Johan Van Huylenbroeck

AFLP markers as a tool to reconstruct complex relationships: A case study in Rosa (Rosaceae)

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in Rosa, using UPGMA clustering, Wagner parsimony, and Bayesian inference. All trees were well resolved, but many of the deeper branches were weakly supported. The cluster analysis showed that the rose cultivars can be separated into a European and an Oriental cluster, each being related to different wild species. The phylogenetic analyses showed that (1) two of the four subgenera (Hulthemia and Platyrhodon) do not deserve subgeneric status; (2) section Carolinae should be merged with sect. Cinnamomeae; (3) subsection Rubigineae is a monophyletic group within sect. Caninae, making sect. Caninae paraphyletic; and (4) there is little support for the distinction of the five other subsections within sect. Caninae. Comparison of the trees with morphological classifications and with previous molecular studies showed that all methods yielded reliable trees. Bayesian inference proved to be a useful alternative to parsimony analysis of AFLP data. Because of their genome-wide sampling, AFLPs are the markers of choice to reconstruct (species) relationships in evolutionary complex groups.

Progress in plant protoplast research

Abstract In this review we focus on recent progress in protoplast regeneration, symmetric and asymmetric hybridization and novel technology developments. Regeneration of new species and improved culture techniques opened new horizons for practical breeding in a number of crops. The importance of protoplast sources and embedding systems is discussed. The study of reactive oxygen species effects and DNA (de)condensation, along with thorough phytohormone monitoring, are in our opinion the most promising research topics in the further strive for rationalization of protoplast regeneration. Following, fusion and fragmentation progress is summarized. Genomic, transcriptomic and proteomic studies have led to better insights in fundamental processes such as cell wall formation, cell development and chromosome rearrangements in fusion products, whether or not obtained after irradiation. Advanced molecular screening methods of both genome and cytoplasmome facilitate efficient screening of both symmetric and asymmetric fusion products. We expect that emerging technologies as GISH, high resolution melting and next generation sequencing will pay major contributions to our insights of genome creation and stabilization, mainly after asymmetric hybridization. Finally, we demonstrate agricultural valorization of somatic hybridization through enumerating recent introgression of diverse traits in a number of commercial crops.

Occurrence of viable unreduced pollen in a Begonia collection

Polyploidy is widespread in plants and has played a major role in the evolution and diversification of the plant kingdom. Unreduced (2n) gametes are an interesting tool for polyploidisation and the creation of genetic variation in plant breeding. Especially in ornamentals, polyploidisation can broaden attractive features within a species. A Begonia collection was screened on the occurrence of 2n pollen with the aid of four different techniques: pollen size measurement, flow cytometric analysis of nuclei isolated from germinated and non germinated pollen, investigation of the microsporogenesis and analysis of progeny. In ten of the 70 screened genotypes (B. dregei, B. pearcei, B. ‘Anna Christina’, B. ‘Bubbles’, B. ‘Florence Rita’, B. ‘Orococo’, B. ‘Rubaiyat’, B. ‘Spatflacier’, B. ‘Tamo’ and B276), large pollen were observed with a rather spherical than normal ellipsoidal shape. Flow cytometric data proved that these aberrantly shaped pollen were associated with 2n ploidy levels, although they were not always viable. Meiotic aberrations in these large pollen producers resulted mainly in dyads although also monads, triads and polyads were observed. Successful crosses were obtained with B. dregei, B. ‘Orococo’, and B276 as pollinators; DNA content had increased in all or a part of the progeny. The results show that the occurrence of 2n pollen is not a rare phenomenon in Begonia.

Exploitation of flow cytometry for plant breeding

In this review, the different applications of flow cytometry in plant breeding are highlighted. Four main breeding related purposes can be distinguished for flow cytometry: (i) Characterisation of available plant material, including screening of possible parent plants for breeding programs as well as evaluation of population biodiversity; (ii) Offspring screening after interspecific, interploidy or aberrant crosses; (iii) Ploidy level determination after haploidization and polyploidization treatments and (iv) Particle sorting, that allows separation of plant cells based on morphological or fluorescent characteristics. An overview and discussion of these various applications indicates that flow cytometry is a relatively quick, cheap and reliable tool for many breeding related objectives.

Histogenic analysis of chemically induced mixoploids in Spathiphyllum wallisii

Tetraploids and mixoploids were induced in several Spathiphyllum wallisii genotypes through in vitro application of mitosis inhibitors. Flow cytometry of leaves enabled the identification of sectorial hybrids, whereas microscopic nuclei research combined with root flow cytometry was required to provide insights in the histogenic composition of the mixoploids and to identify periclinal chimeras. Microscopic observation of epidermal or parenchymatic cell areas or the average cell thickness did not allow unequivocal characterization. However, direct visualization of diploid and tetraploid cells was enabled by analysis of nuclear areas. Root analysis was performed by classical fluorescence of metaphase chromosome spreads. This study showed the necessity of microscopic techniques for a thorough characterization of mixoploids. It also demonstrated the potential of the combination of these techniques with flow cytometry to unravel the exact effect of mitosis inhibitors on monocot plants. Finally, it might help to optimize the tetraploidization efficiency.

Change in Auxin and Cytokinin Levels Coincides with Altered Expression of Branching Genes during Axillary Bud Outgrowth in Chrysanthemum

In the production and breeding of Chrysanthemum sp., shoot branching is an important quality aspect as the outgrowth of axillary buds determines the final plant shape. Bud outgrowth is mainly controlled by apical dominance and the crosstalk between the plant hormones auxin, cytokinin and strigolactone. In this work the hormonal and genetic regulation of axillary bud outgrowth was studied in two differently branching cut flower Chrysanthemum morifolium (Ramat) genotypes. C17 is a split-type which forms an inflorescence meristem after a certain vegetative period, while C18 remains vegetative under long day conditions. Plant growth of both genotypes was monitored during 5 subsequent weeks starting one week before flower initiation occurred in C17. Axillary bud outgrowth was measured weekly and samples of shoot apex, stem and axillary buds were taken during the first two weeks. We combined auxin and cytokinin measurements by UPLC-MS/MS with RT-qPCR expression analysis of genes involved in shoot branching regulation pathways in chrysanthemum. These included bud development genes (CmBRC1, CmDRM1, CmSTM, CmLsL), auxin pathway genes (CmPIN1, CmTIR3, CmTIR1, CmAXR1, CmAXR6, CmAXR2, CmIAA16, CmIAA12), cytokinin pathway genes (CmIPT3, CmHK3, CmRR1) and strigolactone genes (CmMAX1 and CmMAX2). Genotype C17 showed a release from apical dominance after floral transition coinciding with decreased auxin and increased cytokinin levels in the subapical axillary buds. As opposed to C17, C18 maintained strong apical dominance with vegetative growth throughout the experiment. Here high auxin levels and decreasing cytokinin levels in axillary buds and stem were measured. A differential expression of several branching genes accompanied the different hormonal change and bud outgrowth in C17 and C18. This was clear for the strigolactone biosynthesis gene CmMAX1, the transcription factor CmBRC1 and the dormancy associated gene CmDRM1, that all showed a decreased expression in C17 at floral transition and an increased expression in C18 with continuous vegetative growth. These results offer a case study for Chrysanthemum, showing an altered cytokinin to auxin balance and differential gene expression between vegetative growth with apical dominance and transition to generative growth with loss of apical dominance and axillary bud outgrowth. This suggests a conservation of several aspects of the hormonal and genetical regulation of bud outgrowth in Chrysanthemum. Furthermore, 15 previously uncharacterised genes in chrysanthemum, were described in this study. Of those genes involved in axillary bud outgrowth we identified CmDRM1, CmBRC1 and CmMAX1 as having an altered expression preceding axillary bud outgrowth, which could be useful as markers for bud activity.

Karyotype analysis and visualization of 45S rRNA genes using fluorescence in situ hybridization in aroids (Araceae).

Abstract Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species Anthurium andraeanum Linden ex André, 1877, Monstera deliciosa Liebmann, 1849, Philodendron scandens Koch & Sello, 1853, Spathiphyllum wallisii Regel, 1877, Syngonium auritum (Linnaeus, 1759) Schott, 1829 and Zantedeschia elliottiana (Knight, 1890) Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendron scandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllum wallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthurium andraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschia elliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies.

Screening for broad mite susceptibility in Rhododendron simsii hybrids

Broad mite Polyphagotarsonemus latus, is a key pest of pot azalea (Rhododendron simsii Planch hybrids). No information on variation in susceptibility or potential tolerance within these plants is available to date. We evaluated a selection of 32 Rhododendron cultivars, mainly R. simsii hybrids, for susceptibility to broad mite. The plants were artificially infested in a greenhouse by surrounding each azalea with four broad mite-infested English ivy plants (Hedera helix). Broad mite infestation was evaluated by counting the number of broad mites per shoot tip and assigning a damage rate. Results indicated a comparable infestation rate expressed as the number of mites on all cultivars at the initial stage of the experiments. Correlations between the average damage rate and the number of broad mites per shoot tip on all cultivars at different time intervals were significantly positive, although low, in each experiment, with R2-values of 0.14 and 0.61. At the end of the experiments significant differences in susceptibility between the evaluated cultivars were observed. The cultivars, ‘Emil De Coninck’ and ‘Mont Blanc’ were rated as the most susceptible, whereas ‘Mistral’ and its bud sport ‘Elien’ were tolerant towards the broad mite, because damage rates were low and very few broad mites were found.

Development and validation of a three‐step detection protocol for broad mites (Polyphagotarsonemus latus) in pot azalea (Rhododendron simsii hybrids)

protocol for broad mites (Polyphagotarsonemus latus) in pot azalea (Rhododendron simsii hybrids) Els Mechant*, Gil Luypaert, Bart Van Delsen, Els Pauwels, Johan Witters, Johan Van Huylenbroeck & Bruno Gobin PCS –Ornamental Plant Research, Schaessestraat 18, 9070 Destelbergen, Belgium, Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Applied Genetics and Breeding, Caritasstraat 21, 9090Melle, Belgium, and Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Crop Protection, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium

Branching gene expression during chrysanthemum axillary bud outgrowth regulated by strigolactone and auxin transport

Shoot branching is essential in ornamental chrysanthemum production and determines final plant shape and quality. Auxin is associated with apical dominance to indirectly inhibit bud outgrowth. Two non-mutually exclusive models exist for indirect auxin inhibition. Basipetal auxin transport inhibits axillary bud outgrowth by limiting auxin export from buds to stem (canalization model) or by increasing strigolactone levels (second messenger model). Here we analyzed bud outgrowth in treatments with auxin (IAA), strigolactone (GR24) and auxin transport inhibitor (NPA) using a split-plate bioassay with isolated chrysanthemum stem segments. Besides measuring bud length, dividing cell percentage was measured with flow cytometry and RT-qPCR was used to monitor expression levels of genes involved in auxin transport (CmPIN1) and signaling (CmAXR2), bud dormancy (CmBRC1, CmDRM1) and strigolactone biosynthesis (CmMAX1, CmMAX3). Treatments over a 5-day period showed bud outgrowth in the control and inhibition with IAA and IAA + GR24. Bud outgrowth in the control coincided with high dividing cell percentage, decreased expression of CmBRC1 and CmDRM1 and increased CmPIN1 expression. Inhibition by IAA and IAA + GR24 coincided with low dividing cell percentage and unchanged or increased expressions of CmBRC1, CmDRM1 and CmPIN1. Treatment with GR24 showed restricted bud outgrowth that was counteracted by NPA. This restricted bud outgrowth was still concomitant with a high dividing cell percentage and coincided with decreased expression of dormancy genes. These results indicate incomplete inhibition of bud outgrowth by GR24 treatment and suggest involvement of auxin transport in the mechanism of bud inhibition by strigolactones, supporting the canalization model.