The Promising Potential of Triploidy in Date Palm (Phoenix dactylifera L.) Breeding.

Date palms are a vital part of oasis ecosystems and are an important source of income in arid and semi-arid areas. Crossbreeding is limited due to the long juvenile stage of date palms and their dioecious nature. The aim of this study was to create triploid date palms to obtain larger and seedless fruits and to increase resilience to abiotic stresses. A tetraploid date palm mutant was crossed with a diploid male palm, yielding hundreds of seeds suspected of containing triploid embryos. Six years after planting, four palms with confirmed triploidy reached maturity. They are phenotypically distinct from diploids, with a thicker rachis, thinner spines, wider and longer midleaf spines, and a longer apical spine. They were classified as sterile bisexual, sterile male and fertile female. One of the latter produced very tasty dates with a very small seed, which is promising for the marketability and profitability of date palm fruits. This first report on triploid date palms provides a way in which to make a significant leap forward in date palm breeding. Given the vigor and fruit quality of female triploid date palms, compared to their diploid counterparts, they will be the target of breeding programs and may spearhead new oases.

Unravelling hybridization in Phytophthora using phylogenomics and genome size estimation.

The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors. Phytophthora hybrids therefore pose a serious threat to agriculture as well as to natural ecosystems. Early and correct identification of hybrids is therefore essential for adequate plant protection but this is hampered by the limitations of morphological and traditional molecular methods. Identification of hybrids is also important in evolutionary studies as the positioning of hybrids in a phylogenetic tree can lead to suboptimal topologies. To improve the identification of hybrids we have combined genotyping-by-sequencing (GBS) and genome size estimation on a genus-wide collection of 614 Phytophthora isolates. Analyses based on locus- and allele counts and especially on the combination of species-specific loci and genome size estimations allowed us to confirm and characterize 27 previously described hybrid species and discover 16 new hybrid species. Our method was also valuable for species identification at an unprecedented resolution and further allowed correct naming of misidentified isolates. We used both a concatenation- and a coalescent-based phylogenomic method to construct a reliable phylogeny using the GBS data of 140 non-hybrid Phytophthora isolates. Hybrid species were subsequently connected to their progenitors in this phylogenetic tree. In this study we demonstrate the application of two validated techniques (GBS and flow cytometry) for relatively low cost but high resolution identification of hybrids and their phylogenetic relations.

Can Knowledge of Genetic Distances, Genome Sizes and Chromosome Numbers Support Breeding Programs in Hardy Geraniums?

Breeding programs in ornamentals can be facilitated by integrating knowledge of phylogenetic relatedness of potential parents along with other genomic information. Using AFLP, genetic distances were determined for 59 Geranium genotypes, comprising 55 commercial cultivars of the three subgenera of a total collection of 61 Geranium genotypes. A subgroup of 45 genotypes, including intragroup and intergroup hybrids, were selected and further characterized for genome sizes and chromosome numbers. The variation in genome size ranged from 1.51 ± 0.01 pg/2C to 12.94 ± 0.07 pg/2C. The chromosome numbers ranged from 26 to 108-110 with some hybrids showing an aberrant number of chromosomes based on their parents' constitution. All chromosome numbers of Geranium are an even number, which presumes that unreduced gametes occur in some cross combinations. Overall, parental difference in genome size and chromosome number were not limiting for cross compatibility. Good crossing compatibility was correlated to a Jaccard similarity coefficient as parameter for parental relatedness of about 0.5. Additionally, parent combinations with high differences in the DNA/chromosome value could not result in a successful cross. We expect that our results will enable breeding programs to overcome crossing barriers and support further breeding initiatives.

Hybridization of Oxalis corniculata and O. dillenii in their non-native range.

Many species have been introduced beyond their native ranges and many have become global weeds. Human mediated dispersal has removed the geographic isolation of these species, reversing millions of years of independent evolution. Examples are the Oxalis species in section Corniculatae where several species have become invasive. Here we characterize and formally describe a hybrid between O. dillenii and O. corniculata, which occurs spontaneously in Belgium and Japan. Oxalis corniculata is probably native to Japan, but both species are alien to Belgium and O. dillenii is native to North America. We formally name this hybrid as Oxalis × vanaelstii. Although this hybrid is sterile, it is nevertheless vigorous and perennial. Both parent species grow as weeds in gardens; therefore, it is likely to be more common than currently appreciated in countries where these species co-occur.

Evaluation of genome size and quantitative features of the dolipore septum as taxonomic predictors for the Serendipita 'williamsii' species complex.

Despite multiple taxonomic revisions, several uncertainties at the genus and species level remain to be resolved within the Serendipitaceae family (Sebacinales). This volatile classification is attributed to the limited number of available axenic cultures and the scarcity of useful morphological traits. In the current study, we attempted to discover alternative taxonomic markers not relying on DNA sequences to differentiate among the closely related members of our Congolese Serendipita isolate collection and the reference strains S. indica (syn. Piriformospora indica) and S. williamsii (syn. P. williamsii). We demonstrated that nuclear distribution across hyphal cells and genome size (determined by flow cytometry) did not have enough resolving power, but quantitative and qualitative variations in the ultrastructure of the dolipore septa investigated by transmission electron microscopy did provide useful markers. Multivariate analysis revealed that subtle differences in ultrastructural characteristics of the parenthesome and the attached endoplasmic reticulum are most relevant when studying this fungal group. Moreover, the observed clustering pattern showed that there might be more diversity amongst the Congolese isolates within the S. 'williamsii' species complex than previously anticipated based on molecular data. Altogether, our results provide novel perspectives on the use of integrative approaches to support sebacinoid and Serendipitaceae taxonomy.

Artificial Chromosome Doubling in Allotetraploid Calendula officinalis.

Calendula officinalis L. is known as an ornamental plant as well as a source of biochemical compounds used in cosmetics and industry. C. officinalis has a complex karyotype. Published chromosome numbers differ between 2n = 4x = 28 or 32. We have estimated genome sizes in nine commercial cultivars and evaluated the ploidy level by karyotyping and fluorescent in situ hybridization (FISH) using 5S and 45S rDNA loci. The detection of chromosome sets of two rather than four homologues would suggest that C. officinalis has an allotetraploid background. In addition, four signals for 45S but only two for 5S were found by using FISH. Artificial chromosome doubling is a common technique in plant breeding, as polyploidization results in several consequences for plant growth and development. Especially the suggested allotetraploid background in C. officinalis is interesting when examining the effect of chromosome doubling on the plant phenotype. Here we describe chromosome doubling of three allotetraploid cultivars of C. officinalis, 'Nova,' 'WUR 1553-7' and 'Orange Beauty'. Three antimitotic agents - colchicine, oryzalin and trifluralin - were used in different concentrations to find the combination of the best agent and the best dosage to obtain octaploids. For all three cultivars a few octaploids were obtained. A concentration of 200 and 400 ppm of colchicine was most efficient for chromosome doubling in 'Nova' and 'Orange Beauty,' respectively. For 'WUR 1553-7' the treatment with 20 ppm oryzalin was also effective. Cell numbers and first observations of the phenotype in the chromosome doubled plants show thicker leaves and bigger cells, as commonly observed after ploidy doubling. Due to the low number of chromosome doubled plants obtained more elaborate phenotyping will be performed on following generations cultivated under field conditions.

Cold Acclimation and Deacclimation of Two Garden Rose Cultivars Under Controlled Daylength and Temperature.

Low temperature stress is an important abiotic stress for garden roses in northern regions. Two garden rose cultivars ('Dagmar Hastrup' and 'Chandos Beauty') were selected to study the role of dehydrin and of carbohydrate metabolism during cold acclimation and deacclimation under the controlled daylength and temperature. The presence of bud dormancy was also observed as this could prevent budburst during warm spells. Both cultivars showed a similar changing pattern of cold acclimation and deacclimation and did not differ in their lowest LT50 values. Dehydrin (RhDHN5) was up-regulated by low temperatures and not by dehydration stress as the stem water content remained stable during the treatments. Total soluble sugars accumulated with a transient up-regulation of RhBAM3 (a key gene in starch hydrolysis) for 'Dagmar Hastrup' at 2°C and a strong expression under both 2 and -3°C for 'Chandos Beauty'. At 2 and -3°C, raffinose and stachyose strongly accumulated though the up-regulation of RhRS6 and RhGK differed in the cultivars. Although similar cold hardiness levels were reached, carbohydrate metabolism in response to cold stress is different in the two cultivars. Increasing the temperature after a cold period resulted in fast deacclimation as found by the downregulation of RhDHN5 and RhBAM3, the decrease of raffinose and stachyose. Bud endodormancy was hardly present in both cultivars.

Somaclonal Variation in Chrysanthemum × morifolium Protoplast Regenerants.

Chrysanthemum × morifolium protoplasts were isolated and regenerated to assess possible protoclonal variation in the regenerants. After a preliminary screening of the potential of different regeneration systems for protoplast regeneration, we produced a series of cut chrysanthemum 'Arjuna' leaf protoplast regenerants through liquid culture. Regenerants (54) were vegetatively propagated and grown under a commercial production system in 2 different seasons. All screened regenerants were significantly affected with regard to either flower number, flower size, flower weight, leaf weight, stalk weight, or plant size. A significant plant size reduction in 43/52 and 48/49 regenerants for both seasons was the most recorded effect. Also a reduction in flowering induction time up to 10 days, altered flower types and colors were observed. Differences between growing seasons were notable. Possible molecular backgrounds including genome size variation and commercial applications in breeding of chrysanthemum are discussed.

In the name of the rose: a roadmap for rose research in the genome era.

The recent completion of the rose genome sequence is not the end of a process, but rather a starting point that opens up a whole set of new and exciting activities. Next to a high-quality genome sequence other genomic tools have also become available for rose, including transcriptomics data, a high-density single-nucleotide polymorphism array and software to perform linkage and quantitative trait locus mapping in polyploids. Rose cultivars are highly heterogeneous and diverse. This vast diversity in cultivated roses can be explained through the genetic potential of the genus, introgressions from wild species into commercial tetraploid germplasm and the inimitable efforts of historical breeders. We can now investigate how this diversity can best be exploited and refined in future breeding work, given the rich molecular toolbox now available to the rose breeding community. This paper presents possible lines of research now that rose has entered the genomics era, and attempts to partially answer the question that arises after the completion of any draft genome sequence: 'Now that we have "the" genome, what's next?'. Having access to a genome sequence will allow both (fundamental) scientific and (applied) breeding-orientated questions to be addressed. We outline possible approaches for a number of these questions.

Seasonal changes in cold hardiness and carbohydrate metabolism in four garden rose cultivars.

We studied metabolic adaptations to cold stress in roses and identified genes in the carbohydrate pathway during acclimation and deacclimation. A field experiment with four rose cultivars belonging to different USDA plant hardiness zones was set up in Melle, Belgium (51° 0' N, 3° 48' E). The more cold-hardy cultivars ('Dagmar Hastrup' and 'John Cabot') reached their lowest LT50 value in December, indicating a rapid acclimation after the first occurrence of frost. Less cold-hardy cultivars ('Abraham Darby' and 'Chandos Beauty') reached their lowest LT50 in January/February when exposed to prolonged freezing temperatures. A cell dehydration pattern was found in the less cold-hardy cultivars 'Abraham Darby' and 'Chandos Beauty'. The expression of dehydrins (RhDHN5 and RhDNH6) was up-regulated during November-January. Carbohydrate metabolism is highly involved in cold acclimation in roses. Starch decreased from November towards January in all four cultivars and the hydrolysis of starch by the ß-amylolytic pathway (BAM, DPE2) was identified in 'Dagmar Hastrup' from November to January. Oligosaccharides correlated with cold hardiness in three cultivars although no significant upregulation in RhMIPS and RhRS6, key genes in their biosynthesis, was found. Higher sucrose levels were found during acclimation in hardy cultivars, although transcript levels of RhINV2 was more prominent in 'Chandos Beauty'.

Prediction of Lime Tolerance in Rhododendron Based on Herbarium Specimen and Geochemical Data.

Rhododendrons are typically known to be calcifuges that cannot grow well in lime soils. Data on lime tolerance of different taxa in Rhododendron are scarce. Habitats of naturally distributed specimens of genus Rhododendron were compiled as Chinese text-based locations from the Chinese Virtual Herbarium. The locations were then geocoded into latitude/longitude pairs and subsequently connected to soil characteristics including pH and CaCO3 from the Harmonized World Soil Database (HWSD). Using the upper quartile values of pH > 7.2 and CaCO3 > 2% weight in topsoil as threshold, we predicted the lime tolerant taxa. A dataset of 31,146 Rhododendron specimens including the information on taxonomy, GPS locations and soil parameters for both top- and subsoil was built. The majority of the specimens were distributed in soils with moderately acidic pH and without presence of CaCO3. 76 taxa with potential lime tolerance were predicted out of 525 taxa. The large scale data analysis based on combined data of geocoded herbarium specimens and HWSD allows identification of valuable Rhododendron species, subspecies or botanical varieties with potential tolerance to lime soils with higher pH. The predicted tolerant taxa are valuable resources for an in-depth evaluation of lime tolerance or for further use in horticulture and breeding.

The Variable Effect of Polyploidization on the Phenotype in Escallonia.

To induce new variation within the Escallonia genus, chromosome doubling was performed in E. rubra, E. rosea, and E. illinita, three important species within this genus of mainly evergreen woody ornamental species. Obtained tetraploids and diploid controls were analyzed for rooting capacity, leaf and flower characteristics, and plant architecture using image analysis and cold tolerance. In the present study, a breeders' collection of 23 accessions was characterized cytogenetically and described morphologically. All analyzed species and cultivars were diploid (2n = 2x = 24), with exception of E. pendula, a tetraploid. Today, breeding in Escallonia is limited to lucky finds in seedling populations and few efforts in interspecific hybridization. Three selected Escallonia species underwent an in vitro chromosome doubling with both oryzalin and trifluralin applied as either a continuous or shock treatment. The treatments successfully induced polyploids in all three species. Image analysis revealed that tetraploid E. rosea had decreased shoot length (from 3.8 to 1.3 cm), higher circularity and more dense growth habit compared to diploids. No significant changes in cold tolerance were seen. Tetraploid E. illinita did not differ in shoot length, but an increased outgrowth of axillary buds on the main axis led to denser plants. For tetraploid E. rubra, an increase in plant height (from 4.9 to 5.5 cm) was observed together with a large decrease in circularity and density due to a more polar outgrowth of branches on the main axis. E. rubra tetraploids bore larger flowers than diploids and had an increased cold tolerance (from -7.7 to -11.8°C). Leaf width and area of tetraploids increased for both E. illinita and E. rubra, while a decrease was seen in E. rosea genotypes. For all three species, the rooting capacity of the tetraploids did not differ from the diploids. We conclude that the effect of polyploidization on Escallonia was highly variable and species dependent.

Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves.

This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [(2)H6] (+)-2-cis-4-trans-abscisic acid, [(2)H6] (±)-jasmonic acid, and [(2)H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm(3) Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 µg/g, 0.00015 and 0.00030 µg/g, and 0.0089 and 0.018 µg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.

Host adaptation and speciation through hybridization and polyploidy in Phytophthora.

It is becoming increasingly evident that interspecific hybridization is a common event in phytophthora evolution. Yet, the fundamental processes underlying interspecific hybridization and the consequences for its ecological fitness and distribution are not well understood. We studied hybridization events in phytophthora clade 8b. This is a cold-tolerant group of plant pathogenic oomycetes in which six host-specific species have been described that mostly attack winter-grown vegetables. Hybrid characterization was done by sequencing and cloning of two nuclear (ITS and Ypt1) and two mitochondrial loci (Cox1 and Nadh1) combined with DNA content estimation using flow cytometry. Three different mtDNA haplotypes were recovered among the presumed hybrid isolates, dividing the hybrids into three types, with different parental species involved. In the nuclear genes, additivity, i.e. the presence of two alleles coming from different parents, was detected. Hybrid isolates showed large variations in DNA content, which was positively correlated with the additivity in nuclear loci, indicating allopolyploid hybridization followed by a process of diploidization. Moreover, indications of homeologous recombination were found in the hybrids by cloning ITS products. The hybrid isolates have been isolated from a range of hosts that have not been reported previously for clade 8b species, indicating that they have novel pathogenic potential. Next to this, DNA content measurements of the non-hybrid clade 8b species suggest that polyploidy is a common feature of this clade. We hypothesize that interspecific hybridization and polyploidy are two linked phenomena in phytophthora, and that these processes might play an important and ongoing role in the evolution 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 F(1) 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 F(1) generation.

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status.

Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology.

Patterns of hybridization between diploid and derived allotetraploid species of Dactylorhiza (Orchidaceae) co-occurring in Belgium.

PREMISE OF THE STUDY: Although the potential for gene flow between species with large differences in chromosome numbers has long been recognized, only few studies have thoroughly investigated in situ hybridization across taxa with different ploidy levels. We combined morphological, cytological, and genetic marker data with pollination experiments to investigate the degree, direction, and spatial pattern of hybridization between the diploid Dactylorhiza incarnata and its tetraploid derivative, D. praetermissa. METHODS: To identify hybrids, 169 individuals were genotyped using AFLPs and morphologically characterized. Individuals were clustered on the basis of their AFLP profile using the program Structure. To reduce the dimensionality of the plant-trait matrix, PCA was applied. The origin of suspected hybrid individuals was verified using flow cytometry. An AMOVA and spatial autocorrelation analysis were used to indirectly infer the extent of gene flow. KEY RESULTS: Only five individuals were regarded as putative hybrids on the basis of the AFLP data; all had been assigned to the D. praetermissa morphotype. Only one had deviating DNA content and was presumably a triploid. High Φ(ST) values between different subpopulations and significant spatial genetic structure were observed, suggesting localized gene flow. CONCLUSIONS: Using combined data to study hybridization between D. incarnata and D. praetermissa, very few unequivocal hybrids were observed. We propose several non-mutually exclusive explanations. Localized pollen flow, in combination with different microhabitat preferences, is probably one of the reasons for the low frequency of hybrids. Also, the triploid first-generation hybrids may experience difficulties in successful establishment, as a result of genic incompatibilities.

Aberrant genome size and instability of Phytophthora ramorum oospore progenies.

The functionality of the sexual cycle in the heterothallic pathogen Phytophthora ramorum, causal agent of Sudden Oak Death, has recently been demonstrated. Sexual reproduction could create genotypic variation and increase the pathogen's ability to adapt to other host plants or changing environments. Genetic characterization using co-dominant microsatellite markers and flow cytometry of single-oospore progeny of crosses between a European A1 isolate and North American or European A2 isolates revealed a considerable number of non-Mendelian inheritance events. This includes inheritance of more than two alleles at a locus and non-inheritance of alleles from one parent at another locus. The progenies were mitotically unstable: zoospore and hyphal tip derivatives of the progenies showed genotypic rearrangements and phenotypic variation. Flow cytometry confirmed variation and instability in DNA content of the single-oospore progenies. This indicates that single-oospore progenies not only display aberrant genomic and phenotypic variation due to meiotic irregularities, but also extra variation as a result of post-meiotic genomic rearrangements.

Genome size variation in Begonia.

The genome sizes of a Begonia collection comprising 37 species and 23 hybrids of African, Asiatic, Middle American, and South American origin were screened using flow cytometry. Within the collection, 1C values varied between 0.23 and 1.46 pg DNA. Genome sizes were, in most cases, not positively correlated with chromosome number, but with pollen size. A 12-fold difference in mean chromosome size was found between the genotypes with the largest and smallest chromosomes. In general, chromosomes from South American genotypes were smaller than chromosomes of African, Asian, or Middle American genotypes, except for B. boliviensis and B. pearcei. Cytological chromosome studies in different genotypes showed variable chromosome numbers, length, width, and total chromosome volume, which confirmed the diversity in genome size. Large secondary constrictions were present in several investigated genotypes. These data show that chromosome number and structure exhibit a great deal of variation within the genus Begonia, and likely help to explain the large number of taxa found within the genus.