Outcrossing Plant Species Research Articles

Development of Microsatellite Markers for Tanacetum cinerariifolium (Trevis.) Sch. Bip., a Plant with a Large and Highly Repetitive Genome.

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch. Bip.) is an outcrossing plant species (2n = 18) endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repeatability and large genome (1C-value = 9.58 pg) our previous attempts to develop microsatellite markers using the traditional method were unsuccessful. Now we have used Illumina paired-end whole genome sequencing and developed a specific procedure to obtain useful microsatellite markers. A total of 796,130,142 high-quality reads (approx. 12.5× coverage) were assembled into 6,909,675 contigs using two approaches (de novo assembly and joining of overlapped pair-end reads). A total of 31,380 contigs contained one or more microsatellite sequences, of which di-(59.7%) and trinucleotide (25.9%) repeats were the most abundant. Contigs containing microsatellites were filtered according to various criteria to achieve better yield of functional markers. After two rounds of testing, 17 microsatellite markers were developed and characterized in one natural population. Twelve loci were selected for preliminary genetic diversity analysis of three natural populations. Neighbor-joining tree, based on the proportion of shared alleles distances, grouped individuals into clusters according to population affiliation. The availability of codominant SSR markers will allow analysis of genetic diversity and structure of natural Dalmatian pyrethrum populations as well as identification of breeding lines and cultivars.

A bulked segregant analysis tool for out-crossing species (BSATOS) and QTL-based genomics-assisted prediction of complex traits in apple

IntroductionGenomic heterozygosity, self-incompatibility, and rich-in somatic mutations hinder the molecular breeding efficiency of outcrossing plants. ObjectivesWe attempted to develop an efficient integrated strategy to identify quantitative trait loci (QTLs) and trait-associated genes, to develop gene markers, and to construct genomics-assisted prediction (GAP) modes. MethodsA novel protocol, bulked segregant analysis tool for out-crossing species (BSATOS), is presented here, which is characterized by taking full advantage of all segregation patterns (including AB × AB markers) and haplotype information. To verify the effectiveness of the protocol in dealing with the complex traits of outbreeding species, three apple cross populations with 9,654 individuals were adopted. ResultsBy using BSATOS, 90, 60, and 77 significant QTLs were identified successfully and candidate genes were predicted for apple fruit weight (FW), fruit ripening date (FRD), and fruit soluble solid content (SSC), respectively. The gene-based markers were developed and genotyped for 1,396 individuals in a training population, including 145 Malus accessions and 1,251 F1 plants of the three full-sib families. GAP models were trained using marker genotype effect estimates of the training population. The prediction accuracy was 0.7658, 0.6455, and 0.3758 for FW, FRD, and SSC, respectively. ConclusionThe BSATOS and GAP models provided a convenient and efficient methodology for candidate gene mining and molecular breeding in out-crossing plant species. The BSATOS pipeline can be freely downloaded from: https://github.com/maypoleflyn/BSATOS.

Evolution of Autonomous Selfing in Marginal Habitats: Spatiotemporal Variation in the Floral Traits of the Distylous Primula wannanensis.

Outcrossing plant species are more likely to exhibit autonomous selfing in marginal habitats to ensure reproduction under conditions of limited pollinator and/or mate availability. Distyly is a classical paradigm that promotes outcrossing; however, little is known about the variation in floral traits associated with distylous syndrome in marginal populations. In this study, we compared the variation in floral traits including stigma and anther height, corolla tube length, herkogamy, and corolla diameter between the central and peripheral populations of the distylous Primula wannanensis, and assessed the variation of floral traits at early and late florescence stages for each population. To evaluate the potential consequences of the variation in floral traits on the mating system, we investigated seed set in each population under both open-pollinated and pollinator-excluded conditions. The flower size of both short- and long-styled morphs was significantly reduced in late-opening flowers compared with early opening flowers in both central and peripheral populations. Sex-organ reciprocity was perfect in early opening flowers; however, it was largely weakened in the late-opening flowers of peripheral populations compared with central populations. Of these flowers, disproportionate change in stigma height (elongated in S-morph and shortened in L-morph) was the main cause of reduced herkogamy, and seed set was fairly high under pollinator-excluded condition. Our results provide empirical support for the hypothesis on the evolution of delayed autonomous selfing in marginal populations of distylous species. Unsatisfactory pollinator service is likely to have promoted reproductive assurance of distylous plants with largely reduced herkogamy mimicking “homostyles.”

Horsenettle (Solanum carolinense) fruit bacterial communities are not variable across fine spatial scales.

Fruit house microbial communities that are unique from the rest of the plant. While symbiotic microbial communities complete important functions for their hosts, the fruit microbiome is often understudied compared to other plant organs. Fruits are reproductive tissues that house, protect, and facilitate the dispersal of seeds, and thus they are directly tied to plant fitness. Fruit microbial communities may, therefore, also impact plant fitness. In this study, we assessed how bacterial communities associated with fruit of Solanum carolinense, a native herbaceous perennial weed, vary at fine spatial scales (<0.5 km). A majority of the studies conducted on plant microbial communities have been done at large spatial scales and have observed microbial community variation across these large spatial scales. However, both the environment and pollinators play a role in shaping plant microbial communities and likely have impacts on the plant microbiome at fine scales. We collected fruit samples from eight sampling locations, ranging from 2 to 450 m apart, and assessed the fruit bacterial communities using 16S rRNA gene amplicon sequencing. Overall, we found no differences in observed richness or microbial community composition among sampling locations. Bacterial community structure of fruits collected near one another were not more different than those that were farther apart at the scales we examined. These fine spatial scales are important to obligate out-crossing plant species such as S. carolinense because they are ecologically relevant to pollinators. Thus, our results could imply that pollinators serve to homogenize fruit bacterial communities across these smaller scales.

Genome-Wide Prediction of Complex Traits in Two Outcrossing Plant Species Through Deep Learning and Bayesian Regularized Neural Network.

Genomic selection models were investigated to predict several complex traits in breeding populations of Zea mays L. and Eucalyptus globulus Labill. For this, the following methods of Machine Learning (ML) were implemented: (i) Deep Learning (DL) and (ii) Bayesian Regularized Neural Network (BRNN) both in combination with different hyperparameters. These ML methods were also compared with Genomic Best Linear Unbiased Prediction (GBLUP) and different Bayesian regression models [Bayes A, Bayes B, Bayes Cπ, Bayesian Ridge Regression, Bayesian LASSO, and Reproducing Kernel Hilbert Space (RKHS)]. DL models, using Rectified Linear Units (as the activation function), had higher predictive ability values, which varied from 0.27 (pilodyn penetration of 6 years old eucalypt trees) to 0.78 (flowering-related traits of maize). Moreover, the larger mini-batch size (100%) had a significantly higher predictive ability for wood-related traits than the smaller mini-batch size (10%). On the other hand, in the BRNN method, the architectures of one and two layers that used only the pureline function showed better results of prediction, with values ranging from 0.21 (pilodyn penetration) to 0.71 (flowering traits). A significant increase in the prediction ability was observed for DL in comparison with other methods of genomic prediction (Bayesian alphabet models, GBLUP, RKHS, and BRNN). Another important finding was the usefulness of DL models (through an iterative algorithm) as an SNP detection strategy for genome-wide association studies. The results of this study confirm the importance of DL for genome-wide analyses and crop/tree improvement strategies, which holds promise for accelerating breeding progress.

Multi-Trait Genomic Prediction Improves Predictive Ability for Dry Matter Yield and Water-Soluble Carbohydrates in Perennial Ryegrass.

In perennial ryegrass (Lolium perenne L), annual and seasonal dry matter yield (DMY) and nutritive quality of herbage are high-priority traits targeted for improvement through selective breeding. Genomic prediction (GP) has proven to be a valuable tool for improving complex traits and may be further enhanced through the use of multi-trait (MT) prediction models. In this study, we evaluated the relative performance of MT prediction models to improve predictive ability for DMY and key nutritive quality traits, using two different training populations (TP1, n = 463 and TP2, n = 517) phenotyped at multiple locations. MT models outperformed single-trait (ST) models by 24% to 59% for DMY and 67% to 105% for nutritive quality traits, such as low, high, and total WSC, when a correlated secondary trait was included in both the training and test set (MT-CV2) or in the test set alone (MT-CV3) (trait-assisted genomic selection). However, when a secondary trait was included in training set and not the test set (MT-CV1), the predictive ability was not statistically significant (p > 0.05) compared to the ST model. We evaluated the impact of training set size when using a MT-CV2 model. Using a highly correlated trait (rg = 0.88) as the secondary trait in the MT-CV2 model, there was no loss in predictive ability for DMY even when the training set was reduced to 50% of its original size. In contrast, using a weakly correlated secondary trait (rg = 0.56) in the MT-CV2 model, predictive ability began to decline when the training set size was reduced by only 11% from its original size. Using a ST model, genomic predictive ability in a population unrelated to the training set was poor (rp = −0.06). However, when using an MT-CV2 model, the predictive ability was positive and high (rp = 0.76) for the same population. Our results demonstrate the first assessment of MT models in forage species and illustrate the prospects of using MT genomic selection in forages, and other outcrossing plant species, to accelerate genetic gains for complex agronomical traits, such as DMY and nutritive quality characteristics.

Genetic diversity and structure of a rare endemic cactus and an assessment of its genetic relationship with a more common congener.

Endemic, obligate outcrossing plant species with narrow geographic distributions and disjunct populations are prone to loss of genetic diversity. Simultaneously, delineating clear species boundaries is important for targeted conservation efforts. The rare and endemic cactus, Sclerocactus brevihamatus subsp. tobuschii (SBT), has a parapatric relationship with Sclerocactus brevihamatus subsp. brevihamatus (SBB) but genetic distance between the two taxa is unknown. We: (1) developed taxon-specific polymorphic microsatellites, (2) assessed genetic diversity within and among nine populations of SBT, and within one population of SBB, and (3) estimated the genetic relationship between the two subspecies. Within-population genetic diversity of SBT was moderate to high (mean Ho = 0.37; mean He = 0.59). Indirect estimate of inbreeding corrected for null alleles (Fis-INEst) was low for SBT, ranging from 0.03 to 0.14 (mean Fis-INEst = 0.07). Genetic differentiation among populations of SBT was low based on Fst (0.08) and AMOVA (ФPT = 0.10). Lack of genetic and spatial correlation in SBT populations coupled with the presence of private alleles and bottleneck events in several populations suggests that reproductive isolation is occurring but that sufficient time may not have yet passed to manifest strong differentiation. Cluster analyses segregated the 10 populations into three distinct groups, and separated SBB genotypes clearly. Results suggest that while hybridization between the two subspecies may occur, SBT is clearly differentiated genetically from SBB to retain its current taxonomic status.

Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa.

The CRISPR/Cas9 technique was successfully used to edit the genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa). RNA-guided genome engineering using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. Successful application and validation of the CRISPR technique in a multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa. Because of the complex features of the alfalfa genome, we first used droplet digital PCR (ddPCR) for high-throughput screening of large populations of CRISPR-modified plants. Based on the results of genome editing rates obtained from the ddPCR screening, plants with relatively high rates were subjected to further analysis by restriction enzyme digestion/PCR amplification analyses. PCR products encompassing the respective small guided RNA target locus were then sub-cloned and sequenced to verify genome editing. In summary, we successfully applied the CRISPR/Cas9 technique to edit the SPL9 gene in a multiplex genome, providing some insights into opportunities to apply this technology in future alfalfa breeding. The overall efficiency in the polyploid alfalfa genome was lower compared to other less-complex plant genomes. Further refinement of the CRISPR technology system will thus be required for more efficient genome editing in this plant.

Does origin always matter? Evaluating the influence of nonlocal seed provenances for ecological restoration purposes in a widespread and outcrossing plant species.

For restoration purposes, nature conservation generally enforces the use of local seed material based on the “local‐is‐best” (LIB) approach. However, in some cases recommendations to refrain from this approach have been made. Here we test if a common widespread species with no obvious signs of local adaptation may be a candidate species for abandoning LIB during restoration. Using 10 microsatellite markers we compared population genetic patterns of the generalist species Daucus carota in indigenous and formerly restored sites (nonlocal seed provenances). Gene diversity overall ranged between H e = 0.67 and 0.86 and showed no significant differences between the two groups. Hierarchical AMOVA and principal component analysis revealed very high genetic population admixture and negligible differentiation between indigenous and restored sites (F CT = 0.002). Moreover, differentiation between groups was caused by only one outlier population, where inbreeding effects are presumed. We therefore conclude that the introduction of nonlocal seed provenances in the course of landscape restoration did not jeopardize regional species persistence by contributing to inbreeding or outbreeding depressions, or any measurable adverse population genetic effect. On the basis of these results, we see no obvious objections to the current practice to use the 10‐fold cheaper, nonlocal seed material of D. carota for restoration projects.

Nectar Attracts Foraging Honey Bees with Components of Their Queen Pheromones.

Floral nectar often contains chemicals that are deterrent to pollinators, presenting potential challenges to outcrossing plant species. Plants may be able to co-opt pollinator chemical signals to mitigate the negative effects of nectar deterrent compounds on pollination services. We found that buckwheat (Fagopyrum esculentum) and Mexican sunflower (Tithonia diversifolia) produce nectar with abundant phenolics, including three components of the Apis honeybee queen mandibular pheromone (QMP). In addition, these nectars contain a non-pheromonal phenolic, chlorogenic acid (CA), which was toxic to honeybees, and T. diversifolia nectar also contained isochlorogenic acid (IA). Fresh nectar or solutions containing nectar phenolics reduced Apis individual feeding compared to sucrose solutions. However, freely foraging bees preferred solutions with QMP components to control solutions, and QMP components over-rode or reversed avoidance of CA and IA. Furthermore, prior exposure to the presence or just the odor of QMP components removed the deterrent effects of CA and IA. By mimicking the honey bee pheromone blend, nectar may maintain pollinator attraction in spite of deterrent nectar compounds.

Factor analytic and reduced animal models for the investigation of additive genotype-by-environment interaction in outcrossing plant species with application to a Pinus radiata breeding programme

Modelling additive genotype-by-environment interaction is best achieved with the use of factor analytic models. With numerous environments and for outcrossing plant species, computation is facilitated using reduced animal models. The development of efficient plant breeding strategies requires a knowledge of the magnitude and structure of genotype-by-environment interaction. This information can be obtained from appropriate linear mixed model analyses of phenotypic data from multi-environment trials. The use of factor analytic models for genotype-by-environment effects is known to provide a reliable, parsimonious and holistic approach for obtaining estimates of genetic correlations between all pairs of trials. When breeding for outcrossing species the focus is on estimating additive genetic correlations and effects which is achieved by including pedigree information in the analysis. The use of factor analytic models in this setting may be computationally prohibitive when the number of environments is moderate to large. In this paper, we present an approach that uses an approximate reduced animal model to overcome the computational issues associated with factor analytic models for additive genotype-by-environment effects. The approach is illustrated using a Pinus radiata breeding dataset involving 77 trials, located in environments across New Zealand and south eastern Australia, and with pedigree information on 315,581 trees. Using this approach we demonstrate the existence of substantial additive genotype-by-environment interaction for the trait of stem diameter measured at breast height. This finding has potentially significant implications for both breeding and deployment strategies. Although our approach has been developed for forest tree breeding programmes, it is directly applicable for other outcrossing plant species, including sugarcane, maize and numerous horticultural crops.

Conservation genetics and breeding system of Penstemon debilis (Plantaginaceae), a rare beardtongue endemic to oil shale talus in western Colorado, USA

AbstractRare species of plants are especially vulnerable to extinction when populations are few, have small numbers of individuals, and are fragmented. Such conditions lead to a reduction in gene flow and genetic diversity, and encourage inbreeding depression. We conducted a study of the reproductive biology and population genetics of Penstemon debilis (Plantaginaceae), a Federally Threatened species endemic to a small region of oil shale extraction in western Colorado, USA. Most of the habitat area is privately owned and undergoing natural gas extraction activities. Penstemon debilis reproduces both vegetatively and as an outcrosser that requires a pollen vector. Moderate levels of inbreeding, but no inbreeding depression, were found within populations of P. debilis. Genetic divergence among the extant populations surveyed was moderate (FST values = 0.069–0.231; Nm = 0.831–3.385) with levels of genetic diversity within populations relatively low compared to congeners with similar modes of pollination and reproductive biology. STRUCTURE analysis revealed three population clusters with some admixture among all extant populations. Genetic diversity within and among P. debilis populations is similar to genetic diversity found for other rare and endemic outcrossing plant species. Our results are consistent with a pattern of recent population fragmentation or low levels of pollen‐mediated gene flow among populations in close proximity to one another. Conservation of P. debilis will require cooperative management strategies between private landowners, government agencies, and concerned NGOs to preserve habitat for this rare species.

The relative importance of solitary bees and syrphid flies as pollinators of two outcrossing plant species in the New Zealand alpine

AbstractPollinators vary in their relative contribution to the conspecific pollen deposited onto receptive stigmas, because of variation in both visitation rate and effectiveness of pollen transfer. Syrphid flies and short‐tongued solitary bees are common flower visitors in alpine New Zealand, yet their relative importance as pollinators is unknown. We measured pollinator performance of the New Zealand alpine endemics Hylaeus matamoko (Hymenoptera: Colletidae) and Allograpta spp. (Diptera: Syrphidae) on two New Zealand alpine herbs, Ourisia glandulosa (Plantaginaceae) and Wahlenbergia albomarginata (Campanulaceae). Ourisia glandulosa received visits by solitary bees and syrphid flies at equal frequencies, whereas W. albomarginata was mostly visited by H. matamoko. Based on single‐visit pollen deposition to virgin stigmas, H. matamoko was a much more effective pollinator than Allograpta spp., delivering 10 times as much pollen per visit to O. glandulosa stigmas and 3 times as much to W. albomarginata stigmas. By multiplying visitation frequency by single‐visit pollen deposition, we estimated that H. matamoko performed 90% and 95% of the pollination of O. glandulosa and W. albomarginata, respectively. Although H. matamoko bees are short‐tongued and small in size, they are critically important to plant reproductive success in the New Zealand alpine. These bees contributed most of the pollination, even to a species that received just as many visits by flies, underscoring the need to consider per‐visit effectiveness as well as visitation rate in assessing the importance of different pollinators.

A hidden Markov model approach to multilocus linkage analysis in a full-sib family

Statistical packages for constructing genetic linkage maps in inbred lines are well developed and applied extensively, while linkage analysis in outcrossing species faces some statistical challenges because of their complicated genetic structures. In this article, we present a multilocus linkage analysis via hidden Markov models for a linkage group of markers in a full-sib family. The advantage of this method is the simultaneous estimation of the recombination fractions between adjacent markers that possibly segregate in different ratios, and the calculation of likelihood for a certain order of the markers. When the number of markers decreases to two or three, the multilocus linkage analysis becomes traditional two-point or three-point linkage analysis, respectively. Monte Carlo simulations are performed to show that the recombination fraction estimates of multilocus linkage analysis are more accurate than those just using two-point linkage analysis and that the likelihood as an objective function for ordering maker loci is the most powerful method compared with other methods. By incorporating this multilocus linkage analysis, we have developed a Windows software, FsLinkageMap, for constructing genetic maps in a full-sib family. A real example is presented for illustrating linkage maps constructed by using mixed segregation markers. Our multilocus linkage analysis provides a powerful method for constructing high-density genetic linkage maps in some outcrossing plant species, especially in forest trees.

Fine scale genetic structure in the wild ancestor of maize (Zea maysssp.parviglumis)

Analysis of fine scale genetic structure in continuous populations of outcrossing plant species has traditionally been limited by the availability of sufficient markers. We used a set of 468 SNPs to characterize fine-scale genetic structure within and between two dense stands of the wild ancestor of maize, teosinte (Zea mays ssp. parviglumis). Our analyses confirmed that teosinte is highly outcrossing and showed little population structure over short distances. We found that the two populations were clearly genetically differentiated, although the actual level of differentiation was low. Spatial autocorrelation of relatedness was observed within both sites but was somewhat stronger in one of the populations. Using principal component analysis, we found evidence for significant local differentiation in the population with stronger spatial autocorrelation. This differentiation was associated with pronounced shifts in the first two principal components along the field. These shifts corresponded to changes in allele frequencies, potentially due to local topographical features. There was little evidence for selection at individual loci as a contributing factor to differentiation. Our results demonstrate that significant local differentiation may, but need not, co-occur with spatial autocorrelation of relatedness. The present study represents one of the most detailed analyses of local genetic structure to date and provides a benchmark for future studies dealing with fine scale patterns of genetic diversity in natural plant populations.

Population genetics and breeding system ofTupistra pingbianensis(Liliaceae), a naturally rare plant endemic to SW China

Abstract The levels and partitioning of genetic diversity and inbreeding depression were investigated inTupistra pingbianensis, a narrow endemic of southeast Yunnan, China, characterized by a naturally fragmented distribution due to extreme specialization on a rare habitat type. Here genetic diversity and patterns of genetic variation within and among 11 populations were analyzed using amplified fragment length polymorphism markers with 97 individuals across its whole geographical range. High levels of genetic variation were revealed both at the species level (P99= 96.012%;Ht= 0.302) and at the population level (P99= 51.41%;Hs= 0.224). Strong genetic differentiation among populations was also detected (FST= 0.2961; θII= 0.281), which corresponded to results reported for typical animal‐pollinated, mixed selfing, and outcrossing plant species. This result was consistent with mating patterns detected by our pollination experiments. The indirect estimate of gene flow based on θIIwas low (Nm= 0.64). Special habitat and its life history traits might play an important role in shaping the genetic diversity and the genetic structure of this species. A pollination experiment also failed to detect significant inbreeding depression upon F1fruit set, seed weight, and germinate rate fitness‐traits. As a naturally rare species,T. pingbianensisis not seriously genetically impoverished and likely to have adapted to tolerating a high level of inbreeding early in its history, we propose this species need only periodic monitoring to ensure their continued persistence, but not intervention to remain viable.

Sampling strategies for whole genome association studies in aquaculture and outcrossing plant species

A number of farmed species are characterized by breeding populations of large full-sib families, including aquaculture species and outcrossing plant species. Whole genome association studies in such species must account for stratification arising from the full-sib family structure to avoid high rates of false discovery. Here, we demonstrate the value of selective genotyping strategies which balance the contribution of families across high and low phenotypes to greatly reduce rates of false discovery with a minimal effect on power.

Early inbreeding depression in the sexually polymorphic plant Dianthus sylvestris (Caryophyllaceae): Effects of selfing and biparental inbreeding among sex morphs

Predominantly outcrossing plant species are expected to accumulate recessive deleterious mutations, which can be purged when in a homozygous state following selfing. Individuals may vary in their genetic load because of different selfing histories, which could lead to differences in inbreeding depression among families. Lineage-dependent inbreeding depression can appear in gynodioecious species if obligatory outcrossed females are more likely to produce female offspring and if partially selfing hermaphrodites are more likely to produce hermaphrodites. We investigated inbreeding depression at the zygote, seed, and germination stages in the gynomonoecious-gynodioecious Dianthus sylvestris, including pure-sexed plants and a mixed morph. We performed hand-pollinations on 56 plants, belonging to the three morphs, each receiving 2-3 cross treatments (out-, sib- and self-pollination) on multiple flowers. Effects of cross treatments varied among stages and influenced seed provisioning, with sibling competition mainly occurring within outcrossed fruits. We found significant inbreeding depression for seed mass and germination and cumulative early inbreeding depression varied greatly among families. Among sex morphs, we found that females and hermaphrodites differed in biparental inbreeding depression, whereas uniparental was similar for all. Significant inbreeding depression levels may play a role in female maintenance in this species, and individual variation in association with sex-lineages proclivity is discussed.

Patch Size and Landscape Effects on Pollinators and Seed Set of the Horseshoe Vetch, Hippocrepis comosa, in an Agricultural Landscape of Central Europe

Agricultural land use intensification has caused a considerable decline of once common semi-natural habitats leaving only small and isolated habitat remnants within a homogeneous landscape matrix. Populations of flowering plant species in these remaining habitat fragments are also declining and can not offer sufficient resources for potential pollinators. The loss of pollinators and consequential pollination limitation is especially severe for obligately out-crossing plant species. In this study, pollination and reproductive success of Hippocrepis comosa was examined. Hippocrepis comosa L is a self-incompatible, declining plant species occurring on fragmented remnants in semi-natural calcareous grassland habitats in an agricultural landscape in Southern Lower Saxony, Germany. Plant-pollinator interactions were analysed in small, medium, and large plant patches in 15 differently sized calcareous grassland fragments surrounded by landscape matrices of differing complexity. Flower visitors were observed during three 15-minute observations, pollen supplementation experiments were performed to test for pollination limitation, and fruits from the plant patches were collected to calculate seed set. Hippocrepis comosa showed evidence of pollination limitation because hand-pollinated flowers set more seeds per inflorescence than open-pollinated flowers and seed set was strongly reduced by decreased visitation rates. Pollinators were most abundant in large patches and in diverse landscapes. Visitation rate was not affected by patch size. Hence, in order to maintain plant-pollinator interactions in an agricultural landscape, conservation efforts should include the preservation of calcareous grasslands that can support sufficiently large patches of a plant species and offer ample rewards for pollinators and the establishment of a diverse agricultural landscape around the semi-natural habitats that will further enhance pollinator abundance.

OneMap: software for genetic mapping in outcrossing species

OneMap is an environment for constructing linkage maps of outcrossing plant species, using full-sib families derived from two outbred parents. The analyses are performed using a novel methodology based on the maximum likelihood approach for simultaneous estimation of linkage and linkage phases (Wu et al. 2002), which has been successfully applied to sugarcane (Garcia et al. 2006). It is implemented as a set of functions for the freely distributed software R, and handles pairwise marker analysis, marker ordering and map refinement. The software is freely available at http://www.ciagri.usp.br/ approximately aafgarci/OneMap/.