Genetic Relationship of Brassicaceae Hybrids with Various Resistance to Blackleg Is Disclosed by the Use of Molecular Markers

Brassica napus is an important oil source. Its narrow gene pool can be widened by interspecific hybridization with the Brassicaceae species. One of the agronomically important traits, that can be transferred through the hybridization, is the resistance to blackleg, a dangerous disease mainly caused by Leptosphaeria maculans. Hybrid individuals can be analyzed with various molecular markers, including Simple Sequence Repeats (SSR). We investigated the genetic similarity of 32 Brassicaceae hybrids and 19 parental components using SSR markers to reveal their genetic relationship. Furthermore, we compared the field resistance to blackleg of the interspecific progenies. The tested set of 15 SSR markers proved to be useful in revealing the genetic distances in the Brassicaceae hybrids and species. However, genetic similarity of the studied hybrids could not be correlated with the level of field resistance to L. maculans. Moreover, our studies confirmed the usefulness of the Brassicaceae hybrids in terms of blackleg management.     

Differential organellar inheritance in Passiflora’s (Passifloraceae) subgenera

Four chloroplast (cp), one mitochondrial (mt), and one ribosomal nuclear (ITS) DNA regions were studied in four artificial and one natural interspecific Passiflora hybrids. The ITS results confirmed their hybrid origin and all mtDNAs were maternally inherited. The same, however, was not true for cpDNA. The four hybrids (three artificial and one natural) derived from species of the Passiflora subgenus showed a cpDNA paternal inheritance, while the one involving taxa of the Decaloba subgenus gave evidence of maternal transmission. These results are of significance for the ongoing studies which are being performed on the molecular evolution of this genus and furnish important background for investigations aimed at clarifying the factors which determine cpDNA inheritance.     

Potentials and limitations of the cross-species transfer of nuclear microsatellite marker in six species belonging to three sections of the genus Populus L.

The genus Populus is classified into six different sections, and depending on the declaration of hybrids, the number of species varies between 22 and 85. Species within one section, and sometimes between sections, are crossable to each other, resulting in many naturally but also artificially produced hybrids. Morphological attributes for a clone characterisation are often difficult to evaluate when different poplar species or even hybrids are crossed; thus, molecular markers are needed to characterise the different species. Taking advantage of the large microsatellite resource developed for Populus trichocarpa, however, amplification of these microsatellite markers in other Populus species either often fails, or in the case of amplification, unrelated genomic regions are amplified. To meet this obvious problem of the species transferability of microsatellite markers, in total, 305 microsatellite loci, mainly from P. trichocarpa but also few from Populus tremuloides and Populus nigra, were tested for their transferability to diverse genotypes of six species belonging to three sections of the genus Populus. Ultimately, 209 microsatellite loci could be amplified with varying sizes in the different species. The PCR products of selected loci were separated in a polyacrylamide gel and sequenced to assure that the expected loci were derived from the database genome of P. trichocarpa. The present results constitute a large study for microsatellite transferability for Populus species. The documented microsatellite loci can be applied to species-, hybrid- and clone-specific diagnostic approaches or as universal markers for comprehensive ecological studies.     

Selection of generally applicable SSR markers for evaluation of genetic diversity and identity in Lilium

There are approximately 100 species and 10,000 cultivars of Lilium and in general their phylogeny is understood. Difficulties remain, however, in understanding the breeding relationships of cultivars and commercial hybrids. One solution to this problem is to identify a selection of validated and transferable SSR markers for use in genotyping. Although over 100 Lilium SSRs have been developed, they have not been validated for use with broad populations. Here, were-evaluated 112 SSRs with 69 lily accessions from different sources, and selected 70 SSRs as easy to score, transferable and polymorphic in all accessions tested. Based on the marker data from 70 SSRs, two main clusters were established for 69 accessions using TREECON, one includes Asiatic hybrids, Longiflorum × Asiatic hybrids and Asiatic local landraces (Lilium brownii, L. brownii var. giganteum, Lilium pumilum, Lilium davidii var. unicolor and Lilium lancifolium), the other is composed primarily of Oriental hybrids and Oriental × Trumpet hybrids, which is in agreement with previous studies and the breeding pedigree. The utility of the 70 SSR markers for establishing parentage and taxon identity of landraces was validated. Our study offers valuable information and validated markers for Lilium systematic classification and the establishment of identity.     

High polymorphism and resolution in targeted fingerprinting with combined β-tubulin introns

Tubulin-Based-Polymorphism (TBP) was originally introduced as a novel method for assaying genetic diversity in plants. TBP is based on polymorphism resulting from the PCR-mediated amplification of the first intron in the coding region of the β-tubulin gene family. Although, the method was successful in genetic assessment of some plant species and varieties, it suffered from low number of molecular markers due to limited variation in the first intron of β-tubulin gene family. We have now rectified this limitation by introducing the second intron of the β-tubulin genes as a valuable source of molecular markers. We show that the combined use of the two introns substantially increases the number of molecular markers and results in a reliable assessment of species/varieties relationships. After a preliminary validation on Brassica, this new combinatorial method was tested on species of Eleusine and Arachis. For both, reliable assessment of species relationships were obtained that were consistent with recently published studies resulting from more elaborated methods including DNA sequencing. Combinatorial TBP is a reliable, reproducible, simple, fast, and easy to score method that is very useful for breeding programs and species and variety assessments.     

Karyomorphology and GC-rich heterochromatin pattern in Passiflora (Passifloraceae) wild species from Decaloba and Passiflora subgenera

Thirteen wild species of Passiflora were analyzed using conventional and CMA/DA/DAPI staining to evaluate the karyotype diversity between and within the subgenus Decaloba and Passiflora. The karyotypic features indicate that both subgenera have a conserved chromosome number, as reported before for several species. Submetacentric (sm) chromosomes were found in species from both subgenera, suggesting that sm chromosomes are not restricted to a particular subgenus. The analysis of the karyotypic heterogeneity enabled to distribute the species in three groups, but with no support to phylogenetic and taxonomic levels. The application of fluorochromes allowed for the visualization of CMA⁺/DAPI⁻ blocks, which in our studies always correlated with the occurrence of satellites, showing that occurrence of two chromosome pairs with satellites per cell is a characteristic shared by some species from both subgenera. This feature does not always have relationship with the basic chromosome number. The data found in this study will help to understand the phylogeny, cytotaxonomy, and evolution of the genus Passiflora showing that karyotypic variation can be seen between and within the subgenus Decaloba and Passiflora.     

Vacuolar remodelling mediates touch-induced androgynophore movement in Passiflora (Subg. Decaloba, Sect. Xerogona) flowers

Some species of the genus Passiflora (sect. Xerogona) have flowers that exhibit a mechanically-induced movement of the androgynophore that is probably involved in pollination. Despite the many reports of touch-induced movements of floral parts, the studies concerning anatomical, ultrastructural and molecular aspects of the plant movements are restricted to the vegetative parts. Rapid plant movements are highly dependent on turgor changes of a particular flexible tissue formed by specialized cells capable of losing and gaining water rapidly. Thigmotropic androgynophores of four species of Passiflora from section Xerogona were analyzed at cellular and subcellular levels. Our results show that the movement is due to a vacuolar remodelling in a group of parenchymatous cells at the base of the androgynophore. After the movement, plasmolyzed and multivacuolated cells are present at the stimulated side and turgid and univacuolated cells at the opposite side. The results suggest that the mechanisms leading to the androgynophore movement in Passiflora are, in general, analogous to those reported for the movements of legume pulvinus, motile stamen filaments and guard cells of stomata, pointing to conserved cellular mechanisms of plant movement.     

Evolutionary,genetic, environmental and hormonal-induced plasticity in the fate of organs arising from axillary meristems in Passiflora spp.

Tendrils can be found in different plant species. In legumes such as pea, tendrils are modified leaves produced by the vegetative meristem but in the grape vine, a same meristem is used to either form a tendril or an inflorescence. Passiflora species originated in ecosystems in which there is dense vegetation and competition for light. Thus climbing on other plants in order to reach regions with higher light using tendrils is an adaptive advantage. In Passiflora species, after a juvenile phase, every leaf has a subtending vegetative meristem, and a separate meristem that forms both flowers and a tendril. Thus, flowers are formed once a tendril is formed yet whether or not this flower will reach bloom depends on the environment. For example, in Passiflora edulis flowers do not develop under shaded conditions, so that tendrils are needed to bring the plant to positions were flowers can develop. This separate meristem generally forms a single tendril in different Passiflora species yet the number and position of flowers formed from the same meristem diverges among species. Here we display the variation among species as well as variation within a single species, P. edulis. We also show that the number of flowers within a specific genotype can be modulated by applying Cytokinins. Finally, this separate meristem is capable of transforming into a leaf-producing meristem under specific environmental conditions. Thus, behind what appears to be a species-specific rigid program regarding the fate of this meristem, our study helps to reveal a plasticity normally restrained by genetic, hormonal and environmental constraints.     

Taxonomic Identification of Mediterranean Pines and Their Hybrids Based on the High Resolution Melting (HRM) and trnL Approaches: From Cytoplasmic Inheritance to Timber Tracing

Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples.     

Novel polymorphic microsatellite loci for distinguishing rock bass (<Emphasis Type="Italic">Ambloplites rupestris</Emphasis>), Roanoke bass (<Emphasis Type="Italic">Ambloplites cavifrons</Emphasis>), and their hybrids

The rock bass (Ambloplites rupestris) is a popular sport-fish native to the Mississippi and Great Lakes basins of North America. The species has been widely introduced outside its native range, including into Atlantic-slope streams of Virginia where it may hybridize with an imperiled, similar-looking congener, the Roanoke bass (Ambloplites cavifrons). In this study, we identified and evaluated novel molecular markers to facilitate identification of these species and study the extent of hybridization. Using molecular libraries developed from A. rupestris, we identified a suite of candidate nuclear microsatellite loci, synthesized primer sets, and tested these markers for amplification and polymorphism in populations of both species. We then calculated standard diversity statistics within and differentiation statistics between species, the latter providing an indication of marker power for distinguishing the species and their hybrids. Additionally, we evaluated our efficiency for identifying hybrids by classifying simulated genotypes of known ancestry. Eleven loci were polymorphic (2–22 alleles per locus) and reliably amplified in both species. Multilocus genetic differentiation between A. cavifrons and A. rupestris was quite high (F _ST  = 0.66; D _LR  = 19.3), indicating the high statistical power of this marker set for species and hybrid identification. Analyses of simulated data suggested these markers reliably distinguish between hybrids and non-hybrids, as well as between F1 hybrids and backcrossed individuals. This panel of 11 loci should prove useful for understanding patterns of hybridization between A. rupestris and A. cavifrons. As the first microsatellite markers developed for Ambloplites, these markers also should prove broadly useful for population genetic studies of this genus.     

Reproductive isolation and hybridization in sympatric populations of three Dactylorhiza species (Orchidaceae) with different ploidy levels

Background and Aims

The potential for gene exchange between species with different ploidy levels has long been recognized, but only a few studies have tested this hypothesis in situ and most of them focused on not more than two co-occurring species. In this study, we examined hybridization patterns in two sites containing three species of the genus Dactylorhiza (diploid D. incarnata and D. fuchsii and their allotetraploid derivative D. praetermissa).

Methods

To compare the strength of reproductive barriers between diploid species, and between diploid and tetraploid species, crossing experiments were combined with morphometric and molecular analyses using amplified fragment length polymorphism markers, whereas flow cytometric analyses were used to verify the hybrid origin of putative hybrids.

Key Results

In both sites, extensive hybridization was observed, indicating that gene flow between species is possible within the investigated populations. Bayesian assignment analyses indicated that the majority of hybrids were F₁ hybrids, but in some cases triple hybrids (hybrids with three species as parents) were observed, suggesting secondary gene flow. Crossing experiments showed that only crosses between pure species yielded a high percentage of viable seeds. When hybrids were involved as either pollen-receptor or pollen-donor, almost no viable seeds were formed, indicating strong post-zygotic reproductive isolation and high sterility.

Conclusions

Strong post-mating reproductive barriers prevent local breakdown of species boundaries in Dactylorhiza despite frequent hybridization between parental species. However, the presence of triple hybrids indicates that in some cases hybridization may extend the F₁ generation.     

Phenotypic plasticity in chemical defence of butterflies allows usage of diverse host plants

Host plant specialization is a major force driving ecological niche partitioning and diversification in insect herbivores. The cyanogenic defences of Passiflora plants keep most herbivores at bay, but not the larvae of Heliconius butterflies, which can both sequester and biosynthesize cyanogenic compounds. Here, we demonstrate that both Heliconius cydno chioneus and H. melpomene rosina have remarkable plasticity in their chemical defences. When feeding on Passiflora species with cyanogenic compounds that they can readily sequester, both species downregulate the biosynthesis of these compounds. By contrast, when fed on Passiflora plants that do not contain cyanogenic glucosides that can be sequestered, both species increase biosynthesis. This biochemical plasticity comes at a fitness cost for the more specialist H. m. rosina, as adult size and weight for this species negatively correlate with biosynthesis levels, but not for the more generalist H. c. chioneus. By contrast, H. m rosina has increased performance when sequestration is possible on its specialized host plant. In summary, phenotypic plasticity in biochemical responses to different host plants offers these butterflies the ability to widen their range of potential hosts within the Passiflora genus, while maintaining their chemical defences.     

Genetic Diversity in Cymbopogon Species using PCR-Based Functional Markers

Genetic diversity among 25 accessions (involving 8 species, 2 interspecific hybrids and one hybrid mutant) of medicinally important genus Cymbopogon was assessed using 17 PCR-based functional markers, that were designed from members of three different multigene families. We developed 16 primer pairs from two multigene families, 8 primer pairs each from cytochrome P450 and UDP-glucosyltransferase (UGT); one primer pair was derived from 5S rRNA gene family. A total of 119 fragments were visualized, of which 108 (91%) were polymorphic. The level of diversity among different taxa/accessions observed during the present study was, however, low relative to the diversity level obtained due to RAPD markers in two earlier studies. The pattern of genetic diversity neither matched with the known taxonomic classification, nor did it always match with the distribution of chemical constituents of the essential oils available in these accessions. Thus, present investigation though revealed poor correlation between the molecular and chemical diversity, these gene-based markers may prove useful in the development of perfect markers for association mapping of genes involved in controlling agronomically important traits.     

Photosynthetic responses of ornamental passion flower hybrids to varying light intensities

Wild species and hybrids of passion flower are cultivated in many countries for their ornamental value, and used for landscaping as indoor and garden plants. However, knowledge of physiological responses of passion flower to changes in light level is limited. In this paper, the relationship among low (25 %), medium (50 %), and high light level (75 %) and photosynthetic efficiency was investigated on hybrids of Passiflora ‘Aninha’ and Passiflora ‘Priscilla’. Hybrids of P. ‘Aninha’ grown under 25 % showed higher net photosynthetic rate and light-saturated rate of net photosynthesis, while for hybrids of P. ‘Priscilla’ the highest values were observed at 50 %. For P. ‘Aninha’ and P. ‘Priscilla’ the highest level of light (75 %) was not favorable as regards the maximization of stomatal conductance and contributed to reduction in transpiration rate. The values of maximal quantum yield of photosystem II photochemistry indicated efficient conversion of photosynthetically active radiation, being higher at 25 % light level and decreased progressively with the increase in light level. On the whole, leaf gas exchange parameters and chlorophyll fluorescence indicated higher photosynthetic efficiency at low- and medium-light levels for the two studied hybrids. Summing up, hybrids of P. ‘Aninha’ and P. ‘Priscilla’, showed adaptability to the shade, and could be used for landscaping projects of indoor.     

Morphological identification of Lucilia sericata,Lucilia cuprina and their hybrids (Diptera,Calliphoridae)

Hybrids of Lucilia sericata and Lucilia cuprina have been shown to exist in previous studies using molecular methods, but no study has shown explicitly that these hybrids can be identified morphologically. Published morphological characters used to identify L. sericata and L. cuprina were reviewed, and then scored and tested using specimens of both species and known hybrids. Ordination by multi-dimensional scaling indicated that the species were separable, and that hybrids resembled L. cuprina, whatever their origin. Discriminant function analysis of the characters successfully separated the specimens into three unambiguous groups – L. sericata, L. cuprina and hybrids. The hybrids were morphologically similar irrespective of whether they were from an ancient introgressed lineage or more modern. This is the first evidence that hybrids of these two species can be identified from their morphology. The usefulness of the morphological characters is also discussed and photographs of several characters are included to facilitate their assessment.     

Exploring the role of auxin in the androgynophore movement in Passiflora

The flowers of the species belonging to the genus Passiflorashow a range of features that are thought to have arisen as adaptations to different pollinators. Some Passiflora species belonging to the subgenus Decaloba sect. Xerogona, show touch-sensitive motile androgynophores. We tested the role of auxin polar transport in the modulation of the androgynophore movement by applying auxin (IAA) or an inhibitor of auxin polar transport (NPA) in the flowers. We recorded the movement of the androgynophore during mechano-stimulation and analyzed the duration, speed, and the angle formed by the androgynophore before and after the movement, and found that both IAA and NPA increase the amplitude of the movement in P. sanguinolenta. We hypothesize that auxin might have a role in modulating the fitness of these Decaloba species to different pollination syndromes and demonstrate that an interspecific hybrid between insect- and hummingbird-pollinated Xerogona species present a heterosis effect on the speed of the androgynophore movement.     

Determinants of growth rate on chemically heterogeneous host plants by specialist insects

Nine Passiflora species growing sympatrically in a Costa Rican rain forest were screened for the presence of alkaloids, tannins, cyanogenic compounds, saponins and cardenolides. The first three chemical classes were found to be present and the Passiflora species could be classified into five groups according to the presence/absence of each chemical class. In order to assess the effects of this chemical variability on specialist herbivores, larvae of Heliconius ismenius and H. melpomane were experimentally introduced to seven species of Passiflora host plants growing naturally in the rain forest. Survival and growth rates were subsequently monitored at 2 day intervals and were found to be independent of each other. Larval growth rate and larval survivorship were not significantly affected by cyanogenesis in the host plants, nor by the presence of alkaloids, tannins and non-tanning phenolic compounds. Larval growth rate was found to be correlated to percentage nitrogen in the host plants, but was independent of host water content and non-structural carbohydrate content. Although these insects would be classified as host-specific by most authors, the results indicate the presence of a sufficiently general detoxication apparatus to consume many different classes of potential toxins with no apparent ill effects. We conclude that Passiflora natural product diversity does not cause major deleterious effects in Heliconius growth characteristics and that these effects are not the major component maintaining the current pattern of larval feeding specialization in this plant-herbivore system.