Oene Dolstra

The potential of C4 grasses for cellulosic biofuel production.

With the advent of biorefinery technologies enabling plant biomass to be processed into biofuel, many researchers set out to study and improve candidate biomass crops. Many of these candidates are C4 grasses, characterized by a high productivity and resource use efficiency. In this review the potential of five C4 grasses as lignocellulosic feedstock for biofuel production is discussed. These include three important field crops—maize, sugarcane and sorghum—and two undomesticated perennial energy grasses—miscanthus and switchgrass. Although all these grasses are high yielding, they produce different products. While miscanthus and switchgrass are exploited exclusively for lignocellulosic biomass, maize, sorghum, and sugarcane are dual-purpose crops. It is unlikely that all the prerequisites for the sustainable and economic production of biomass for a global cellulosic biofuel industry will be fulfilled by a single crop. High and stable yields of lignocellulose are required in diverse environments worldwide, to sustain a year-round production of biofuel. A high resource use efficiency is indispensable to allow cultivation with minimal inputs of nutrients and water and the exploitation of marginal soils for biomass production. Finally, the lignocellulose composition of the feedstock should be optimized to allow its efficient conversion into biofuel and other by-products. Breeding for these objectives should encompass diverse crops, to meet the demands of local biorefineries and provide adaptability to different environments. Collectively, these C4 grasses are likely to play a central role in the supply of lignocellulose for the cellulosic ethanol industry. Moreover, as these species are evolutionary closely related, advances in each of these crops will expedite improvements in the other crops. This review aims to provide an overview of their potential, prospects and research needs as lignocellulose feedstocks for the commercial production of biofuel.

Identification of QTLs influencing combustion quality in Miscanthus sinensis Anderss. II. Chlorine and potassium content

Abstract Chlorine and potassium content are important traits related to combustion quality of Miscanthus species. These traits were analysed in a cross between F1.1 and F1.7 entries of Miscanthus sinensis Anderss, both lines offspring of the cross between MS-90-2 and MS-88-110. Quantitative trait locus (QTL) analyses were performed on a previous linkage map constructed with the offspring cross mapping strategy. The mapqtl 4.0 package was used to perform QTL analyses. Six potential QTLs were detected with data collected over a 2-year period. Of these, four were associated with chlorine and two with potassium. These results could be used as an initial step to develop a marker-aided selection programme for biomass with low mineral content.

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.

Identification of QTLs influencing agronomic traits in Miscanthus sinensis Anderss. I. Total height, flag-leaf height and stem diameter.

We have developed the first quantitative trait locus (QTL) analyses for agronomic traits in a cross between F1.1 (P1) and F1.7 (P7) entries of Miscanthus sinensis Anderss. Both lines are offspring of the cross between MS-90-2 and MS-88-110. A map based on random amplified polymorphic DNA markers previously constructed was used to perform the QTL analyses. This map was developed using a new mapping strategy that has been designated offspring cross. Eleven QTLs were detected for height, panicle height and diameter using the programme mapqtl 4.0 and the multiple QTL method. QTL significance was determined using several analyses, including Kruskal-Wallis analyses, empirical determination of LOD critical values using permutation tests, QTLs validation with field data over 2 years and co-localization of QTLs for correlated traits. The results obtained could be the first step in developing a marker-assisted selection programming in this species for biomass production.

The mode of inheritance in tetraploid cut roses

Tetraploid hybrid tea roses (Rosa hybrida) represent most of the commercial cultivars of cut roses and form the basis for breeding programmes. Due to intensive interspecific hybridizations, modern cut roses are complex tetraploids for which the mode of inheritance is not exactly known. The segregation patterns of molecular markers in a tetraploid mapping population of 184 genotypes, an F1 progeny from a cross of two heterozygous parents, were investigated for disomic and tetrasomic inheritance. The possible occurrence of double reduction was studied as well. We can exclude disomic inheritance, but while our observations are more in line with a tetrasomic inheritance, we cannot exclude that there is a mixture of both inheritance modes. Two novel parental tetraploid linkage maps were constructed using markers known from literature, combined with newly generated markers. Comparison with the integrated consensus diploid map (ICM) of Spiller et al. (Theor Appl Genet 122:489–500, 2010) allowed assigning numbers to each of the linkage groups of both maps and including small linkage groups. So far, the possibility of using marker-assisted selection in breeding of tetraploid cut roses and of other species with a tetrasomic or partly tetrasomic inheritance, is still limited due to the difficulties in establishing marker-trait associations. We used these tetraploid linkage maps to determine associations between markers, two morphological traits and powdery mildew resistance. The knowledge on inheritance and marker-trait associations in tetraploid cut roses will be of direct use to cut rose breeding.

Identification of QTLs associated with yield and its components in Miscanthus sinensis Anderss

Yield and its components (stem-, leaf- and top-yield) were analyzed in a population derived from the cross between F1.1 andF1.7 entries of Miscanthus sinensisAnders. Both lines are offspring of the cross between MS-90-2 and MS-88-110. The aim of this work was to identify QTLs for yield and its components suitable to develop a Marker Aided Selection (MAS) program in M. sinensis.QTL analyses were performed using a previous linkage map based on RAPD markers which was constructed using a new mapping strategy named ‘offspring cross’ that is useful for mapping in forest and fruit trees. The MapQTL 4.0 package was used to perform QTL analyses. Twenty potential QTLs were detected over two years of analyses. Out of these, 6 were associated with yield,8 with stem-yield, 2 with leaf-yield and 4with top-yield. These results constitute an initial step to develop a MAS program for biomass production.

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.

Impact of drought stress on growth and quality of miscanthus for biofuel production

Miscanthus has a high potential as a biomass feedstock for biofuel production. Drought tolerance is an important breeding goal in miscanthus as water deficit is a common abiotic stress and crop irrigation is in most cases uneconomical. Drought may not only severely reduce biomass yields, but also affect biomass quality for biofuel production as cell wall remodeling is a common plant response to abiotic stresses. The quality and plant weight of 50 diverse miscanthus genotypes were evaluated under control and drought conditions (28 days no water) in a glasshouse experiment. Overall, drought treatment decreased plant weight by 45%. Drought tolerance – as defined by maintenance of plant weight – varied extensively among the tested miscanthus genotypes and ranged from 30% to 110%. Biomass composition was drastically altered due to drought stress, with large reductions in cell wall and cellulose content and a substantial increase in hemicellulosic polysaccharides. Stress had only a small effect on lignin content. Cell wall structural rigidity was also affected by drought conditions; substantially higher cellulose conversion rates were observed upon enzymatic saccharification of drought‐treated samples with respect to controls. Both cell wall composition and the extent of cell wall plasticity under drought varied extensively among all genotypes, but only weak correlations were found with the level of drought tolerance, suggesting their independent genetic control. High drought tolerance and biomass quality can thus potentially be advanced simultaneously. The extensive genotypic variation found for most traits in the evaluated miscanthus germplasm provides ample scope for breeding of drought‐tolerant varieties that are able to produce substantial yields of high‐quality biomass under water deficit conditions. The higher degradability of drought‐treated samples makes miscanthus an interesting crop for the production of second‐generation biofuels in marginal soils.

Evaluation of Miscanthus sinensis biomass quality as feedstock for conversion into different bioenergy products

Miscanthus is a promising fiber crop with high potential for sustainable biomass production for a biobased economy. The effect of biomass composition on the processing efficiency of miscanthus biomass for different biorefinery value chains was evaluated, including combustion, anaerobic digestion and enzymatic saccharification for the production of bioethanol. Biomass quality and composition was analyzed in detail using stem and leaf fractions of summer (July) and winter (March) harvested biomass of eight compositionally diverse Miscanthus sinensis genotypes. Genotype performance in tests for enzymatic saccharification, anaerobic digestion and combustion differed extensively. The variation between the best and the worst performing genotype was 18% for biogas yield (ml g−1 dm) and 42% for saccharification efficiency (glucose release as %dm). The ash content of the best performing genotype was 62% lower than that of the genotype with the highest ash content and showed a considerably high ash melting temperature during combustion. Variation between genotypes in biomass quality for the different thermochemical bioconversion processes was shown to be strongly correlated to differences in biomass composition. The most important traits that contributed favorably to biogas yields and saccharification efficiency were a high content of trans‐ferulic acid, a high ratio of para‐coumaric acid to lignin and a low lignin content. Additionally, a high content of hemicellulosic polysaccharides positively affected saccharification efficiency. Low contents of ash and inorganic elements positively affect biomass quality for combustion and low potassium and chloride contents contributed to a higher ash melting temperature. These results demonstrate the potential for optimizing and exploiting M. sinensis as a multipurpose lignocellulosic feedstock, particularly for bioenergy applications.