Birte Boelt

The development of sequence-tagged sites (STSs) in Lolium perenne L. : the application of primer sets derived from other genera

Abstract Genetic analysis, particularly the development of genetic linkage maps in forage grass species, lags well behind other members of the Poaceae. Comparative mapping within this family has revealed extensive conservation in gene and marker synteny among chromosomes of diverse genera. Recently, the ability to transfer mapped STS markers between barley and wheat has been demonstrated. The transfer of mapped STS markers between cereals and forage grasses could provide PCR-based markers for comparative mapping in these species providing they amplify homologous sequences. In this study, primers derived from three barley genes of defined function and a gene from Phalaris coerulescens were used to amplify homologous fragments in Lolium perenne. Primers derived from two barley and two oat cDNA clones were also tested along with eight barley and two Triticum tauchii STS markers. Twenty one primer pairs derived from 18 loci were tested. Eleven primer pairs (52%) amplified homologous sequences in L. perenne from ten (55%) of the loci targetted. Thirteen new STS markers were generated in L. perenne, of which ten have been mapped in barley or rye and amplify homologous sequences in L. perenne.

Breeding for Grass Seed Yield

Seed yield is a trait of major interest for forage and turf grass species and has received increasing attention since seed multiplication is economically relevant for novel grass cultivars to compete commercially.

Optimal Sample Size for Predicting Viability of Cabbage and Radish Seeds Based on near Infrared Spectra of Single Seeds

The effects of the number of seeds in a training sample set on the ability to predict the viability of cabbage or radish seeds are presented and discussed. The supervised classification method extended canonical variates analysis (ECVA) was used to develop a classification model. Calibration sub-sets of different sizes were chosen randomly with several iterations and using the spectral-based sample selection algorithms DUPLEX and CADEX. An independent test set was used to validate the developed classification models. The results showed that 200 seeds were optimal in a calibration set for both cabbage and radish data. The misclassification rates at optimal sample size were 8%, 6% and 7% for cabbage and 3%, 3% and 2% for radish respectively for random method (averaged for 10 iterations), DUPLEX and CADEX algorithms. This was similar to the misclassification rate of 6% and 2% for cabbage and radish obtained using all 600 seeds in the calibration set. Thus, the number of seeds in the calibration set can be reduced by up to 67% without significant loss of classification accuracy, which will effectively enhance the cost-effectiveness of NIR spectral analysis. Wavelength regions important for the discrimination between viable and non-viable seeds were identified using interval ECVA (iECVA) models, ECVA weight plots and the mean difference spectrum for viable and non-viable seeds.

Effects of elevated CO2 and temperature on seed quality

SUMMARY Successful crop production depends initially on the availability of high-quality seed. By 2050 global climate change will have influenced crop yields, but will these changes affect seed quality? The present review examines the effects of elevated carbon dioxide (CO2) and temperature during seed production on three seed quality components: seed mass, germination and seed vigour. In response to elevated CO2, seed mass has been reported to both increase and decrease in C3 plants, but not change in C4 plants. Increases are greater in legumes than non-legumes, and there is considerable variation among species. Seed mass increases may result in a decrease of seed nitrogen (N) concentration in non-legumes. Increasing temperature may decrease seed mass because of an accelerated growth rate and reduced seed filling duration, but lower seed mass does not necessarily reduce seed germination or vigour. Like seed mass, reported seed germination responses to elevated CO2 have been variable. The reported changes in seed C/N ratio can decrease seed protein content which may eventually lead to reduced viability. Conversely, increased ethylene production may stimulate germination in some species. High-temperature stress before developing seeds reach physiological maturity (PM) can reduce germination by inhibiting the ability of the plant to supply the assimilates necessary to synthesize the storage compounds required for germination. Nothing is known concerning the effects of elevated CO2 on seed vigour. However, seed vigour can be reduced by high-temperature stress both before and after PM. High temperatures induce or increase the physiological deterioration of seeds. Limited evidence suggests that only short periods of high-temperature stress at critical seed development stages are required to reduce seed vigour, but further research is required. The predicted environmental changes will lead to losses of seed quality, particularly for seed vigour and possibly germination. The seed industry will need to consider management changes to minimize the risk of this occurring.

Fructan accumulation and transcription of candidate genes during cold acclimation in three varieties of Poa pratensis.

Poa pratensis, a type species for the grass family (Poaceae), is an important cool season grass that accumulates fructans as a polysaccharide reserve. We studied fructan contents and expression of candidate fructan metabolism genes during cold acclimation in three varieties of P. pratensis adapted to different environments: Northern Norway, Denmark, and the Netherlands. Fructan content increased significantly during cold acclimation and varieties showed significant differences in the level of fructan accumulation. cDNA sequences of putative fructosyltransferase (FT), fructan exohydrolase (FEH), and cold acclimation protein (CAP) genes were identified and cloned. In agreement with a function in fructan biosynthesis, transcription of a putative sucrose:fructan 6-fructosyltransferase (Pp6-SFT) gene was induced during cold acclimation and fructan accumulation in all three P. pratensis varieties. Transcription of putative PpFEH and PpCAP genes was also induced by cold acclimation; however, transcription of these two genes was several-fold higher in the variety from Norway compared to the other two varieties. The results presented here suggest that Pp6-SFT is involved in fructan biosynthesis in P. pratensis. FEHs have previously been suggested to be involved in fructan biosynthesis and freezing tolerance, and induced expression of PpFEH during fructan accumulation could also suggest a role in fructan biosynthesis. However, based on the different PpFEH transcription rates among varieties and similar expression of PpFEH and PpCAP, we suggest that PpFEH is more likely to be involved in mediating freezing tolerance, e.g., by regulating the cell osmotic potential through fructan degradation.

Viability Prediction of Ricinus cummunis L. Seeds Using Multispectral Imaging

The purpose of this study was to highlight the use of multispectral imaging in seed quality testing of castor seeds. Visually, 120 seeds were divided into three classes: yellow, grey and black seeds. Thereafter, images at 19 different wavelengths ranging from 375–970 nm were captured of all the seeds. Mean intensity for each single seed was extracted from the images, and a significant difference between the three colour classes was observed, with the best separation in the near-infrared wavelengths. A specified feature (RegionMSI mean) based on normalized canonical discriminant analysis, were employed and viable seeds were distinguished from dead seeds with 92% accuracy. The same model was tested on a validation set of seeds. These seeds were divided into two groups depending on germination ability, 241 were predicted as viable and expected to germinate and 59 were predicted as dead or non-germinated seeds. This validation of the model resulted in 96% correct classification of the seeds. The results illustrate how multispectral imaging technology can be employed for prediction of viable castor seeds, based on seed coat colour.

Use of partial least squares discriminant analysis on visible-near infrared multispectral image data to examine germination ability and germ length in spinach seeds

Because of the difficulties in obtaining homogenous germination of spinach seeds for baby leaf production, the possibility of using partial least squares discriminant analysis (PLS‐DA) on features extracted from multispectral images of spinach seeds was investigated. The objective has been to discriminate between different seed sizes, as well as to predict germination ability and germ length. Images of 300 seeds including small, medium, and large seeds were taken, and the seeds were examined for germination ability and germ length. PLS‐DA loadings plots were used to reduce the multidimensional image features to a few important features. The PLS‐DA prediction resulted in an independent test set not only providing discrimination of seed size but also demonstrating how germination ability and germ length vary according to seed size. The result indicated that larger seeds had both a significantly higher germination potential and germ length compared with smaller seeds. The variable importance for projection method showed that the near infrared (NIR) wavelength region is important for germination predictability. However, the PLS‐DA model did not improve when only the NIR region was used. Copyright

Fructan metabolism and changes in fructan composition during cold acclimation in perennial ryegrass

Perennial ryegrass (Lolium perenne L.) produces high levels of fructans as a mixture of oligosaccharides and polysaccharides with different degrees of polymerization (DP). The present study describes the analysis of the compositional changes in the full spectrum of fructans, fructan distribution between above ground biomass (top) and the roots, and the transcription of candidate genes involved in fructan metabolism during cold acclimation in perennial ryegrass variety “Veyo” and ecotype “Falster” from distinct geographical origins. We observed changes in fructan composition and induction of low-DP fructans, especially DP = 4, in both the top and the roots of “Veyo” and “Falster” in response to low-temperature stress. The accumulation of DP > 50 fructans was only apparent in the top tissues where the Lp1-FFT expression is higher compared to the roots in both “Veyo” and “Falster.” Our results also show the accumulation and depolymerization of fructans with different DP, together with the induction of genes encoding fructosyltransferases and fructan exohydrolases in both “Veyo” and “Falster” during cold acclimation, supporting the hypothesis that fructan synthesis and depolymerization occurring simultaneously. The ecotype “Falster,” adapted to cold climates, increased total fructan content and produced more DP > 7 fructans in the roots than the variety “Veyo,” adapted to warmer climates. This indicates that high-DP fructan accumulation in roots may be an adaptive trait for plant recovery after abiotic stresses.

Legume Seed Production Meeting Market Requirements and Economic Impacts

The seed is the carrier of the genetic improvements brought about by modern plant breeding, and seed production is carried out in accordance with certification systems to guarantee consistent high quality. In forage legumes, breeding efforts are primarily related to the vegetative development of the plant, although the commercial success of an agronomically superior cultivar is dependent on a reliable supply of competitively priced seed. In seed production of the three most important forage legumes, alfalfa (Medicago sativa L.), white clover (Trifolium repens L.), and red clover (Trifolium pratense L.), crop management techniques are applied to stimulate reproductive development in order to obtain high seed yields. These include a low plant density, manipulation of canopy size to avoid lodging and shading of fruiting organs, synchronization of flowering with pollinating insects as well as controlling pests. High seed yield is correlated to inflorescence density and seed yield per inflorescence, traits which should be selected for in breeding populations as moderate to high heritability has been found. However, seed yield is a genetically complex trait and in the perennial, insect-pollinated forage legumes it is further highly influenced by environmental conditions and crop management factors. Further investigations into the use of plant growth regulators and an improved understanding of the interaction between pollinators and the seed crop might improve future seed yields. There is likely to be an increasing emphasis on the role of forage legumes in producing high-quality meat and milk, combined with the requirement to reduce the environmental footprint of grassland agriculture. A high forage legume seed yield is a prerequisite to meet market requirements for new, improved cultivars and hence achieve the economic impacts of modern plant breeding for a better livelihood and environment.

Changes in Lolium perenne transcriptome during cold acclimation in two genotypes adapted to different climatic conditions

BackgroundActivation of numerous protective mechanisms during cold acclimation is important for the acquisition of freezing tolerance in perennial ryegrass (Lolium perenne L.). To elucidate the molecular mechanisms of cold acclimation in two genotypes (‘Veyo’ and ‘Falster’) of perennial ryegrass from distinct geographical origins, we performed transcriptome profiling during cold acclimation using RNA-Seq.MethodsWe cold-acclimated plants from both genotypes in controlled conditions for a period of 17 days and isolated Total RNA at various time points for high throughput sequencing using Illumina technology. RNA-seq reads were aligned to genotype specific references to identify transcripts with significant changes in expression during cold acclimation.ResultsThe genes induced were involved in protective mechanisms such as cell response to abiotic stimulus, signal transduction, redox homeostasis, plasma membrane and cell wall modifications, and carbohydrate metabolism in both genotypes. ‘Falster’ genotype, adapted to cold climates, showed a stronger transcriptional differentiation during cold acclimation, and more differentially expressed transcripts related to stress, signal transduction, response to abiotic stimulus, and metabolic processes compared to ‘Veyo’. ‘Falster’ genotype also showed an induction of more transcripts with sequence homology to fructosyltransferase genes (FTs) and a higher fold induction of fructan in response to low-temperature stress. The circadian rhythm network was perturbed in the ‘Veyo’ genotype adapted to warmer climates.ConclusionIn this study, the differentially expressed genes during cold acclimation, potentially involved in numerous protective mechanisms, were identified in two genotypes of perennial ryegrass from distinct geographical origins. The observation that the circadian rhythm network was perturbed in ‘Veyo’ during cold acclimation may point to a low adaptability of ‘Veyo’ to low temperature stresses. This study also revealed the transcriptional mechanisms underlying carbon allocation towards fructan biosynthesis in perennial ryegrass.