J. De Riek

AFLP based alternatives for the assessment of Distinctness, Uniformity and Stability of sugar beet varieties

Abstract  Three approaches for addressing criteria for Distinctness, Uniformity and Stability (DUS) assessment by means of AFLP data are presented. AFLP data were obtained for three consecutive seed deliveries of 15 sugar beet varieties that were under investigation for the official Belgian list (’93, ’94 and ’95). In total, 696 AFLP markers were scored on 1350 plants. As a first approach, a cluster analysis based on Nei’s standard genetic distances between varieties and/or seed deliveries was made. Three major groups put together varieties belonging to corresponding breeding programmes. Statistical procedures, involving bootstrapping and random sampling of subsets of markers, were applied to test the reproducibility of the ordinations and the redundancy present in the data set. In a second approach, the genetic structure inferred by varieties and seed deliveries was submitted to an Analysis of Molecular Variance (AMOVA). Major genetic variation was attributed to individual plant differences within seed deliveries. Differences among seed deliveries seemed to be as important as differences among varieties or breeding programmes. Individual plant data were used for assignment tests. The computation of the assignment was based on the ranking of individual genotypes to one other (based on Jaccard similarity coefficients). The distribution over the accessions for each variety or seed delivery was used to check what group of plants each individual is genetically most similar to. Varieties were classified according to the degree to which the distribution over the different accessions was mainly allocated to their appropriate seed deliveries (from the same variety) or cross- allocated to other varieties. Criteria for DUS-evaluation could be set by each of the approaches; it is discussed in what way the result obtained differs and agrees.

Validation of criteria for the selection of AFLP markers to assess the genetic variation of a breeders' collection of evergreen azaleas

Abstract Fluorescent AFLP and automated data analysis were employed to assess the genetic conformity within a breeders’ collection of evergreen azaleas. The study included 75 genotypes of Belgian pot azaleas (Rhododendron simsii Planch. hybrids), Kurume and Hirado azaleas and wild ancestor species from the Tsutsusi subgenus. Fluorescent detection and addition of an internal size standard to each lane enabled the automated scoring of each fragment arising from a single AFLP primer combination (PC). The use of three PCs generated an initial data set with a total of 648 fragments ranging from 70 bp to 450 bp. Different marker selection thresholds for average fluorescent signal intensity and marker frequency were used to create eight extra restricted data subsets. Pairwise plant genetic similarity was calculated for the nine data sets using the Simple Matching coefficient (symmetrical, including double-zeros) and Jaccard coefficient (asymmetrical, excluding double zeros). The averages, the ranges and the correlation to one other (Mantel analysis) were compared for the obtained similarity matrices. This revealed the sensitivity of ordinations obtained by both similarity coefficients for the presence of weak or intensive markers or for the degree of polymorphism of the markers. For 34 cultivars, pedigree information (at maximum to the fifth ancestor generation) was available. Genetic similarity by descent (kinship coefficient) was turned into a genetic distance and correlated to the genetic conformity, as revealed by the different selections of AFLP markers (Mantel analysis). Use of a Simple Matching coefficient with no or moderate selection to signal intensity and excluding rare and abundant markers gave the best correlation with pedigree. Finally, the ordination of the studied genotypes by means of dendrograms and principal co-ordinate analysis was confronted with known or accepted relationships based on geographical origin, parentage and morphological characters. Genotypes could be assigned to three distinct groups: pot azaleas, Kurume azaleas and Hirado azaleas. Wild ancestor species appeared to be more related to the Japanese azaleas. Intermediate cultivars could be typified as crossings with Kurume or Hirado azaleas or with wild species.

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.

Sugar and starch metabolism during ex vitro rooting and acclimatization of micropropagated Spathiphyllum ‘Petite’ plantlets

Abstract Sugar and starch metabolism during ex vitro rooting and acclimatization of micropropagated Spathiphyllum ‘Petite’ plantlets were studied to uncover in what way photosynthesis and carbohydrate reserves, accumulated during tissue culture, sustain plantlets regrowth and root formation. We followed carbon partitioning between sugars (sucrose, glucose and fructose) and starch and the changes in the activities of the key enzymes involved, including sucrose- P -synthase, sucrose synthase, acid invertase and ADPG pyrophosphorylase. It was concluded that during acclimatization of tissue cultured Spathiphyllum plantlets, photoassimilates accumulate before root emergence. Afterwards, carbohydrates were mainly translocated to these growing sinks. This was supported by the elevated levels of sucrose metabolising enzymes. Sink demands at the end of the light period gradually became saturated when plants were fully acclimatized. This resulted in a down regulation of sucrose- P -synthase and an activation of ADPG pyrophosphorylase, and at that moment a further starch accumulation started.

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).

Genetic relationships among Hibiscus syriacus, Hibiscus sinosyriacus and Hibiscus paramutabilis revealed by AFLP, morphology and ploidy analysis

The genetic relationships between and within Hibiscus syriacus, Hibiscus sinosyriacus and Hibiscus paramutabilis, three winter hardy Hibiscus species native to China, were analysed by the amplified fragment length polymorphisms (AFLP) technique and by morphological evaluation of flower and leaf characteristics. Both methods clearly discriminated between the three species. H. sinosyriacus was classified as an intermediate form between H. paramutabilis and H. syriacus; however, it showed a higher similarity to H. paramutabilis. The different H. syriacus cultivars could be identified by AFLP analysis, while also leaf indices clustered the different cultivars in small groups. Flow cytometry showed that both triploid and tetraploid cultivars occur in the H. syriacus assortment. In general triploid cultivars had larger flowers compared to diploid cultivars.The genetic relationships between and within Hibiscus syriacus, Hibiscus sinosyriacus and Hibiscus paramutabilis, three winter hardy Hibiscus species native to China, were analysed by the amplified fragment length polymorphisms (AFLP) technique and by morphological evaluation of flower and leaf characteristics. Both methods clearly discriminated between the three species. H. sinosyriacus was classified as an intermediate form between H. paramutabilis and H. syriacus; however, it showed a higher similarity to H. paramutabilis. The different H. syriacus cultivars could be identified by AFLP analysis, while also leaf indices clustered the different cultivars in small groups. Flow cytometry showed that both triploid and tetraploid cultivars occur in the H. syriacus assortment. In general triploid cultivars had larger flowers compared to diploid cultivars.

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.

GENETIC DIVERSITY AND POPULATION STRUCTURE OF SCLEROTINIA SPECIES FROM EUROPEAN RED CLOVER CROPS

SUMMARY Sclerotinia trifoliorum Erikks. is an ascomycete fungus that causes rot in red clover crops, a disease difficult to control as no completely resistant red clover varieties are available. Breeding programs aimed at improving the resistance of red clover varieties are hindered by insufficient knowledge about the genetic diversity of S. trifoliorum in Europe. Therefore, the genetic diversity among 192 Sclerotinia isolates from red clover crops from 12 European countries was investigated using mycelial compatibility grouping and AFLP analysis. Phylogenetic analysis revealed that two Sclerotinia species are present on red clover: S. trifoliorum in most locations and S. sclerotiorum in 2 out of 25 locations. Species identification was confirmed using a test based on the β-tubulin gene we constructed. No subpopulations within the S. trifoliorum species were found, nor correlation between genetic and geographical distance. Most of the total variation (79.2%) was detected within S. trifoliorum populations, whereas a lower variation (20.8%) occurred among populations. The average F ST value was 0.208, indicating that S. trifoliorum isolates from red clover should be regarded as one population close to panmixis with a very low degree of population differentiation. Future research would be useful to compare the aggressiveness of S. trifoliorum and S. sclerotiorum on red clover plants and to determine the correlation between the isolates’ aggressiveness and their genetic diversity.

Acclimatization of micropropagated roses in multilayer-cells: effect of different stage III conditions and CO2-enrichment

Recently, interest in the photosynthetic capacities of tissue cultured plants and shoots has been growing. It is often claimed that at the end of the in vitro period the shoots or plants should be as photoautotrophic as possible, so that acclimatization under greenhouse conditions occurs with maximum survival of plants. According to Kozai[6] complete photoautotrophic micropropagation can be a solution. To obtain plants that are able to grow on a sugar free medium, the external CO2-concentration and the light intensities in the culture rooms must be enhanced. Other results [1, 2] suggest that a sufficient carbon reserve in the form of a starch accumulation in the chloroplasts is more important; increased starch accumulation has been observed on media with a higher sucrose content. This carbon reserve can be used during the first days after transplantation to recover and to initiate growth.

Variation and Heritability of α-Linolenic Acid Content and Rumen Escape Protein Fraction in Fodder Grass and Clover

α-Linolenic acid (C18:3) in forages enhances ω-3 fatty acid and conjugated linoleic acid content in milk and meat of ruminants with beneficial effects on human health. An increase of the fraction of rumen escape protein (REP) in grass and clover may reduce nitrogen losses by cattle. To determine the variation of C18:3 and REP, field plot trials under conservation management were set up. The trials included cultivars of perennial ryegrass, Italian ryegrass, meadow fescue, tall fescue, timothy, cocksfoot and red and white clover. For the grasses we found the highest C18:3 content in timothy and the lowest in Italian ryegrass. Tetraploid ryegrass varieties had on average a higher C18:3 content than diploid varieties. The linolenic acid content in white clover was higher than in red clover and the content in both clovers was higher than in the grasses. For all species the C18:3 content was highly positively correlated with the protein content. Among the grasses the REP was highest in cocksfoot and tall fescue and lowest in perennial ryegrass. Diploid ryegrass cultivars had a higher REP fraction than tetraploid cultivars. The REP in red clover was higher than in white clover and the REP in both clovers was lower than in the grasses. REP was highly negatively correlated with dry matter digestibility and the content of digestible protein. For the estimation of the heritability we determined the C18:3 content and REP of 300 single plants of each of the species perennial ryegrass, tall fescue and red and white clover, grown in pots. We carried out positive and negative selections for both parameters in the four species. The results of their offspring suggested a moderate to good heritability of both parameters and opportunities for breeding. Improved grass/clover mixtures may be an important source of C18:3 in ruminant feeding. However because of the negative correlation between REP and the digestible protein content, breeding for improved nitrogen efficiency in cattle by increasing the fraction of rumen escape protein in the forage is not obvious.