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PubMed2001

Evidence for local outbreeding depression in the Mediterranean island endemic Anchusa crispa Viv. (Boraginaceae).

In this study we examined the relative effects of inbreeding and outbreeding on offspring fitness over two generations in Anchusa crispa Viv., a rare species which on Corsica (France) occurs in small populations composed of patches of few individuals. Self- and outcross-pollinations were carried out in a single population and F(1) progeny grown to flowering. Plants grown from selfing and outcrossing were then selfed or outcrossed to produce F(2) individuals in four combinations of pollination treatments over two generations, i.e. self + self, self + outcross, outcross + self and outcross + outcross. In the F(1) generation, selfed progeny had a significantly greater number of cymes per plant than outcrossed progeny (P=0.006). Plants from two generations of selfing had fewer seeds per fruit (P=0.06) but a significantly greater survival rate (P < 0.001) and a greater number of cymes (P=0.06) than those from two generations of outcrossing. Selfed F(2) from outcrossed F(1) had a significantly greater number of cymes (P < 0.01) than outcrossed F(2) from outcrossed F(1). In the comparison between selfed and outcrossed F(2) from selfed F(1) the former had a significantly greater survival rate (P < 0.001), but the latter significantly more flowers per cyme (P < 0.05). Together, these results indicate that outbreeding depression may occur on a very local scale within populations of this rare endemic species, with important implications for the conservation of rare plants.

Boraginaceae↗
PubMed2000

Monitoring gene flow from transgenic sugar beet using cytoplasmic male-sterile bait plants.

One of the most discussed environmental effects associated with the use of transgenic plants is the flow of genes to plants in the environment. The flow of genes may occur through pollen since it is the reproductive system that is designed for gene movement. Pollen-mediated gene escape is hard to control in mating plants. Pollen from a wind pollinator can move over distances of more than 1000 m. To investigate the efficiency of transgenic pollen movement under realistic environmental conditions, the use of bait plants might be an effective tool. In this study, cytoplasmic male-sterile (CMS) sugar beets were tested with regard to their potential for monitoring transgene flow. As the pollen source, transgenic sugar beets were used that express recombinant DNA encoding viral (beet necrotic yellow vein virus) resistance, and antibiotic (kanamycin) and herbicide (glufosinate) tolerance genes. In a field trial, the effectiveness of a hemp (Cannabis sativa) stripe containment strategy was tested by measuring the frequency of pollinated CMS bait plants placed at different distances and directions from a transgenic pollen source. The results demonstrated the ineffectiveness of the containment strategy. Physiological and molecular tests confirmed the escape and production of transgenic offspring more than 200 m behind the hemp containment. Since absolute containment is unlikely to be effective, the CMS-bait plant detection system is a useful tool for other monitoring purposes.

Aminobutyrates↗
PubMed2002

Microsatellite diversity and genetic structure of fragmented populations of the rare, fire-dependent shrub Grevillea macleayana.

Recent habitat loss and fragmentation superimposed upon ancient patterns of population subdivision are likely to have produced low levels of neutral genetic diversity and marked genetic structure in many plant species. The genetic effects of habitat fragmentation may be most pronounced in species that form small populations, are fully self-compatible and have limited seed dispersal. However, long-lived seed banks, mobile pollinators and long adult lifespans may prevent or delay the accumulation of genetic effects. We studied a rare Australian shrub species, Grevillea macleayana (Proteaceae), that occurs in many small populations, is self-compatible and has restricted seed dispersal. However, it has a relatively long adult lifespan (c. 30 years), a long-lived seed bank that germinates after fire and is pollinated by birds that are numerous and highly mobile. These latter characteristics raise the possibility that populations in the past may have been effectively large and genetically homogeneous. Using six microsatellites, we found that G. macleayana may have relatively low within-population diversity (3.2-4.2 alleles/locus; Hexp = 0.420-0.530), significant population differentiation and moderate genetic structure (FST = 0.218) showing isolation by distance, consistent with historically low gene flow. The frequency distribution of allele sizes suggest that this geographical differentiation is being driven by mutation. We found a lack mutation-drift equilibrium in some populations that is indicative of population bottlenecks. Combined with evidence for large spatiotemporal variation of selfing rates, this suggests that fluctuating population sizes characterize the demography in this species, promoting genetic drift. We argue that natural patterns of pollen and seed dispersal, coupled with the patchy, fire-shaped distribution, may have restricted long-distance gene flow in the past.

Alleles↗
PubMed2002

Complex patterns of mating revealed in a Eucalyptus regnans seed orchard using allozyme markers and the neighbourhood model.

The neighbourhood model apportions offspring of individual mother plants to self-fertilization, outcrossing to males within a circumscribed area around the mother plant (the neighbourhood), and outcrossing to males outside the neighbourhood. Formerly the model was applied only to haploid pollen gametes in the offspring of conifers, but is extended so that it can be used with genotypic data from diploid offspring of both angiosperms and gymnosperms. In addition, it is shown that the mating parameters can be estimated without independent estimates of allele frequencies in the pollen pools outside the neighbourhood; thus the model might be applied effectively to natural populations exposed to unknown external pollen sources. Parameters of the neighbourhood mating model were estimated for a 10-year-old seed orchard population of the insect-pollinated tree, Eucalyptus regnans, in southeast Australia, which contained a mixture of two geographical provenances (Victoria and Tasmania). The mating patterns revealed were complex. Crosses between trees of the same provenance occurred three times more often than crosses between trees of different provenances. Levels of self-fertilization and patterns of mating within neighbourhoods were influenced by provenance origin, crop fecundity and orchard position (central vs. edge) of mother trees. Gene dispersal, however, was extensive, with approximately 50% of effective pollen gametes coming from males more than 40 m away from mother trees (average distance between neighbouring trees was 7.4 m). Thus, insect pollinators are efficient promoters of cross-fertilization in this orchard, with the result that the effective number of males mating with each female is large.

Australia↗
PubMed2003

Genetic effects of habitat fragmentation in Viola pubescens (Violaceae), a perennial herb with chasmogamous and cleistogamous flowers.

The reproductive biology of a plant species is important in the response of populations to habitat fragmentation, especially if plant-pollinator interactions are disrupted. The genetic effects of forest fragmentation were examined in the common understorey herb Viola pubescens, a species that produces self-pollinated cleistogamous (CL) flowers and potentially outcrossing chasmogamous (CH) flowers. Using allozymes, we measured genetic variation in different sized populations. These were located in woodlots of various sizes (0.5-40.5 ha) and distances from one another (0.3-46 km) within the agricultural landscape of central Ohio in the Midwestern United States. Changes in forest cover of each woodlot within the past 180 years were determined from historical sources and aerial photographs. Woodlot and population sizes were significantly and positively correlated with measures of genetic variation (A, P, HO and HE), with variation highest in populations in the largest woodlot population and lowest in the smallest woodlot population. Most large woodlots resulted from fluctuations in forest cover over the past 60 years, while smaller fragments remained the same size. Overall, populations in Crawford County were genetically differentiated from one another (theta = 0.34), but there was no relationship between genetic and geographical distance. Preliminary evidence for a single year indicated a high rate of outcrossing in most populations. Despite the CH/CL reproductive advantage and apparent outcrossing, populations of V. pubescens in small woodlots remain susceptible to potentially detrimental effects of fragmentation such as genetic drift and reduced levels of genetic variation.

Cluster Analysis↗
PubMed1999

The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis.

The Arabidopsis AtSUC1 protein has previously been characterized as a plasma membrane H+-sucrose symporter. This paper describes the sites of AtSUC1 gene expression and AtSUC1 protein localization and assigns specific functions to this sucrose transporter in anther development and pollen tube growth. RNase protection assays revealed AtSUC1 expression exclusively in floral tissue, which was confirmed by analyses of AtSUC1 promoter-beta-glucuronidase (GUS) plants. In situ hybridizations identified AtSUC1 expression in anther connective tissue, in funiculi and in fully developed pollen grains. Indirect immuno-fluorescence analyses with anti-AtSUC1 antiserum confirmed AtSUC1 protein localization in the connective tissue and funiculi. In mature pollen grains, however, despite high AtSUC1 mRNA levels no AtSUC1 protein was found. Only after pollination of stylar papillae was AtSUC1 protein detected inside the pollen and later inside the growing pollen tubes, suggesting a translation of pre-existing AtSUC1 mRNA after pollination. Pollen germination analyses underlined the important role of sucrose for pollen tube growth. The data presented suggest a role of AtSUC1 in the controlled dehiscence of Arabidopsis anthers. It is postulated that an important function of AtSUC1 is the cell-specific modulation of water potentials.

Amino Acid Sequence↗
PubMed2000

FLP-mediated recombination for use in hybrid plant production.

We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites. Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.

Arabidopsis↗
PubMed2000

Aquaporin PIP genes are not expressed in the stigma papillae in Brassica oleracea.

The pollen grains of angiosperms are usually desiccated at maturity. Following pollination, pollen hydrates on the stigma surface before germination takes place. Rehydration is an essential step for the success of pollination and depends on the movement of water from the stigmatic cells. This water flow has been shown to be biologically regulated, and components of both pollen and stigma surfaces have been demonstrated to play a role in the control of pollen hydration. Regulation of water transport between animal or plant cells involves membrane proteins, designated aquaporins, which possess water-channel activity. Such molecules may be candidates for controlling pollen hydration, and consequently we investigated whether aquaporins are present in the pollen and stigma cells in Brassica oleracea. Here, we report the identification of two new aquaporin genes, Bo-PIP1b1 and Bo-PIP1b2, which are highly homologous to PIP1b from Arabidopsis thaliana. Both Bo-PIP1b1 and Bo-PIP1b2 proteins are active water channels when expressed in Xenopus oocytes. Expression of Bo-PIP1b1 and Bo-PIP1b2 was observed in reproductive organs as well as in vegetative tissues. Interestingly, the use of a Bo-PIP1b2 cDNA probe revealed that PIP1-like transcripts were not present in the pollen grains or in the stigma papillae, but were present in the stigma cell layers underlying the papillar cells. This observation suggests that water flow between the pollen and stigma papillae may be dependent on aquaporins expressed in cells that are not directly in contact with the pollen grain.

Amino Acid Sequence↗
PubMed2001

Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system.

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.

Base Sequence↗
PubMed2003

Reproductive isolation and hybrid pollen disadvantage in Ipomopsis.

One cause of reproductive isolation is gamete competition, in which conspecific pollen has an advantage over heterospecific pollen in siring seeds, thereby decreasing the formation of F1 hybrids. Analogous pollen interactions between hybrid pollen and conspecific pollen can contribute to post-zygotic isolation. The herbaceous plants Ipomopsis aggregata and I. tenuituba frequently hybridize in nature. Hand-pollination of I. aggregata with pollen from F1 or F2 hybrids produced as many seeds as hand-pollination with conspecific pollen, suggesting equal pollen viability. However, when mixed pollen loads with 50% conspecific pollen and 50% hybrid pollen were applied to I. aggregata stigmas, fewer than half of the seeds had hybrid sires. Such pollen mixtures are frequently received if plants of the two species and F1 and F2 hybrids are intermixed, suggesting that this advantage of conspecific over hybrid pollen reduces backcrossing and contributes to reproductive isolation.

Colorado↗
PubMedDate not supplied

Environmental implications of gene flow from sugar beet to wild beet--current status and future research needs.

Gene flow via seed or pollen is a basic biological process in plant evolution. The ecological and genetic consequences of gene flow depend on the amount and direction of gene flow as well as on the fitness of hybrids. The assessment of potential risks of transgenic plants should take into account the fact that conventional crops can often cross with wild plants. The precautionary approach in risk management of genetically modified plants (GMPs) may make it necessary to monitor significant wild and weed populations that might be affected by transgene escape. Gene flow is hard to control in wind-pollinated plants like beet (Beta vulgaris). In addition, wild beet populations potentially can undergo evolutionary changes which might expand their geographical distribution. Unintended products of cultivated beets pollinated by wild beets are weed beets that bolt and flower during their first year of planting. Weed beets cause yield losses and can delay harvest. Wild beets are important plant genetic resources and the preservation of wild beet diversity in Europe has been considered in biosafety research. We present here the methodology and research approaches that can be used for monitoring the geographical distribution and diversity of Beta populations. It has recently been shown that a century of gene flow from Beta vulgaris ssp. vulgaris has not altered the genetic diversity of wild Beta vulgaris L. ssp. maritima (L.) Arcang. in the Italian sugar beet seed production area. Future research should focus on the potential evolution of transgenic wild beet populations in comparison to these baseline data. Two monitoring models are presented describing how endpoints can be measured: (1) "Pre-post" crop commercialization against today's baseline and (2) "Parallel" to crop commercialization against GMP free reference areas/ populations. Model 2 has the advantage of taking ongoing changes in genetic diversity and population dynamics into account. Model 1 is more applicable if gene flow is so strong that most areas/populations contain GMPs. Important traits that may change the ecology of populations are genes that confer tolerance to biotic and abiotic stress. An assessment of environmental effects can realistically only be based on endpoints and consequences of gene introgression, which may include economic values of biodiversity in littoral and other ecosystems containing wild beet. In general, there is still a great need to harmonize worldwide monitoring systems by the development of appropriate methods to evaluate the environmental impact of introgressed transgenes.

Beta vulgaris↗
PubMed2004

Phenology, sex ratio, and spatial distribution among dioecious species of Trichilia (Meliaceae).

The flowering, sex ratio, and spatial distribution of four dioecious species of Trichilia (Meliaceae) were studied in a semi-deciduous forest in southeastern Brazil. All reproductive trees (T. clausseni, T. pallida and T. catigua) with dbh > or = 5 cm within a 1-ha plot were collected, sexed, mapped and, for individuals of each species, the distances to the nearest neighbour of the same and opposite sex were measured. For the shrub species T. elegans (dbh < 5 cm), all reproductive individuals were sampled randomly in 10 samples of 10 x 10 m. The reproductive phenology was observed at weekly to monthly intervals from May 1988 to January 1990. The species are strictly dioecious, did not present any sex-mixed trees or sex switching during the study, and sex ratio did not differ significantly from 1 : 1. The size distributions and the relative size variation were not significantly different between sexes. There was no significant segregation or clumping between individuals of either sex and no fruit production without pollination. Onset of flowering and flowering peak were synchronous between male and female plants for all species studied. Flower synchrony was related to outcrossing and pollinator attraction rather than climatic factors.

Flowers↗
PubMed2004

Reduced fecundity in small populations of the rare plant Gentianopsis ciliate (Gentianaceae).

Habitat destruction is the main cause for the biodiversity crisis. Surviving populations are often fragmented, i.e., small and isolated from each other. Reproduction of plants in small populations is often reduced, and this has been attributed to inbreeding depression, reduced attractiveness for pollinators, and reduced habitat quality in small populations. Here we present data on the effects of fragmentation on the rare, self-compatible perennial herb Gentianopsis ciliata (Gentianaceae), a species with very small and presumably well-dispersed seeds. We studied the relationship between population size, plant size, and the number of flowers produced in 63 populations from 1996-1998. In one of the years, leaf and flower size and the number of seeds produced per fruit was studied in a subset of 25 populations. Plant size, flower size, and the number of seeds per fruit and per plant increased with population size, whereas leaf length and the number of flowers per plant did not. The effects of population size on reproduction and on flower size remained significant if the effects were adjusted for differences in plant size, indicating that they could not be explained by differences in habitat quality. The strongly reduced reproduction in small populations may be due to pollination limitation, while the reduced flower size could indicate genetic effects.

Biodiversity↗
PubMed2002

Release of allergens as respirable aerosols: A link between grass pollen and asthma.

BACKGROUND: Asthma incidence has long been linked to pollen, even though pollen grains are too large to penetrate into the airways where asthmatic responses originate. Pollen allergens found in small, respirable particles have been implicated in a number of asthma epidemics, particularly ones following rainfall or thunderstorms. OBJECTIVE: The aim of this study was to determine how pollen allergens form the respirable aerosols necessary for triggering asthma. METHODS: Flowering grasses were humidified and then dried in a controlled-environment chamber connected to a cascade impactor and an aerosol particle counter. Particles shed from the flowers were analyzed with high-resolution microscopy and immunolabeled with rabbit anti-Phl p 1 antibody, which is specific for group 1 pollen allergens. RESULTS: Contrary to what has been reported in other published accounts, most of the pollen in this investigation remained on the open anthers of wind pollinated plants unless disturbed-eg, by wind. Increasing humidity caused anthers to close. After a cycle of wetting and drying followed by wind disturbance, grasses flowering within a chamber produced an aerosol of particles that were collected in a cascade impactor. These particles consisted of fragmented pollen cytoplasm in the size range 0.12 to 4.67 microm; they were loaded with group 1 allergens. CONCLUSION: Here we provide the first direct observations of the release of grass pollen allergens as respirable aerosols. They can emanate directly from the flower after a moisture-drying cycle. This could explain asthmatic responses associated with grass pollination, particularly after moist weather conditions.

Aerosols↗
PubMed2004

Quantum speciation in Aegilops: molecular cytogenetic evidence from rDNA cluster variability in natural populations.

Data are presented on quantum speciation in the Sitopsis section of the genus Aegilops (Poaceae, Monocotyledones). Two small, peripheral, isolated, wild populations of annual cross-pollinated Ae. speltoides and annual self-pollinated Ae. sharonensis are located 30 m apart on different soil types. Despite the close proximity of the two species and their close relatedness, no mixed groups are known. Comparative molecular cytogenetic analysis based on the intrapopulation variability of rRNA-encoding DNA (rDNA) chromosomal patterns of individual Ae. speltoides geno-types revealed an ongoing dynamic process of permanent chromosomal rearrangements. Chromosomal mutations can arise de novo and can be eliminated. Analysis of the progeny of the investigated genotypes testifies that inheritance of de novo rDNA sites happens frequently. Heterologous recombination and/or transposable elements-mediated rDNA transfer seem to be the mechanisms for observed chromosomal repatterning. Consequently, several modified genomic forms, intermediate between Ae. speltoides and Ae. sharonensis, permanently arise in the studied wild population of Ae. speltoides, which make it possible to recognize Ae. sharonensis as a derivative species of Ae. speltoides, as well as to propose rapidness and canalization of quantum speciation in Sitopsis species.

Chromosomes, Plant↗
PubMed2001

Negative frequency-dependent selection maintains a dramatic flower color polymorphism in the rewardless orchid Dactylorhiza sambucina (L.) Soo.

The orchid Dactylorhiza sambucina shows a stable and dramatic flower-color polymorphism, with both yellow- and purple-flowered individuals present in natural populations throughout the range of the species in Europe. The evolutionary significance of flower-color polymorphisms found in many rewardless orchid species has been discussed at length, but the mechanisms responsible for their maintenance remain unclear. Laboratory experiments have suggested that behavioral responses by pollinators to lack of reward availability might result in a reproductive advantage for rare-color morphs. Consequently, we performed an experiment varying the relative frequency of the two color morphs of D. sambucina to test whether rare morph advantage acted in the natural habitat of the species. We show here clear evidence from this manipulative experiment that rare-color morphs have reproductive advantage through male and female components. This is the first demonstration, to our knowledge, that negative frequency-dependent selection through pollinator preference for rare morphs can cause the maintenance of a flower-color polymorphism.

Color↗
PubMed1970

Temporal phenotypic heterogeneity as a substrate for selection.

The interaction of temporal phenotypic heterogeneity with a population level fitness function was explored through the use of a simple model. The model, based upon experimental data, assumes that the exploitation of pollinators, and seed-set within plant populations are dependent upon the frequency of a pink flower morph in populations composed of pink and white morphs. An increase in pink frequency is accompanied by an increase in seed-set for both morphs. The model also assumes that seed-set within a population is the same for pink and white individuals. In perennials and annuals with long-lived seeds, fluctuations in morph balance from one season to another may lead to reproductive pulses or drags, depending upon which morph was in excess. The reproductive differential which stems from different levels of pollinator service leads to an increase in pink frequency and an attendant fitness gain. The perennial habit and long-lived seeds provide a population with a memory which permits the current population image to compete with images of previous years. As a consequence, fitness gains may be achieved in the absence of interplant or interpopulation selection.

Journal Article↗
PubMed1984

Estimation of mating system parameters when outcrossing events are correlated.

Many plant species have mating systems characterized by a mixture of self-fertilization and outcrossing. Statistical estimation of the outcrossing rate has relied on a model of the mating process that assumes that successive outcross events within a family arise from independent draws of pollen from the total population of male plants. Although this assumption is likely to be most appropriate for wind-pollinated plants, it is not appropriate in certain insect-pollinated plants. An alternative model is developed that assumes that successive outcross events within a family involve pollen drawn from a single male parent. The estimation of the parameters that index this model is outlined and a procedure for calculating the variances of the parameter estimates is presented. Monte Carlo simulations of the sampling processes assumed by each model are also presented. The simulations show that application of the incorrect estimation model to data can lead to a large bias in parameter estimates.

Journal Article↗