sqRT-PCR) showed that expression of BrSKS13 is higher in fertile buds than in sterile buds. Quantitative RT-PCR (qRT-PCR) and in situ hybridization showed that BrSKS13 is highly expressed in fertile anthers, peaking at pollen-maturation stage VI, but is weakly expressed in other tissues and floral organs. Expression patterns of BrSKS13 promoter::GUS reporter fusions in Arabidopsis showed that the BrSKS13 promoter drives expression of the GUS gene only in anthers. The relative expression of Brsks13 in fertile buds was higher than in sterile buds for all other MAMS lines of Chinese cabbage examined. These results suggest that BrSKS13 affects pollen development. In situ hybridization analysis of flower stigmas at different times after pollination showed that BrSKS13 expression was first observed in stigmas and immature seeds at 1 h after pollination, and the signal intensity in seeds increased with increasing maturity. Compared to Col-0, A. thaliana sks13 mutant plants have shorter and fewer siliques, shriveled pollen grains, pollen tube abnormalities, and reduced seed number. The phenotype of sks13 mutant was recovered by over-expressing BrSKS13. Our results suggest that BrSKS13 affects pollen development and the pollination/fertilization process, and will enable further study of the genetic mechanisms underlying MAMS in Chinese cabbage., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sheepgrass ( Leymus chinensis (Trin.) Tzvel.) is an economically and ecologically important forage in the grass family. Self-incompatibility (SI) limits its seed production due to the low seed-setting rate after self-pollination. However, investigations into the molecular mechanisms of sheepgrass SI are lacking. Therefore, microscopic observation of pollen germination and pollen tube growth, as well as transcriptomic analyses of pistils after self- and cross-pollination, were performed. The results indicated that pollen tube growth was rapidly inhibited from 10 to 30 min after self-pollination and subsequently stopped but preceded normally after cross-pollination. Time course comparative transcriptomics revealed different transcriptome dynamics between self- and cross-pollination. A pool of SI-related signaling genes and pathways was generated, including genes related to calcium (Ca 2+ ) signaling, protein phosphorylation, plant hormone, reactive oxygen species (ROS), nitric oxide (NO), cytoskeleton, and programmed cell death (PCD). A putative SI response molecular model in sheepgrass was presented. The model shows that SI may trigger a comprehensive calcium- and phytohormone-dominated signaling cascade and activate PCD, which may explain the rapid inhibition of self-pollen tube growth as observed by cytological analyses. These results provided new insight into the molecular mechanisms of sheepgrass (grass family) SI.

Key Message: We developed a novel Agrobacterium-mediated anther transformation for Wucai in planta, and in this procedure, the male germ line was the predominant target. Wucai (Brassica campestris L.), a variant of non-heading Chinese cabbage, is widely cultured in China and only improved by classic breeding methods. Here, a novel and efficient in planta Agrobacterium-mediated anther transformation method is developed based on the optimization of several factors that affect anther transformation. After optimization, transformation with the manual pollination application led to increased transient GUS expression in anthers (reaching 91.59%) and the transformation efficacies in planta (0.59-1.56% for four commercial cultivars). The stable integration and inheritance of the transgenes were further examined by molecular and genetic analyses. Three T 2 transgenic lines presented a segregation ratio of 3:1, which was consistent with the Mendelian feature of a single dominant gene. In addition, the GUS histochemical assay and genetic crossing analysis revealed that the male germ line was the predominant target in this transformation. This optimized transformation system could provide a useful tool for both the improvement of cultivar qualities and investigation of functional genes in Wucai.

Social bees are important insect pollinators of wildflowers and agricultural crops, making their reported declines a global concern. A major factor implicated in these declines is the widespread use of neonicotinoid pesticides. Indeed, recent research has demonstrated that exposure to low doses of these neurotoxic pesticides impairs bee behaviours important for colony function and survival. However, our understanding of the molecular-genetic pathways that lead to such effects is limited, as is our knowledge of how effects may differ between colony members. To understand what genes and pathways are affected by exposure of bumblebee workers and queens to neonicotinoid pesticides, we implemented a transcriptome-wide gene expression study. We chronically exposed Bombus terrestriscolonies to either clothianidin or imidacloprid at field-realistic concentrations while controlling for factors including colony social environment and worker age. We reveal that genes involved in important biological processes including mitochondrial function are differentially expressed in response to neonicotinoid exposure. Additionally, clothianidin exposure had stronger effects on gene expression amplitude and alternative splicing than imidacloprid. Finally, exposure affected workers more strongly than queens. Our work demonstrates how RNA-Seq transcriptome profiling can provide detailed novel insight on the mechanisms mediating pesticide toxicity to a key insect pollinator., (© 2019 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

A PACLOBUTRAZOL-RESISTANCE ( PRE ) gene family, consisting of six genes in Arabidopsis thaliana , encodes a group of helix-loop-helix proteins that act in the growth-promoting transcriptional network. To delineate the specific role of each of the PRE genes in organ growth, we took a reverse genetic approach by constructing high order pre loss-of-function mutants of Arabidopsis thaliana . In addition to dwarf vegetative growth, some double or high order pre mutants exhibited defective floral development, resulting in reduced fertility. While pre2pre5 is normally fertile, both pre2pre6 and pre5pre6 showed reduced fertility. Further, the reduced fertility was exacerbated in the pre2pre5pre6 mutant, indicative of the redundant and critical roles of these PREs. Self-pollination assay and scanning electron microscopy analysis showed that the sterility of pre2pre5pre6 was mainly ascribed to the reduced cell elongation of anther filament, limiting access of pollens to stigma. We found that the expression of a subset of flower-development related genes including ARGOS , IAA19 , ACS8, and MYB24 was downregulated in the pre2pre5pre6 flowers. Given these results, we propose that PREs, with unequal functional redundancy, take part in the coordinated growth of floral organs, contributing to successful autogamous reproduction in Arabidopsis thaliana.

Ovules contain the female gametophytes which are fertilized during pollination to initiate seed development. Thus, the number of ovules that are produced during flower development is an important determinant of seed crop yield and plant fitness. Mutants with pleiotropic effects on development often alter the number of ovules, but specific regulators of ovule number have been difficult to identify in traditional mutant screens. We used natural variation in Arabidopsis accessions to identify new genes involved in the regulation of ovule number. The ovule numbers per flower of 189 Arabidopsis accessions were determined and found to have broad phenotypic variation that ranged from 39 ovules to 84 ovules per pistil. Genome-Wide Association tests revealed several genomic regions that are associated with ovule number. T-DNA insertion lines in candidate genes from the most significantly associated loci were screened for ovule number phenotypes. The NEW ENHANCER of ROOT DWARFISM (NERD1) gene was found to have pleiotropic effects on plant fertility that include regulation of ovule number and both male and female gametophyte development. Overexpression of NERD1 increased ovule number per fruit in a background-dependent manner and more than doubled the total number of flowers produced in all backgrounds tested, indicating that manipulation of NERD1 levels can be used to increase plant productivity., Competing Interests: The authors have declared that no competing interests exist.

The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides. To address this question, we sequenced the genome of the red mason bee, Osmia bicornis, the most abundant and economically important solitary bee species in Central Europe. We show that O. bicornis lacks the CYP9Q subfamily of P450s but, despite this, exhibits low acute toxicity to the N-cyanoamidine neonicotinoid thiacloprid. Functional studies revealed that variation in the sensitivity of O. bicornis to N-cyanoamidine and N-nitroguanidine neonicotinoids does not reside in differences in their affinity for the nicotinic acetylcholine receptor or speed of cuticular penetration. Rather, a P450 within the CYP9BU subfamily, with recent shared ancestry to the Apidae CYP9Q subfamily, metabolises thiacloprid in vitro and confers tolerance in vivo. Our data reveal conserved detoxification pathways in model solitary and eusocial bees despite key differences in the evolution of specific pesticide-metabolising enzymes in the two species groups. The discovery that P450 enzymes of solitary bees can act as metabolic defence systems against certain pesticides can be leveraged to avoid negative pesticide impacts on these important pollinators., Competing Interests: This study received funding from Bayer AG, a manufacturer of neonicotinoid insecticides. Three authors of this study (M. Zaworra, M. Kohler, R. Nauen and B. Buer) are employees of Bayer AG.

Background and Aims: Selection exerted by pollinators on flowers is predicted to occur along two distinct axes. While pollinator attraction to flowers is governed by pollinator preferences, pollen transfer efficiency is mediated by the mechanical fit of pollinators to flower morphology. Although pollinator attraction in sexually deceptive orchids is typically underpinned by floral odour, morphological traits are expected to play a vital role in mechanical fit during floral contact with pollinators., Methods: Here we utilize a comprehensive and novel procedure to test for pollinator-mediated selection through mechanical fit with the flower labellum in the orchid Chiloglottis trapeziformis. This approach combines detailed pollinator observations related to plant reproductive fitness with complementary experimental manipulation and phenotypic selection analysis., Key Results: Experiments with virgin flowers revealed that pollen removal occurs only during vigorous pseudocopulation. This behaviour involves male wasps that grasp the insectiform callus structure on the labellum while probing the labellum tip in a forward orientation. Both orientation and duration of pseudocopulation were significant predictors of pollen removal, confirming a direct relationship between pollinator behaviour and plant fitness. Controlled floral manipulation that either shortened or elongated the distance between the callus and the labellum tip detected no change in pollinator attraction. The duration of pseudocopulation, however, was significantly reduced on flowers with shortened or elongated callus-tip distances, consistent with stabilizing selection. Phenotypic selection analysis confirmed this prediction in natural populations by uncovering evidence for stabilizing selection on the distance between the callus and the labellum tip., Conclusions: Our experimental manipulations and selection analysis in natural populations thus demonstrate stabilizing selection on the distance from the callus to the labellum tip, and illustrate the utility of employing multiple approaches to confirm selection exerted by pollinators on floral form.

Background and Aims: Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions., Methods: The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated., Key Results: Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented., Conclusions: Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades.

Maize often exhibits asynchronous pollination under abiotic and biotic stress conditions; however, the molecular basis of this developmental deficiency has not been elucidated. Tassel development is a key process affecting the anthesis-silking interval (ASI) in maize. In this study, we showed that pollen shedding was delayed and ASI was significantly increased in B73 and Chang7-2 inbred lines under water deficit conditions, which resulted in longer barren tip length and decreased yields under both controlled and field conditions. Comparative transcriptome analysis performed on immature tassels derived from plants grown under well-watered and water deficit conditions identified 1931 and 1713 differentially expressed genes (DEGs) in B73 and Chang7-2, respectively. Further, 28 differentially co-expressed transcription factors were identified across both lines. Collectively, we demonstrated that the molecular regulation of tassel development is associated with water deficit stress at early vegetative stage in maize. This finding extends our understanding of the molecular basis of maize tassel development during abiotic stress., (Copyright © 2018. Published by Elsevier B.V.)

Populations of scarlet Indian paintbrush (Castilleja coccinea) in the Midwestern United States exhibit a bract color polymorphism, with each population having predominantly yellow or scarlet bracts. We investigated a possible mechanism for this maintenance of bract color polymorphism in C. coccinea by conducting hand-pollination experiments in two nearby populations, one predominantly yellow and one predominantly scarlet. The hand-pollination treatments were either self-pollination or cross pollination using pollen from within and between populations. Both color morphs were used as pollen donors for the within and between crosses. We found that both color morphs of C. coccinea were self-compatible. When the scarlet morph was the maternal plant it had higher seed set. When pollinators were excluded, the yellow morph outperformed the scarlet morph in fruit set and seed set. The apparent trade-offs between a higher reproductive output in the scarlet morph and a reproductive assurance advantage in the yellow morph may explain the maintenance of the polymorphism in C. coccinea. While many previous studies have provided evidence for pollinator preference playing a role in floral color polymorphism, the results of the current study indicate that reproductive assurance, which would be important for fluctuations in pollinator abundance or colonizing new areas, may act as a selective agent to maintain such polymorphisms., Competing Interests: The authors have declared that no competing interests exist.

The transition from outcrossing to selfing through the breakdown of distyly to homostyly has occurred repeatedly among families of flowering plants. Homostyles can originate by major gene changes at the S-locus linkage group, or by unlinked polygenic modifiers. Here, we investigate the inheritance of distyly and homostyly in Primula oreodoxa, a subalpine herb endemic to Sichuan, China. Controlled self- and cross-pollinations confirmed that P. oreodoxa unlike most heterostylous species is fully self-compatible. Segregation patterns indicated that the inheritance of distyly is governed by a single Mendelian locus with the short-styled morph carrying at least one dominant S-allele (S-) and long-styled plants homozygous recessive (ss). Crossing data were consistent with a model in which homostyly results from genetic changes at the distylous linkage group, with the homostylous allele (S h ) dominant to the long-styled allele (s), but recessive to the short-styled allele (S). Progeny tests of open-pollinated seed families revealed high rates of intermorph mating in the L-morph but considerable selfing and possibly intramorph mating in the S-morph and in homostyles. S-morph plants homozygous at the S-locus (SS) occurred in several populations but may experience viability selection. The crossing data from distylous and homostylous plants are consistent with either recombination at the S-locus governing distyly, or mutation at gene(s) controlling sex-organ height; both models predict the same patterns of segregation. Recent studies on the molecular genetics of distyly in Primula demonstrating the hemizygous nature of genes at the S-locus make it more likely that homostyles have resulted from mutation rather than recombination.

Sorghum is the fifth most important cereal grain crop after corn, wheat, rice, and pearl millet in the world. Conventional sorghum breeding relies on multiple generations of self-pollination to achieve the adequate levels of homozygosity for hybrid evaluation, which adds several years and great cost to the breeding process. As in maize, doubled haploid (DH) is the key technology to speed up the breeding process in sorghum. Through 3 years of efforts, two haploid inducer lines, SMHI01 and SMHI02, were discovered by screening 4000 germplasms worldwide. These two inducers have been evaluated in different growth environments and have shown to generate haploids at frequency of 1-2%. The putative haploids produced with these two inducers were evaluated and ploidy was confirmed cytologically and biochemically. The discovery of these inducer lines is the first step toward a revolutionary change in sorghum breeding.

Stigmatic papillae develop at the apex of the gynoecium and play an important role as a site of pollination. The papillae in Brassicaceae are of the dry and unicellular type, and more than 15,000 genes are expressed in the papillae; however, the molecular and physiological mechanisms of their development remain unknown. We found that the papillae in Arabidopsis thaliana change their length in response to altered ambient humidity: papillae of flowers incubated under high humidity elongated more than those under normal humidity conditions. Genetic analysis and transcriptome data suggest that an abscisic acid-mediated abiotic stress response mechanism regulates papilla length. Our data suggest a flexible regulation of papilla elongation at the post-anthesis stage, in response to abiotic stress, as an adaptation to environmental conditions.

Artificial pollination of black walnut (Juglans nigra L.) is not practical and timber breeders have historically utilized only open-pollinated half-sib families. An alternate approach called "breeding without breeding," consists of genotyping open-pollinated progeny using DNA markers to identify paternal parents and then constructing full-sib families. In 2014, we used 12 SSR markers to genotype 884 open-pollinated half-sib progeny harvested from two clonal orchards containing 206 trees, comprised of 52 elite timber selections. Seed was harvested in 2011 from each of two ramets of 23 clones, one upwind and one downwind, based on prevailing wind direction from the west-southwest. One orchard was isolated from wild black walnut and composed of forward selections while the other orchard was adjacent to a natural forest containing mature black walnut composed of backward selections. Isolation significantly increased within-orchard pollination (85%) of the progeny from the isolated orchard compared to 42% from the non-isolated orchard. Neither prevailing wind direction nor seed tree position in the orchard affected paternity patterns or wild pollen contamination. Genetic diversity indices revealed that progeny from both orchards were in Hardy-Weinberg equilibrium with very little inbreeding and no selfing. A significant level of inbreeding was present among the forward selected parents, but not the first generation (backward selected) parents. Some orchard clones failed to sire any progeny while other clones pollinated upwards of 20% of progeny., Competing Interests: The authors have declared that no competing interests exist.

Background: Observations of precocious (early bearing) genotypes of walnut (Juglans regia L.) under natural conditions encouraged us to study the origin and genetic control of these fascinating traits., Results: In this study, the self-fertility, progeny performance, and simple sequence repeat (SSR) locus variation of iron walnut (Juglans sigillata Dode), an ecotype of J. regia, were investigated. The average self-pollinated fruit set rate of J. sigillata cv. 'Dapao' (DP) was 7.0% annually from 1979 to 1982. The average germination rate of self-pollinated seeds was 45.2% during the 4-year period. Most progeny had inbreeding depression. Nine representative self-pollinated progeny (SP 1 -SP 9 ), with special or typical traits of DP, were selected. SP 1 -SP 4 were precocious because they initiated flowers as early as 2 years after germination, compared to the 7-10-yr period that is typical of DP. SP 9 had not flowered since 1980. Twelve SSR markers were used to analyze the SP and DP. The genome of SP had a tendency toward high levels of homozygosity. The high levels of homozygosity reported in 18 additional precocious walnut genotypes complemented the results of this study., Conclusions: These results provide evidence of precocious phenotypes and genomes with high levels of homozygosity that might be generated from self-pollinating walnut. This suggests that self-pollination might facilitate the generation of unique homozygous parents for subsequent use in walnut-breeding programs. The results also indicate that more attention should be focused on adequate management of precocious walnut to avoid early depression in the production of nuts.

The process of pollen germination is crucial for flowering plant reproduction, but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood. In this study, we report that a formin family protein, AtFH5, is localized to the vesicles and rotates ahead of Lifeact-mEGFP-labeled actin filaments during pollen germination. The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-labeled vesicles and the polymerization of actin filaments: vesicle-localized AtFH5 promotes actin assembly, and the polymerization and elongation of actin filaments, in turn, is essential for the mobility of AtFH5-labeled vesicles in pollen grains. Taken together, our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

In the Alps phylogeographic studies indicate for small insect-pollinated herbs that climatic fluctuations caused significant population migrations and fragmentations into glacial refugia at the periphery of the Alps. Here we investigate whether this holds also for wind-pollinated and -dispersed species. We therefore analysed the phylogeographic pattern (nuclear and chloroplast dataset) of a clade of the four species of the Helictotrichon parlatorei species group (Poaceae) endemic to the Alps. In contrast to earlier findings for small insect-pollinated herbs no clear barriers to gene flow could be detected in this species group. Instead a few haplotypes are widespread across the entire Alpine region. While the complete absence of a phylogeographic structure in the plastid dataset hints towards very efficient long distance seed dispersal, the moderate phylogeographic structure in the nuclear dataset indicates at least some spatial restriction to pollen dispersal. Rare haplotypes cluster solely in the Western and Southern central Alps and thereby suggest this to be the area of origin for the H. parlatorei species group from where expansion occurred following the presence of calcareous bedrock into the Eastern Alps. We thus conclude that the inclusion of taxa with complementary life-history traits is vital in understanding the glacial history of the Alpine flora., Competing Interests: The authors have declared that no competing interests exist.

Pollen tube growth requires a high amount of metabolic energy and precise targeting toward the ovules. Sugars, especially glucose, can serve as nutrients and as signaling molecules. Unexpectedly, in vitro assays revealed an inhibitory effect of glucose on pollen tube elongation, contradicting the hypothesis that monosaccharide uptake is a source of nutrition for growing pollen tubes. Measurements with Förster resonance energy transfer-based nanosensors revealed that glucose is taken up into pollen tubes and that the intracellular concentration is in the low micromolar range. Pollen tubes of stp4-6-8-9-10-11 sextuple knockout plants generated by crossings and CRISPR/Cas9 showed only a weak response to glucose, indicating that glucose uptake into pollen tubes is mediated mainly by these six monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family. Analyses of HEXOKINASE1 ( HXK1 ) showed a strong expression of this gene in pollen. Together with the glucose insensitivity and altered semi-in vivo growth rate of pollen tubes from hxk1 knockout lines, this strongly suggests that glucose is an important signaling molecule for pollen tubes, is taken up by STPs, and detected by HXK1. Equimolar amounts of fructose abolish the inhibitory effect of glucose indicating that only an excess of glucose is interpreted as a signal. This provides a possible model for the discrimination of signaling and nutritional sugars., (© 2018 American Society of Plant Biologists. All rights reserved.)

Re-domestication of potato into an inbred line-based diploid crop propagated by seed represents a promising alternative to traditional clonal propagation of tetraploid potato, but self-incompatibility has hindered the development of inbred lines. To address this problem, we created self-compatible diploid potatoes by knocking out the self-incompatibility gene S-RNase using the CRISPR-Cas9 system. This strategy opens new avenues for diploid potato breeding and will also be useful for studying other self-incompatible crops.

Simplification by reduction has occurred many times independently in the floral evolution of angiosperms. These reductions have often been attributed to changes in reproductive biology. In the angiosperm plant family Annonaceae, most species have flowers with six petals, and many stamens and carpels. In the genus Monanthotaxis several deviations from this pattern have been observed, including flowers that contain three petals and three stamens only. New DNA sequences were generated for 42 specimens of Monanthotaxis. Five chloroplast markers and two nuclear markers for 72 out of 94 species of Monanthotaxis were used to reconstruct a phylogeny of the genus, which revealed several well-supported, morphologically distinct clades. The evolution of four quantitative and two qualitative floral characters was mapped onto this phylogeny, demonstrating a reduction in flower size and number of flower parts in Monanthotaxis. A large variation in stamen forms and numbers, strong correlations between petal size, stamen and carpel number, combined with a non-gradual mode of evolution and the sympatric co-occurrence of Monanthotaxis species from different clades suggest that the high diversity in the African rainforest of this genus is caused by switches in pollination systems.

Deceptive pollination is key to the species richness of Orchidaceae. However, the genetic basis of species diversification is still under study. Section Trigonopedia is a monophyletic clade of genus Cypripedium distributed in the southwest of China. The species of this section are pollinated by different flies. Pollinator differentiation makes section Trigonopedia an ideal group for studying the genetic basis underlying species diversification. Here, we sequenced the transcriptomes of eight species of the genus Cypripedium, including six co-flowering species of section Trigonopedia and two species outside this section as an outgroup. We reconstructed the phylogeny of the section with the combined 1572 single-copy genes extracted from the eight species and produced a highly resolved tree of the section. Furthermore, we combined substitution rate estimation and differential expression analysis to identify candidate genes, including genes related to floral scent synthesis and environmental adaptation, involved in species differentiation. Field investigations showed that these species have adapted to different habitats. We propose that the species diversification in this section is initiated by floral scent differentiation, followed by habitat differentiation, finally leading to speciation. This study sheds novel light on the diversification of closely related orchid species in the Qinghai-Tibetan region.

Intumescence is a nonpathogenic physiological disorder characterized by leaf blistering. This disorder can affect growth and development in glasshouses and growth chambers and may be confused with pathogenic diseases. We used quantitative trait loci (QTL) analysis to examine the genetic basis of variation in intumescence severity in Eucalyptus globulus, and test for colocation with previously detected QTLs for pathogen susceptibility. QTL analysis used the phenotype means of open-pollinated (OP) families of an outcrossed F 2 mapping family (OP F 3 ; n = 300) of E. globulus and the linkage map constructed in the F 2 . We validate this phenotyping approach for QTL analysis by assessing a trait previously used for QTL discovery in the F 2 and showing the same major QTL was detected with the OP F 3 . For intumescence severity, five putative QTLs were detected across four linkage groups. Four of these did not colocate with previously reported QTLs for fungal pathogen susceptibility in Eucalyptus, suggesting the mechanisms underlying susceptibility to intumescence and to the two fungal pathogens are largely independent. This study demonstrates there is a genetic basis for variation in intumescence severity, reports the first QTL for intumescence severity in plants, and provides a robust framework for investigating the potential mechanisms involved.

Peach (Prunus persica L.) generally exhibits self-pollination, however, they can also be pollinated by other varieties of pollen. Here we found two varieties that are different from other peaches: 'Daifei' and 'Liuyefeitao'. 'Daifei' produces less pollen, which needs artificial pollination, honeybee pollination, and the fruit setting depends on other varieties of peach pollen. 'Liuyefeitao' exhibits strictly self-pollination, hence pollen from other species is rejected. To explore the mechanism of this phenomenon, we performed a high-throughput sequencing of the stigma (including style) of 'Daifei' and 'Liuyefeitao' to explain the rejection mechanism of other varieties of pollen of 'Liuyefeitao' peach. In our study, we found one S gene, and lots of non-S-locus factors such as: F-box proteins, Ub/26S, MAPKs, RLK, and transcription factor were differential expressed between 'Daifei' and 'Liuyefeitao'. We supposed that the strictly self-compatible of 'Liuyefeitao' may result from the synthesis of these factors., Competing Interests: The authors have declared that no competing interests exist.

Identifying drivers of dispersal limitation and genetic differentiation is a key goal in biogeography. We examine patterns of population connectivity and genetic diversity using restriction site-associated DNA sequencing (RADseq) in two bumble bee species, Bombus vosnesenskii and Bombus bifarius, across latitude and altitude in mountain ranges from California, Oregon and Washington, U.S.A. Bombus vosnesenskii, which occurs across a broader elevational range at most latitudes, exhibits little population structure while B. bifarius, which occupies a relatively narrow higher elevation niche across most latitudes, exhibits much stronger population differentiation, although gene flow in both species is best explained by isolation with environmental niche resistance. A relationship between elevational habitat breadth and genetic diversity is also apparent, with B. vosnesenskii exhibiting relatively consistent levels of genetic diversity across its range, while B. bifarius has reduced genetic diversity at low latitudes, where it is restricted to high-elevation habitat. The results of this study highlight the importance of the intersect between elevational range and habitat suitability in influencing population connectivity and suggest that future climate warming will have a fragmenting effect even on populations that are presently well connected, as they track their thermal niches upward in montane systems., (© 2018 John Wiley & Sons Ltd.)

Flowers represent a key innovation during plant evolution. Driven by reproductive optimization, evolution of flower morphology has been central in boosting species diversification. In most cases, this has happened through specialized interactions with animal pollinators and subsequent reduction of gene flow between specialized morphs. While radiation has led to an enormous variability in flower forms and sizes, recurrent evolutionary patterns can be observed. Here, we discuss the targets of selection involved in major trends of pollinator-driven flower evolution. We review recent findings on their adaptive values, developmental grounds and genetic bases, in an attempt to better understand the repeated nature of pollinator-driven flower evolution. This analysis highlights how structural innovation can provide flexibility in phenotypic evolution, adaptation and speciation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

The evolutionary concurrence of intraspecies self-incompatibility (SI) and explosive angiosperm radiation in the Cretaceous have led to the hypothesis that SI was one of the predominant drivers of rapid speciation in angiosperms. Interspecies unilateral incompatibility (UI) usually occurs when pollen from a self-compatible (SC) species is rejected by the pistils of a SI species, while the reciprocal pollination is compatible (UC). Although this SI × SC type UI is most prevalent and viewed as a prezygotic isolation barrier to promote incipient speciation of angiosperms, comparative evidence to support such a role is lacking. We show that SI × SI type UI in SI species pairs is also common in the well-characterized accessions representing the four major lineages of the Arabidopsis genus and is developmentally regulated. This allowed us to reveal a strong correlation between UI strength and species divergence in these representative accessions. In addition, analyses of a SC accession and the pseudo-self-compatible (psc) spontaneous mutant of Arabidopsis lyrata indicate that UI shares, at least, common pollen rejection pathway with SI. Furthermore, genetic and genomic analyses of SI × SI type UI in A. lyrata × A. arenosa species pair showed that two major-effect quantitative trait loci are the stigma and pollen-side determinant of UI, respectively, which could be involved in heterospecies pollen discrimination. By revealing a close link between UI and SI pathway, particularly between UI and species divergence in these representative accessions, our findings establish a connection between SI and speciation. Thus, the pre-existence of SI system would have facilitated the evolution of UI and accordingly promote speciation., (© 2018 John Wiley & Sons Ltd.)

Background and Aims: Capsella is a model genus for studying the transition from outcrossing to selfing, with or without change in ploidy levels. The genomic consequences and changes in reproductive traits (selfing syndrome) associated with these shifts have been studied in depth. However, potential ecological divergence among species of the genus has not been determined. Among ecological traits, competitive ability could be relevant for selfing evolution, as selfing has been shown to be statistically associated with reduced competitiveness in a recent meta-analysis., Methods: We assessed the effect of competition on three Capsella species differing in their mating system and ploidy level. We used an experimental design where fitness related traits were measured in focal individuals with and without competitors., Key Results: The diploid selfer (C. rubella) was most sensitive to competition, whereas the tetraploid selfer (C. bursa-pastoris) performed the best, with the diploid outcrosser (C. grandiflora) being intermediate., Conclusions: These results add to the detailed characterization of Capsella species and highlight the possible roles of ecological context and ploidy in the evolutionary trajectories of selfing species.

Key Message: Superoxide dismutase genes were expressed differentially along with developmental stages of fertilized ovules in Xanthoceras sorbifolium, and the XsMSD gene silencing resulted in the arrest of fertilized ovule development. A very small percentage of mature fruits (ca. 5%) are produced relative to the number of bisexual flowers in Xanthoceras sorbifolium because seeds and fruits are aborted at early stages of development after pollination. Reactive oxygen species (ROS) in plants are implicated in an extensive range of biological processes, such as programmed cell death and senescence. Superoxide dismutase (SOD) activity might be required to regulate ROS homeostasis in the fertilized ovules of X. sorbifolium. The present study identified five SOD genes and one SOD copper chaperone gene in the tree. Their transcripts were differentially expressed along different stages of fertilized ovule development. These genes showed maximum expression in the ovules at 3 days after pollination (DAP), a time point in which free nuclear endosperm and nucleus tissues rapidly develop. The XsCSD1, XsFSD1 and XsMSD contained seven, eight, and five introns, respectively. Analysis of the 5'-flanking region of XsFSD1 and XsMSD revealed many cis-acting regulatory elements. Evaluation of XsMSD gene function based on virus-induced gene silencing (VIGS) indicated that the gene was closely related to early development of the fertilized ovules and fruits. This study suggested that SOD genes might be closely associated with the fate of ovule development (aborted or viable) after fertilization in X. sorbifolium.

Background: Self-incompatibility (SI) is a major barrier that obstructs the breeding process in most horticultural plants including tea plants (Camellia sinensis). The aim of this study was to elucidate the molecular mechanism of SI in tea plants through a high throughput transcriptome analysis., Results: In this study, the transcriptomes of self- and cross-pollinated pistils of two tea cultivars 'Fudingdabai' and 'Yulv' were compared to elucidate the SI mechanism of tea plants. In addition, the ion components and pollen tube growth in self- and cross-pollinated pistils were investigated. Our results revealed that both cultivars had similar pollen activities and cross-pollination could promote the pollen tube growth. In tea pistils, the highest ion content was potassium (K + ), followed by calcium (Ca 2+ ), magnesium (Mg 2+ ) and phosphorus (P 5+ ). Ca 2+ content increased after self-pollination but decreased after cross-pollination, while K + showed reverse trend with Ca 2+ . A total of 990 and 3 common differentially expressed genes (DEGs) were identified in un-pollinated vs. pollinated pistils and self- vs. cross-pollinated groups after 48 h, respectively. Function annotation indicated that three genes encoding UDP-glycosyltransferase 74B1 (UGT74B1), Mitochondrial calcium uniporter protein 2 (MCU2) and G-type lectin S-receptor-like serine/threonine-protein kinase (G-type RLK) might play important roles during SI process in tea plants., Conclusion: Ca 2+ and K + are important signal for SI in tea plants, and three genes including UGT74B1, MCU2 and G-type RLK play essential roles during SI signal transduction.

Both dispersal limitation and environmental sorting can affect genetic variation in populations, but their contribution remains unclear, particularly in microbes. We sought to determine the contribution of geographic distance (as a proxy for dispersal limitation) and phenotypic traits (as a proxy for environmental sorting), including morphology, metabolic ability and interspecific competitiveness, to the genotypic diversity in a nectar yeast species, Metschnikowia reukaufii. To measure genotypic diversity, we sequenced the genomes of 102 strains of M. reukaufii isolated from the floral nectar of hummingbird-pollinated shrub, Mimulus aurantiacus, along a 200-km coastline in California. Intraspecific genetic variation showed no detectable relationship with geographic distance, but could be grouped into three distinct lineages that correlated with metabolic ability and interspecific competitiveness. Despite ample evidence for strong competitive interactions within and among nectar yeasts, a full spectrum of the genotypic and phenotypic diversity observed across the 200-km coastline was represented even at a scale as small as 200 m. Further, more competitive strains were not necessarily more abundant. These results suggest that dispersal limitation and environmental sorting might not fully explain intraspecific diversity in this microbe and highlight the need to also consider other ecological factors such as trade-offs, source-sink dynamics and niche modification., (© 2018 John Wiley & Sons Ltd.)