A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world's most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line ('HapOB') from Rosa chinensis 'Old Blush' and generated a rose genome assembly anchored to seven pseudo-chromosomes (512 Mb with N50 of 3.4 Mb and 564 contigs). The length of 512 Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

Cold storage to overcome dormancy affects the carbohydrate status and photosynthetic capacity of Rhododendron simsii.

Global warming leads to increasing irregular and unexpected warm spells during autumn, and therefore natural chilling requirements to break dormancy are at risk. Controlled cold treatment can provide an answer to this problem. Nevertheless, artificial cold treatment will have consequences for carbon reserves and photosynthesis. In this paper, the effect of dark cold storage at 7 °C to break flower bud dormancy in the evergreen Rhododendron simsii was quantified. Carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht'), semi-early ('M. Marie') and late ('Mw. G. Kint') flowering cultivar showed that carbon loss due to respiration was lowest in 'M. Marie', while 'Mw. G. Kint' was completely depleted of starch reserves at the end of cold treatment. Gene isolation resulted in a candidate gene for sucrose synthase (SUS) RsSus, which appears to be homologous to AtSus3 and had a clear increase in expression in leaves during cold treatment. Photosynthesis measurements on 'Nordlicht' and the late-flowering cultivar 'Thesla' showed that during cold treatment, dark respiration decreased 58% and 63%, respectively. Immediately after cold treatment, dark respiration increased and stabilised after 3 days. The light compensation point followed the same trend as dark respiration. Quantum efficiency showed no significant changes during the first days after cold treatment, but was significantly higher than in plants with dormant flower buds at the start of cold treatment. In conclusion, photosynthesis stabilised 3 days after cold treatment and was improved compared to the level before cold treatment.

Cold storage to overcome dormancy affects the carbon balance of azalea.

Flower bud dormancy in azalea (Rhododendron simsii) is broken by artificial cold treatment and this will have its consequences on carbon reserves and photosynthesis. The effect of cold storage at 7 °C on carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht') and semi-early ('M. Marie) flowering cultivar was quantified. Carbon loss due to respiration was lowest for 'M. Marie'. Photosynthetic measurements on 'Nordlicht' showed that photosynthesis 3 days after cold treatment (plants ready to flower) was improved compared to before cold treatment (plants with dormant flower buds).

Lipid metabolism in mixtures of red clover (Trifolium repens) and perennial ryegrass (Lolium perenne) in lab scale silages and in vitro rumen incubations.

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.

The origin of metamitron resistant Chenopodium album populations in sugar beet.

Chenopodium album L. is a major weed in spring-planted crops in the temperate regions of the world. Since 2000, farmers have reported an unsatisfactory control of this weed in sugar beet fields in Belgium, France and The Netherlands. Frequently, the surviving C. album plants are resistant to metamitron, a key herbicide in this crop. Metamitron resistance in C. album is caused by a Ser264 to Gly mutation in the psbA gene on the chloroplast genome, which prevents binding of metamitron to its target site. This mutation causes also resistance to other herbicides with a similar mode of action, like metribuzin -applied in potato- and atrazine in particular. Atrazine has been applied very frequently in maize in the 1970s and the 1980s, but is now banned in Europe due to environmental reasons. The persistent use of atrazine in maize confronted Belgian and other European farmers in the early 1980s with atrazine resistant C. album with the same Ser264 to Gly mutation. The problems with atrazine resistant C. album disappeared when other herbicides were applied in maize. Unfortunately, this is not the case for metamitron resistant C. album in sugar beet, because no replacement herbicide is readily available. The history of atrazine use in maize brought up a question concerning the origin of the current metamitron resistant C. album populations. Have these populations been selected locally by regular use of metamitron in sugar beet or did the selection occur earlier by atrazine use when maize was grown in the same fields? This would have serious implications regarding the reversibility of herbicide resistance. Therefore, soil samples were collected on 16 fields with different histories: five fields with an organic management over 25 years, two fields with a history of atrazine resistant C. album, five fields with metamitron resistant C. album in sugar beet and four fields which were under permanent grassland for 10 years, preceded by a regular rotation in which sugar beet was a key crop. The seeds of C. album were extracted from the soil and germinated on a germination table. Germinated seeds were allowed to grow in a growth chamber. Metamitron resistance was determined by a chlorophyll fluorescence test and leaf material was sampled for AFLP-analysis. For all fields, estimations were made of the size of the seed bank (i.e. an indirect estimate of population size), the frequency of resistant plants and the genetic diversity of resistant and susceptible populations. The results indicate that herbicide-resistant C. album populations are persistent and maintain their adaptive capacity, challenging future management of metamitron resistant C. album.

Local spread of metamitron resistant Chenopodium album L. patches.

Molecular markers can provide valuable information on the spread of resistant weed biotypes. In particular, tracing local spread of resistant weed patches will give details on the importance of seed migration with machinery, manure, wind or birds. This study investigated the local spread of metamitron resistant Chenopodium album L. patches in the southwest region of the province West-Flanders (Belgium). During the summer of 2009, leaf and seed samples were harvested in 27 patches, distributed over 10 sugar beet fields and 1 maize field. The fields were grouped in four local clusters. Each cluster corresponded with the farmer who cultivated these fields. A cleaved amplified polymorphic sequence (CAPS) procedure identified the Ser264 to Gly mutation in the D1 protein, endowing resistance to metamitron, a key herbicide applied in sugar beet. The majority of the sampled plants within a patch (97% on average) carried this mutation. Amplified fragment length polymorphism (AFLP) analysis was performed with 4 primer pairs and yielded 270 molecular markers, polymorphic for the whole dataset (303 samples). Analysis of molecular variance revealed that a significant part of the genetic variability was attributed to variation among the four farmer locations (12 %) and variation among Chenopodium album patches within the farmer locations (14%). In addition, Mantel tests revealed a positive correlation between genetic distances (linearised phipt between pairs of patches) and geographic distances (Mantel-coefficient significant at p = 0.002), suggesting isolation-by-distance. In one field, a decreased genetic diversity and strong genetic relationships between all the patches in this field supported the hypothesis of a recent introduction of resistant biotypes. Furthermore, genetic similarity between patches from different fields from the same farmer and from different farmers indicated that seed transport between neighbouring fields is likely to have an important impact on the spread of metamitron resistant biotypes.

Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation.

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.

Diversity study on Sclerotinia trifoliorum Erikks., the causal agent of clover rot in red clover crops (Trifolium pratense L.).

Since the 16th century, red clover has been an important crop in Europe. Since the 1940s, the European areal of red clover has been severely reduced, due to the availability of chemical fertilizers and the growing interest in maize. Nowadays there is a growing interest in red clover again, although some setbacks still remain. An important setback is the low persistence of red clover crops. Clover rot, caused by the ascomycete fungus Sclerotinia trifoliorum Erikss., is a major disease in Europe and reduces the persistence of red clover crops severely. The fungus infects clover plants through ascospores in the autumn, the disease develops during the winter and early spring and can kill many plants in this period. In early spring, black sclerotia, serving as surviving bodies, are formed on infected plants. Sclerotia can survive up to 7 years in the soil (Ohberg, 2006). The development of clover rot is highly dependent on the weather conditions: a humid fall, necessary for the germination of the ascospores and an overall warm winter with short periods of frost are favourable for the disease. Cold and dry winters slow the mycelial growth down too much and prevent the disease from spreading. Clover rot is difficult to control and completely resistant red clover varieties have yet to be developed. Because of the great annual variation in disease severity, plant breeders cannot use natural infection as an effective means to screen for resistant material. Breeding for resistant cultivars is being slowed down by the lack of a bio-test usable in breeding programs. When applying artificial infections, it is necessary to have an idea of the diversity of the pathogen. A diverse population will require resistance screening with multiple isolates. The objective of this research is to investigate the genetic diversity among isolates from the pathogen S. trifoliorum from various European countries. We assessed diversity using a species identification test based on the sequence of the beta-tubulin gene, vegetative compatibility grouping and AFLP.

In vitro study of red clover polyphenol oxidase activity, activation, and effect on measured lipase activity and lipolysis.

The goal of this paper was, first, to study the effect of red clover polyphenol oxidase (PPO) activity on protein-bound phenols and measured lipase activity in vitro and, second, to study the effect of PPO activation, measured as an increase in protein-bound phenols, as a result of degrees of damaging (not damaged, crushed, and freeze/thawed) of red clover before wilting on measured enzyme activity and in vitro lipid metabolism when incubated in a phosphate buffer. There was a positive relation between PPO activity and the occurrence of protein-bound phenols with a concomitant decrease in measured lipase activity, indicating a possibility to a direct inhibition of enzymes as a result of protein-bound phenols. Furthermore, damaging can activate PPO in red clover, measured as an increase in protein-bound phenols during wilting [0.7-20.6 nmol of tyrosine equiv (mg of protein)(-1)], again with a concomitant decrease in measured lipase activity [41.3-20.3 mumol of p-nitrophenyl butyrate (PNPB) min(-1) (mg of protein)(-1)]. Lipid metabolism during incubation of these forages in a phosphate buffer with ascorbic acid was only influenced by damaging when wilted for 24 h, with a lower lipolysis in crushed and freeze/thawed (52.9 and 32.6%, respectively, after 8 h of incubation) material compared to all other treatments (on average 60.4% after 8 h of incubation).

Characterisation of powdery mildew resistance in a segregating diploid rose population.

Powdery mildew (Podosphaera pannoso) is one of the most serious fungal diseases on both greenhouse and field grown roses. Improvement of disease resistance is a major selection aim for garden rose breeders. For rose cultivars, being mostly tetraptoid, it is complicated to develop molecular markers for resistance. Hence, a segregating diploid population was established from a cross between 'Yesterday', a commercial available rose variety susceptible to powdery mildew, and R. wichurana, a rose species with resistance to certain isolates of powdery mildew. A progeny of 94 seedlings was planted in the field. The segregation of powdery mildew resistance was studied in this population by means of a bioassay with two different monoconidial isolates of powdery mildew. Based on the response to these inoculations different groups were selected: a first group of genotypes was susceptible to both isolates, other groups were susceptible to one of both isolates and a last group was resistant to both tested isolates. The disease resistance inherits for both isolates in a quantitative way. A genetic map based on AFLP and SSR markers was established and will be used for QTL analysis of powdery mildew resistance.

The use of relative quantitative RT-PCR for expression analysis in azalea flower color sports.

The fastest way to create new azalea (Rhododendron simsii hybrids) cultivars is by making use of flower colour sports, which appear spontaneously on azalea plants. Unfortunately, there is still very little known on how bud sport induction occurs. Therefore, genes coding for two key enzymes of the azalea flavonoid biosynthesis pathway, chalcon synthase (chs) and dihydroflavonol 4-reductase (dfr) that were reported before to be apt for modification by the action of bud sporting, were isolated and characterized. The expression of these two flower colour genes in the petals of azalea flowers will be compared between all 'Hellmut Vogel' flower colour sports. To measure the expression levels of both genes, relative quantitative RT-PCR analysis will be worked out on a real-time PCR machine. The expression of housekeeping genes, which is expected to be the same for all sports, will be used to calculate the relative expression level of the two genes of interest. The optimisation of this technique will be discussed.

Characterization of carotenoid biosynthesis in Rhododendron luteum.

In the color chart of Rhododendron simsii hybrids (Belgian pot azalea), yellow is not yet present, although this color is wanted by growers and consumers. Carotenoid pigments, which are absent in azalea, are responsible for this yellow color of the flower petals. The first objective of this project is characterizing the pathway of carotenoid biosynthesis in the yellow flowers of Rhododendron luteum.