Isabel Roldán-Ruiz

EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass ( Lolium spp.)

BackgroundGenetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.ResultsA set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.ConclusionsThe consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses.

Progress on Optimizing Miscanthus Biomass Production for the European Bioeconomy: Results of the EU FP7 Project OPTIMISC

This paper describes the complete findings of the EU-funded research project OPTIMISC, which investigated methods to optimize the production and use of miscanthus biomass. Miscanthus bioenergy and bioproduct chains were investigated by trialing 15 diverse germplasm types in a range of climatic and soil environments across central Europe, Ukraine, Russia, and China. The abiotic stress tolerances of a wider panel of 100 germplasm types to drought, salinity, and low temperatures were measured in the laboratory and a field trial in Belgium. A small selection of germplasm types was evaluated for performance in grasslands on marginal sites in Germany and the UK. The growth traits underlying biomass yield and quality were measured to improve regional estimates of feedstock availability. Several potential high-value bioproducts were identified. The combined results provide recommendations to policymakers, growers and industry. The major technical advances in miscanthus production achieved by OPTIMISC include: (1) demonstration that novel hybrids can out-yield the standard commercially grown genotype Miscanthus x giganteus; (2) characterization of the interactions of physiological growth responses with environmental variation within and between sites; (3) quantification of biomass-quality-relevant traits; (4) abiotic stress tolerances of miscanthus genotypes; (5) selections suitable for production on marginal land; (6) field establishment methods for seeds using plugs; (7) evaluation of harvesting methods; and (8) quantification of energy used in densification (pellet) technologies with a range of hybrids with differences in stem wall properties. End-user needs were addressed by demonstrating the potential of optimizing miscanthus biomass composition for the production of ethanol and biogas as well as for combustion. The costs and life-cycle assessment of seven miscanthus-based value chains, including small- and large-scale heat and power, ethanol, biogas, and insulation material production, revealed GHG-emission- and fossil-energy-saving potentials of up to 30.6 t CO2eq C ha−1y−1 and 429 GJ ha−1y−1, respectively. Transport distance was identified as an important cost factor. Negative carbon mitigation costs of –78€ t−1 CO2eq C were recorded for local biomass use. The OPTIMISC results demonstrate the potential of miscanthus as a crop for marginal sites and provide information and technologies for the commercial implementation of miscanthus-based value chains.

Analysis of Malus S-RNase gene diversity based on a comparative study of old and modern apple cultivars and European wild apple.

Malus S-RNase genetic diversity was analyzed in Malusxa0×xa0domestica cultivars and compared to European wild apple (Malus sylvestris). Using PCR-based approaches, the S-RNase genotype of 140 M.xa0×xa0domestica cultivars, 196 M. sylvestris trees and 27 M. sylvestris—M.xa0×xa0domestica hybrids was determined. S-RNase allelic richness in M. sylvestris was much higher than in M.xa0×xa0domestica, indicating the negative influence of domestication on S-RNase diversity. Heterogeneity of the S-RNase allelic distribution is much higher in cultivated apple than in wild apple, which shows that breeding leads to strong departure from the expected homogeneity of genes under negative frequency-dependent selection. The majority of the M.xa0×xa0domesticaS-alleles has been found in M. sylvestris as well, which points to strong conservation of the S-locus gene structure. Based on the sequence of all different SCAR-fragments, which comprise both the hypervariable PS1 region and the single intron, S-RNase genetic diversity was further explored. It provided some clues to the occurrence of new S-alleles among the multitude of novel S-RNase sequences that have been identified, which were mostly unique for the group of M. sylvestris individuals. The determination of the S-RNase genotypes of old cultivars and M. sylvestris will enable their introduction into new breeding strategies. As M. sylvestris has become an endangered species in Belgium, the knowledge gathered in this study will be an important tool for selecting useful genotypes for a core collection.

Orthology Guided Assembly in highly heterozygous crops: creating a reference transcriptome to uncover genetic diversity in Lolium perenne

Despite current advances in next-generation sequencing data analysis procedures, de novo assembly of a reference sequence required for SNP discovery and expression analysis is still a major challenge in genetically uncharacterized, highly heterozygous species. High levels of polymorphism inherent to outbreeding crop species hamper De Bruijn Graph-based de novo assembly algorithms, causing transcript fragmentation and the redundant assembly of allelic contigs. If multiple genotypes are sequenced to study genetic diversity, primary de novo assembly is best performed per genotype to limit the level of polymorphism and avoid transcript fragmentation. Here, we propose an Orthology Guided Assembly procedure that first uses sequence similarity (tBLASTn) to proteins of a model species to select allelic and fragmented contigs from all genotypes and then performs CAP3 clustering on a gene-by-gene basis. Thus, we simultaneously annotate putative orthologues for each protein of the model species, resolve allelic redundancy and fragmentation and create a de novo transcript sequence representing the consensus of all alleles present in the sequenced genotypes. We demonstrate the procedure using RNA-seq data from 14 genotypes of Lolium perenne to generate a reference transcriptome for gene discovery and translational research, to reveal the transcriptome-wide distribution and density of SNPs in an outbreeding crop and to illustrate the effect of polymorphisms on the assembly procedure. The results presented here illustrate that constructing a non-redundant reference sequence is essential for comparative genomics, orthology-based annotation and candidate gene selection but also for read mapping and subsequent polymorphism discovery and/or read count-based gene expression analysis.

High-throughput phenotyping of lateral expansion and regrowth of spaced Lolium perenne plants using on-field image analysis

BackgroundGenetic studies and breeding of agricultural crops frequently involve phenotypic characterization of large collections of genotypes grown in field conditions. These evaluations are typically based on visual observations and manual (destructive) measurements. Robust image capture and analysis procedures that allow phenotyping large collections of genotypes in time series during developmental phases represent a clear advantage as they allow non-destructive monitoring of plant growth and performance. A L. perenne germplasm panel including wild accessions, breeding material and commercial varieties has been used to develop a low-cost, high-throughput phenotyping tool for determining plant growth based on images of individual plants during two consecutive growing seasons. Further we have determined the correlation between image analysis-based estimates of the plant’s base area and the capacity to regrow after cutting, with manual counts of tiller number and measurements of leaf growth 2xa0weeks after cutting, respectively. When working with field-grown plants, image acquisition and image segmentation are particularly challenging as outdoor light conditions vary throughout the day and the season, and variable soil colours hamper the delineation of the object of interest in the image. Therefore we have used several segmentation methods including colour-, texture- and edge-based approaches, and factors derived after a fast Fourier transformation. The performance of the procedure developed has been analysed in terms of effectiveness across different environmental conditions and time points in the season.ResultsThe procedure developed was able to analyse correctly 77.2xa0% of the 24,048 top view images processed. High correlations were found between plant’s base area (image analysis-based) and tiller number (manual measurement) and between regrowth after cutting (image analysis-based) and leaf growth 2xa0weeks after cutting (manual measurement), with r values up to 0.792 and 0.824, respectively. Nevertheless, these relations depend on the origin of the plant material (forage breeding lines, current forage varieties, current turf varieties, and wild accessions) and the period in the season.ConclusionsThe image-derived parameters presented here deliver reliable, objective data, complementary to the breeders’ scores, and are useful for genetic studies. Furthermore, large variation was shown among genotypes for the parameters investigated.

Effects of dietary lambda-cyhalothrin exposure on bumblebee survival, reproduction, and foraging behavior in laboratory and greenhouse

Bumblebees of Bombus terrestris are indispensible pollinators for ecosystems and for various agricultural crops. Unfortunately, bumblebees are challenged by various stress factors including insecticide applications. Today sublethal effects of various insecticides need to be thoroughly investigated to allow their combined use with pollinators and other beneficial organisms. In this study, we used lambda-cyhalothrin as a model pyrethroid insecticide and investigated lethal and sublethal effects by different dilutions, ranging from 1/10 to 1/100 of its maximum field recommended dosage (MFRC, 37.5xa0ppm), with the use of a chronic toxicity tests in the laboratory and in flight cages in the greenhouse. In the laboratory, small microcolonies with five bumblebee workers with one being pseudo-queen were used, while in the greenhouse we used queen-right mini-hives where the bumblebees need to fly for pyrethroid-contaminated food. We observed strong sublethal effects in the laboratory with treatments of 1/10 and 1/20 of the MFRC: the nest reproduction was reduced by 49 and 32xa0%, respectively, and the sugar water consumption by 36xa0%. With free-flying bumblebees, the toxic effects at 1/10 of the MFRC were more pronounced. A mortality of 88xa0±xa08xa0% was observed after only 2xa0weeks, being twice the mortality in the laboratory microcolonies test (43xa0±xa011xa0%). Besides, it should be mentioned that in the greenhouse experiment all queens were dead and most of the workers showed signs of incoordination and convulsion and gradually became apathetic. In conclusion, our results demonstrated the pyrethroid lambda-cyhalothrin with a range of lethal and sublethal effects, both crucial for the development and survival of the B. terrestris colonies. Moreover, this study supports the demand to test insecticide compounds on their safety, especially when the bees have to perform complex tasks such as foraging for their food.

Processes underlying branching differences in fodder crops

Plant architectural characteristics are under strong genetic regulation. Economically important traits for forage crops such as biomass yield, ground cover and persistence can be improved by selecting for particular aerial architectural characteristics. Here, we present an easily applicable method for the spatiotemporal description of branching patterns in red clover (Trifolium pratense) and perennial ryegrass (Lolium perenne), two of the most important forage crops in Europe. A detailed analysis of genotypes with contrasting branching phenotypes demonstrates that in these species different factors are the main determinants of shoot branching characteristics. In red clover, bud outgrowth and to a lesser extent bud formation explain inter-genotype branching differences. In perennial ryegrass, differences in the capacity to form new buds determined largely the differences between forage and turf types. However, when a set of four forage types was compared in a separate experiment, variation in quantity and pace of bud formation and bud outgrowth explained the differences in aerial architecture. In both crops, branching patterns are likely determined by several processes, and highly branched phenotypes can result from the formation of more buds, an increased probability of bud outgrowth, or a combination of these processes. Furthermore, the presence of more buds is partly caused by more bud outgrowth.

In situ quantification of forage grass root biomass, distribution and diameter classes under two N fertilisation rates

Background & aimsRoots are of paramount importance in protecting grassland in numerous ecosystem services e.g. soil organic matter build-up. However, studies that quantified root biomass in grasslands predominantly focused on areas managed less intensively than the management that is common to most North-West European grassland-based farms. To fill this knowledge gap, we compared, root and stubble biomass, the distribution in the soil and root diameter classes of five common European forage grass species grown under intensive management.MethodsOn a 3xa0year old trial comparing yield of five cool season forage grass species at two N fertilization levels (190xa0kgxa0Nxa0ha−1 yr−1 or 300xa0kgxa0Nxa0ha−1 yr−1) we sampled root and stubble biomass until a depth of 90xa0cm deep.ResultsTall fescue (Festuca arundinacea) displayed the highest root and stubble biomass and had the highest mean root diameters of all studied grass species. The total dry biomass below cutting height (stubbleu2009+u2009roots up to a depth of 90xa0cm below the soil surface) varied between 18 and 19 tonnes ha−1 for tall fescue and 10 and 11 tonnes ha−1 for Festulolium at 190xa0kgxa0Nxa0ha−1 yr−1 and 300xa0kgxa0Nxa0ha−1 yr−1, respectively.ConclusionsOur findings emphasize that in intensively managed grassland, root and stubble biomass under a 3xa0years old sward can be as high as 19xa0t DM ha−1. Owing to the high forage and root biomass of tall fescue, this species has a high potential in maintaining several ecosystem services.

A direct assessment of realized seed and pollen flow within and between two isolated populations of the food-deceptive orchid Orchis mascula

Gene flow can counteract the loss of genetic diversity caused by genetic drift in small populations. For this reason, clearly understanding gene flow patterns is of the highest importance across fragmented landscapes. However, gene flow patterns are not only dependent upon the degree of spatial isolation of fragmented populations, but are also dependent upon the life-history traits of the species. Indeed, habitat fragmentation effects appear especially unpredictable for food-deceptive orchid species, because of their highly specialised seed and pollen dispersal mechanisms. In this study we used amplified fragment length polymorphism markers and subsequent parentage and spatial autocorrelation analysis to quantify the extent and the patterns of realized gene flow within and between two adjacent fragmented populations of the food-deceptive Orchis mascula. We observed considerable gene flow between both populations, occurring mainly through pollen dispersal. Seed dispersal, on the other hand, was mainly limited to the first few meters from the mother plant in both populations, although at least one among-population seed dispersal event was observed. This, in turn, resulted in a significant spatial genetic structure for both populations. Although genetic diversity was high in both populations and mainly outcrossing occurred, reproductive output was strongly skewed toward a limited number of successful adult plants. These observed patterns are likely due to the different pollinator behaviour associated with food-deceptive plants. We conclude that these populations can be considered viable under their current fragmented state.