Genome-wide association study for in vitro digestibility and related traits in triticale forage.

BACKGROUND: Triticale is making its way on dairy farms as an alternative forage crop. This requires the availability of high-yielding triticale varieties with good digestibility. Triticale forage breeding mainly focussed on biomass yield, but efforts to improve digestibility are increasing. We previously investigated the interrelationships among different quality traits in soft dough triticale: starch, acid detergent fibre and in vitro digestibility of organic matter (IVOMD) and of neutral detergent fibre (IVNDFD) of the total plant, IVNDFD and Klason lignin of the stems, and ear proportion and stem length. Here we determine the genetic control of these traits, using a genome-wide association (GWAS) approach. A total of 33,231 DArTseq SNP markers assessed in a collection of 118 winter triticale genotypes, including 101 varieties and 17 breeding lines, were used. RESULTS: The GWAS identified a total of 53 significant marker-trait associations (MTAs). The highest number of significantly associated SNP markers (n = 10) was identified for total plant IVNDFD. A SNP marker on chromosome 1A (4211801_19_C/T; 474,437,796 bp) was found to be significantly associated with ear proportion, and plant and stem IVNDFD, with the largest phenotypic variation for ear proportion (R²p = 0.23). Based on MTAs, candidate genes were identified which were of particular relevance for variation in in vitro digestibility (IVD) because they are putatively involved in plasma membrane transport, cytoskeleton organisation, carbohydrate metabolic processes, protein phosphorylation, and sterol and cell wall biogenesis. Interestingly, a xyloglucan-related candidate gene on chromosome 2R, SECCE2Rv1G0126340, was located in close proximity of a SNP significantly associated with stem IVNDFD. Furthermore, quantitative trait loci previously reported in wheat co-localized with significantly associated SNP markers in triticale. CONCLUSIONS: A collection of 118 winter triticale genotypes combined with DArTseq SNP markers served as a source for identifying 53 MTAs and several candidate genes for forage IVD and related traits through a GWAS approach. Taken together, the results of this study demonstrate that the genetic diversity available in this collection can be further exploited for research and breeding purposes to improve the IVD of triticale forage.

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant.

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose-to-glucose conversion of maize stover. Here, we analyzed a field-grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD-deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose-to-glucose conversion efficiency was determined after 10 different thermochemical pre-treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre-treatment. However, the relative increase in glucose yields after alkaline pre-treatment was not higher than the relative increase when no pre-treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p-coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD-deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar-platform biorefineries.

Genetic control of tolerance to drought stress in soybean.

BACKGROUND: Drought stress limits the production of soybean [Glycine max (L.) Merr.], which is the most grown high-value legume crop worldwide. Breeding for drought tolerance is a difficult endeavor and understanding the genetic basis of drought tolerance in soybean is therefore crucial for harnessing the genomic regions involved in the tolerance mechanisms. A genome-wide association study (GWAS) analysis was applied in a soybean germplasm collection (the EUCLEG collection) of 359 accessions relevant for breeding in Europe, to identify genomic regions and candidate genes involved in the response to short duration and long duration drought stress (SDS and LDS respectively) in soybean. RESULTS: The phenotypic response to drought was stronger in the long duration drought (LDS) than in the short duration drought (SDS) experiment. Over the four traits considered (canopy wilting, leaf senescence, maximum absolute growth rate and maximum plant height) the variation was in the range of 8.4-25.2% in the SDS, and 14.7-29.7% in the LDS experiments. The GWAS analysis identified a total of 17 and 22 significant marker-trait associations for four traits in the SDS and LDS experiments, respectively. In the genomic regions delimited by these markers we identified a total of 12 and 16 genes with putative functions that are of particular relevance for drought stress responses including stomatal movement, root formation, photosynthesis, ABA signaling, cellular protection and cellular repair mechanisms. Some of these genomic regions co-localized with previously known QTLs for drought tolerance traits including water use efficiency, chlorophyll content and photosynthesis. CONCLUSION: Our results indicate that the mechanism of slow wilting in the SDS might be associated with the characteristics of the root system, whereas in the LDS, slow wilting could be due to low stomatal conductance and transpiration rates enabling a high WUE. Drought-induced leaf senescence was found to be associated to ABA and ROS responses. The QTLs related to WUE contributed to growth rate and canopy height maintenance under drought stress. Co-localization of several previously known QTLs for multiple agronomic traits with the SNPs identified in this study, highlights the importance of the identified genomic regions for the improvement of agronomic performance in addition to drought tolerance in the EUCLEG collection.

Physiological basis of chilling tolerance and early-season growth in miscanthus.

Background and Aims: The high productivity of Miscanthus × giganteus has been at least partly ascribed to its high chilling tolerance compared with related C4 crops, allowing for a longer productive growing season in temperate climates. However, the chilling tolerance of M. × giganteus has been predominantly studied under controlled environmental conditions. The understanding of the underlying mechanisms contributing to chilling tolerance in the field and their variation in different miscanthus genotypes is largely unexplored. Methods: Five miscanthus genotypes with different sensitivities to chilling were grown in the field and scored for a comprehensive set of physiological traits throughout the spring season. Chlorophyll fluorescence was measured as an indication of photosynthesis, and leaf samples were analysed for biochemical traits related to photosynthetic activity (chlorophyll content and pyruvate, Pi dikinase activity), redox homeostasis (malondialdehyde, glutathione and ascorbate contents, and catalase activity) and water-soluble carbohydrate content. Key Results: Chilling-tolerant genotypes were characterized by higher levels of malondialdehyde, raffinose and sucrose, and higher catalase activity, while the chilling-sensitive genotypes were characterized by higher concentrations of glucose and fructose, and higher pyruvate, Pi dikinase activity later in the growing season. On the early sampling dates, the biochemical responses of M. × giganteus were similar to those of the chilling-tolerant genotypes, but later in the season they became more similar to those of the chilling-sensitive genotypes. Conclusions: The overall physiological response of chilling-tolerant genotypes was distinguishable from that of chilling-sensitive genotypes, while M. × giganteus was intermediate between the two. There appears to be a trade-off between high and efficient photosynthesis and chilling stress tolerance. Miscanthus × giganteus is able to overcome this trade-off and, while it is more similar to the chilling-sensitive genotypes in early spring, its photosynthetic capacity is similar to that of the chilling-tolerant genotypes later on.

Introduction of a natural resource balance indicator to assess soil organic carbon management: Agricultural Biomass Productivity Benefit.

The rising demand for feed and food has put an increasing pressure on agriculture, with agricultural intensification as a direct response. Notwithstanding the higher crop productivity, intensive agriculture management entails many adverse environmental impacts. Worldwide, soil organic carbon (SOC) decline is hereby considered as a main danger which affects soil fertility and productivity. The life cycle perspective helps to get a holistic overview when evaluating the environmental sustainability of agricultural systems, though the impact of farm management on soil quality aspects is often not integrated. In this paper, we introduce an indicator called Agricultural Biomass Productivity Benefit of SOC management (ABB_SOC), which, relying on natural resource consumption, enables to estimate the net effect of the efforts made to attain a better soil quality. Hereby the focus is put on SOC. First, we introduce a framework to describe the SOC trend due to farm management decisions. The extent to which remediation measures are required are used as a measure for the induced SOC losses. Next, ABB_SOC values are calculated as the balance between the natural resource consumption of the inputs (including remediation efforts) and the desired output of arable crop production systems. The models RothC and EU-Rotate_N are used to simulate the SOC evolution due to farm management and the response of the biomass productivity, respectively. The developed indicator is applied on several rotation systems in Flanders, comparing different remediation strategies. The indicator could be used as a base for a method to account for soil quality in life cycle analysis.

Overexpression of GA20-OXIDASE1 impacts plant height, biomass allocation and saccharification efficiency in maize.

Increased biomass yield and quality are of great importance for the improvement of feedstock for the biorefinery. For the production of bioethanol, both stem biomass yield and the conversion efficiency of the polysaccharides in the cell wall to fermentable sugars are of relevance. Increasing the endogenous levels of gibberellic acid (GA) by ectopic expression of GA20-OXIDASE1 (GA20-OX1), the rate-limiting step in GA biosynthesis, is known to affect cell division and cell expansion, resulting in larger plants and organs in several plant species. In this study, we examined biomass yield and quality traits of maize plants overexpressing GA20-OX1 (GA20-OX1). GA20-OX1 plants accumulated more vegetative biomass than control plants in greenhouse experiments, but not consistently over two years of field trials. The stems of these plants were longer but also more slender. Investigation of GA20-OX1 biomass quality using biochemical analyses showed the presence of more cellulose, lignin and cell wall residue. Cell wall analysis as well as expression analysis of lignin biosynthetic genes in developing stems revealed that cellulose and lignin were deposited earlier in development. Pretreatment of GA20-OX1 biomass with NaOH resulted in a higher saccharification efficiency per unit of dry weight, in agreement with the higher cellulose content. On the other hand, the cellulose-to-glucose conversion was slower upon HCl or hot-water pretreatment, presumably due to the higher lignin content. This study showed that biomass yield and quality traits can be interconnected, which is important for the development of future breeding strategies to improve lignocellulosic feedstock for bioethanol production.

Exploiting Rye in Wheat Quality Breeding: The Case of Arabinoxylan Content.

Rye (Secale cereale subsp. cereale L.) has long been exploited as a valuable alternative genetic resource in wheat (Triticum aestivum L.) breeding. Indeed, the introgression of rye genetic material led to significant breakthroughs in the improvement of disease and pest resistance of wheat, as well as a few agronomic traits. While such traits remain a high priority in cereal breeding, nutritional aspects of grain crops are coming under the spotlight as consumers become more conscious about their dietary choices and the food industry strives to offer food options that meet their demands. To address this new challenge, wheat breeding can once again turn to rye to look for additional genetic variation. A nutritional aspect that can potentially greatly benefit from the introgression of rye genetic material is the dietary fibre content of flour. In fact, rye is richer in dietary fibre than wheat, especially in terms of arabinoxylan content. Arabinoxylan is a major dietary fibre component in wheat and rye endosperm flours, and it is associated with a variety of health benefits, including normalisation of glycaemic levels and promotion of the gut microbiota. Thus, it is a valuable addition to the human diet, and it can represent a novel target for wheat-rye introgression breeding.

Variation in potential feeding value of triticale forage among plant fraction, maturity stage, growing season and genotype.

Cereal forages, such as triticale forage, progressively gain interest as alternative crop for maize. The main study objective was to investigate the variation in potential feeding value of triticale forage among maturity stage, growing season and genotype, using total plant and stem fractions. Therefore, near infrared spectroscopy (NIRS) was evaluated as fast screening tool. The prediction ability was good (ratio of prediction to deviation, RPD ≥3.0) for total plant residual moisture, starch, sugars and for stem crude ash (CAsh) and neutral detergent fibre (aNDFom); suitable for screening (2.0 ≤ RPD <3.0) for total plant CAsh, acid detergent fibre (ADFom), in vitro digestibility of organic matter (IVOMD), in vitro digestibility of neutral detergent fibre (IVNDFD) and for stem total lignin (TL) and IVNDFD; poor (1.5 ≤ RPD <2.0) for total plant crude protein, crude fat, aNDFom, lignin (sa) and for stem Klason lignin (KL); unreliable (RPD <1.5) for stem residual moisture and acid soluble lignin (ASL). The evolution in potential feeding value of 36 genotypes harvested at the medium and late milk to the early, soft and hard dough stage was followed. The most important changes occurred between the late milk and early dough stage, with little variation in quality after the soft dough stage. During 2 growing seasons, variation in feeding value of 120 genotypes harvested at the soft dough stage was demonstrated. Interestingly, variation in stem IVNDFD is almost twice as high as for the total plant (CV 12.4% versus 6.6%). Furthermore, Spearman correlations show no link between dry matter yield and digestibility of genotypes harvested at the soft dough stage. Based on linear regression models ADFom appears as main predictor of both plant IVOMD and plant IVNDFD. Stem IVNDFD is particularly determined by KL.

Phenotypic characterization of drought responses in red clover (Trifolium pratense L.).

Introduction: Red clover (Trifolium pratense) is a protein-rich, short-lived perennial forage crop that can achieve high yields, but suffers increasingly from drought in different cultivation areas. Breeding for increased adaptation to drought is becoming essential, but at this stage it is unclear which traits breeders should target to phenotype responses to drought that allow them to identify the most promising red clover genotypes. In this study, we assessed how prolonged periods of drought affected plant growth in field conditions, and which traits could be used to distinguish better adapted plant material. Methods: A diverse panel of 395 red clover accessions was evaluated during two growing seasons. We simulated 6-to-8-week drought periods during two consecutive summers, using mobile rain-out shelters, while an irrigated control field was established in an adjacent parcel. Plant growth was monitored throughout both growing seasons using multiple flights with a drone equipped with RGB and thermal sensors. At various observation moments throughout both growing seasons, we measured canopy cover (CC) and canopy height (CH). The crop water stress index (CWSI) was determined at two moments, during or shortly after the drought event. Results: Manual and UAV-derived measurements for CH were well correlated, indicating that UAV-derived measurements can be reliably used in red clover. In both years, CC, CH and CWSI were affected by drought, with measurable growth reductions by the end of the drought periods, and during the recovery phase. We found that the end of the drought treatment and the recovery phase of approximately 20 days after drought were suitable periods to phenotype drought responses and to distinguish among genotypes. Discussion: Multifactorial analysis of accession responses revealed interactions of the maturity type with drought responses, which suggests the presence of two independent strategies in red clover: 'drought tolerance' and 'drought recovery'. We further found that a large proportion of the accessions able to perform well under well-watered conditions were also the ones that were less affected by drought. The results of this investigation are interpreted in view of the development of breeding for adaptation to drought in red clover.

Physicochemical Characterization of Thirteen Quinoa (Chenopodium quinoa Willd.) Varieties Grown in North-West Europe-Part II.

Quinoa cultivation has gained increasing interest in Europe but more research on the characteristics of European varieties is required to help determine their end use applications. A comparative study was performed on 13 quinoa varieties cultivated under North-West European field conditions during three consecutive growing seasons (2017-2019). The seeds were milled to wholemeal flour (WMF) to evaluate the physicochemical properties. The WMFs of 2019 were characterized by the highest water absorption capacity (1.46-2.06 g/g), while the water absorption index (WAI) between 55 °C (2.04-3.80 g/g) and 85 °C (4.04-7.82 g/g) increased over the years. The WMFs of 2018 had the highest WAI at 95 °C (6.48-9.48 g/g). The pasting profiles were characterized by a high viscosity peak (1696-2560 mPa.s) and strong breakdown (-78-643 mPa.s) in 2017. The peak viscosity decreased in 2018 and 2019 (823-2492 mPa.s), while breakdown (-364-555 mPa.s) and setback (19-1037 mPa.s) increased. Jessie, Summer Red, Rouge Marie, Vikinga, and Zwarte WMFs were characterized by low WAIs and high shear resistance. Bastille WMF developed high viscosities and, along with Faro WMF, showed a high breakdown. The wide variation in physicochemical properties suggests that the potential food applications of WMFs depend on the variety and growing conditions.

Response of a Diverse European Soybean Collection to "Short Duration" and "Long Duration" Drought Stress.

Drought causes significant damage to a high value crop of soybean. Europe has an increasing demand for soybean and its own production is insufficient. Selection and breeding of cultivars adapted to European growth conditions is therefore urgently needed. These new cultivars must have a shorter growing cycle (specifically for adaptation to North-West Europe), high yield potential under European growing conditions, and sufficient drought resistance. We have evaluated the performance of a diverse collection of 359 soybean accessions under drought stress using rain-out shelters for 2 years. The contrasting weather conditions between years and correspondingly the varying plant responses demonstrated that the consequences of drought for an individual accession can vary strongly depending on the characteristics (e.g., duration and intensity) of the drought period. Short duration drought stress, for a period of four to 7 weeks, caused an average reduction of 11% in maximum canopy height (CH), a reduction of 17% in seed number per plant (SN) and a reduction of 16% in seed weight per plant (SW). Long duration drought stress caused an average reduction of 29% in CH, a reduction of 38% in SN and a reduction of 43% in SW. Drought accelerated plant development and caused an earlier cessation of flowering and pod formation. This seemed to help some accessions to better protect the seed yield, under short duration drought stress. Drought resistance for yield-related traits was associated with the maintenance of growth under long duration drought stress. The collection displayed a broad range of variation for canopy wilting and leaf senescence but a very narrow range of variation for crop water stress index (CWSI; derived from canopy temperature data). To the best of our knowledge this is the first study reporting a detailed investigation of the response to drought within a diverse soybean collection relevant for breeding in Europe.

A Genome-Wide Genetic Diversity Scan Reveals Multiple Signatures of Selection in a European Soybean Collection Compared to Chinese Collections of Wild and Cultivated Soybean Accessions.

Targeted and untargeted selections including domestication and breeding efforts can reduce genetic diversity in breeding germplasm and create selective sweeps in crop genomes. The genomic regions at which selective sweeps are detected can reveal important information about signatures of selection. We have analyzed the genetic diversity within a soybean germplasm collection relevant for breeding in Europe (the EUCLEG collection), and have identified selective sweeps through a genome-wide scan comparing that collection to Chinese soybean collections. This work involved genotyping of 480 EUCLEG soybean accessions, including 210 improved varieties, 216 breeding lines and 54 landraces using the 355K SoySNP microarray. SNP calling of 477 EUCLEG accessions together with 328 Chinese soybean accessions identified 224,993 high-quality SNP markers. Population structure analysis revealed a clear differentiation between the EUCLEG collection and the Chinese materials. Further, the EUCLEG collection was sub-structured into five subgroups that were differentiated by geographical origin. No clear association between subgroups and maturity group was detected. The genetic diversity was lower in the EUCLEG collection compared to the Chinese collections. Selective sweep analysis revealed 23 selective sweep regions distributed over 12 chromosomes. Co-localization of these selective sweep regions with previously reported QTLs and genes revealed that various signatures of selection in the EUCLEG collection may be related to domestication and improvement traits including seed protein and oil content, phenology, nitrogen fixation, yield components, diseases resistance and quality. No signatures of selection related to stem determinacy were detected. In addition, absence of signatures of selection for a substantial number of QTLs related to yield, protein content, oil content and phenological traits suggests the presence of substantial genetic diversity in the EUCLEG collection. Taken together, the results obtained demonstrate that the available genetic diversity in the EUCLEG collection can be further exploited for research and breeding purposes. However, incorporation of exotic material can be considered to broaden its genetic base.

Yield and Nutritional Characterization of Thirteen Quinoa (Chenopodium quinoa Willd.) Varieties Grown in North-West Europe-Part I.

The cultivation of quinoa has gained increasing interest in Europe. Different European varieties exist, but more research is required to understand the individual variety characteristics for end-use applications. The objective of this study is to evaluate the agronomic performance of 13 quinoa varieties under North-West European field conditions during three growing seasons (2017-2019). Furthermore, seeds were qualitatively characterized based on characteristics and composition. Yield differed among varieties and growing seasons (0.47-3.42 ton/ha), with lower yields obtained for late-maturing varieties. The saponin content varied from sweet to very bitter. The seeds contained high protein levels (12.1-18.8 g/100 g dry matter), whereas varieties had a similar essential amino acid profile. The main fatty acids were linoleic (53.0-59.8%), α-linolenic (4.7-8.2%), and oleic acid (15.5-22.7%), indicating a high degree of unsaturation. The clustering of varieties/years revealed subtle differences between growing seasons but also reflected the significant interaction effects of variety and year. Most varieties perform well under North-West European conditions, and their nutritional content is well within the values previously described for other cultivation areas. However, optimal yield and quality traits were not combined in one variety, illustrating the importance of breeding for adapted quinoa varieties.

Seasonal Differences in Structural and Genetic Control of Digestibility in Perennial Ryegrass.

Perennial ryegrass is an important forage crop in dairy farming, either for grazing or haying purposes. To further optimise the forage use, this study focused on understanding forage digestibility in the two most important cuts of perennial ryegrass, the spring cut at heading and the autumn cut. In a highly diverse collection of 592 Lolium perenne genotypes, the organic matter digestibility (OMD) and underlying traits such as cell wall digestibility (NDFD) and cell wall components (cellulose, hemicellulose, and lignin) were investigated for 2 years. A high genotype × season interaction was found for OMD and NDFD, indicating differences in genetic control of these forage quality traits in spring versus autumn. OMD could be explained by both the quantity of cell wall content (NDF) and the quality of the cell wall content (NDFD). The variability in NDFD in spring was mainly explained by differences in hemicellulose. A 1% increase of the hemicellulose content in the cell wall (HC.NDF) resulted in an increase of 0.81% of NDFD. In autumn, it was mainly explained by the lignin content in the cell wall (ADL.NDF). A 0.1% decrease of ADL.NDF resulted in an increase of 0.41% of NDFD. The seasonal traits were highly heritable and showed a higher variation in autumn versus spring, indicating the potential to select for forage quality in the autumn cut. In a candidate gene association mapping approach, in which 503 genes involved in cell wall biogenesis, plant architecture, and phytohormone biosynthesis and signalling, identified significant quantitative trait loci (QTLs) which could explain from 29 to 52% of the phenotypic variance in the forage quality traits OMD and NDFD, with small effects of each marker taken individually (ranging from 1 to 7%). No identical QTLs were identified between seasons, but within a season, some QTLs were in common between digestibility traits and cell wall composition traits confirming the importance of hemicellulose concentration for spring digestibility and lignin concentration in NDF for autumn digestibility.

Canonical correlations reveal adaptive loci and phenotypic responses to climate in perennial ryegrass.

Germplasm from perennial ryegrass (Lolium perenne L.) natural populations is useful for breeding because of its adaptation to a wide range of climates. Climate-adaptive genes can be detected from associations between genotype, phenotype and climate but an integrated framework for the analysis of these three sources of information is lacking. We used two approaches to identify adaptive loci in perennial ryegrass and their effect on phenotypic traits. First, we combined Genome-Environment Association (GEA) and GWAS analyses. Then, we implemented a new test based on a Canonical Correlation Analysis (CANCOR) to detect adaptive loci. Furthermore, we improved the previous perennial ryegrass gene set by de novo gene prediction and functional annotation of 39,967 genes. GEA-GWAS revealed eight outlier loci associated with both environmental variables and phenotypic traits. CANCOR retrieved 633 outlier loci associated with two climatic gradients, characterized by cold-dry winter versus mild-wet winter and long rainy season versus long summer, and pointed out traits putatively conferring adaptation at the extremes of these gradients. Our CANCOR test also revealed the presence of both polygenic and oligogenic climatic adaptations. Our gene annotation revealed that 374 of the CANCOR outlier loci were positioned within or close to a gene. Co-association networks of outlier loci revealed a potential utility of CANCOR for investigating the interaction of genes involved in polygenic adaptations. The CANCOR test provides an integrated framework to analyse adaptive genomic diversity and phenotypic responses to environmental selection pressures that could be used to facilitate the adaptation of plant species to climate change.

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

BACKGROUND: Genetic 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. RESULTS: A 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. CONCLUSIONS: The 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.

Artificial Chromosome Doubling in Allotetraploid Calendula officinalis.

Calendula officinalis L. is known as an ornamental plant as well as a source of biochemical compounds used in cosmetics and industry. C. officinalis has a complex karyotype. Published chromosome numbers differ between 2n = 4x = 28 or 32. We have estimated genome sizes in nine commercial cultivars and evaluated the ploidy level by karyotyping and fluorescent in situ hybridization (FISH) using 5S and 45S rDNA loci. The detection of chromosome sets of two rather than four homologues would suggest that C. officinalis has an allotetraploid background. In addition, four signals for 45S but only two for 5S were found by using FISH. Artificial chromosome doubling is a common technique in plant breeding, as polyploidization results in several consequences for plant growth and development. Especially the suggested allotetraploid background in C. officinalis is interesting when examining the effect of chromosome doubling on the plant phenotype. Here we describe chromosome doubling of three allotetraploid cultivars of C. officinalis, 'Nova,' 'WUR 1553-7' and 'Orange Beauty'. Three antimitotic agents - colchicine, oryzalin and trifluralin - were used in different concentrations to find the combination of the best agent and the best dosage to obtain octaploids. For all three cultivars a few octaploids were obtained. A concentration of 200 and 400 ppm of colchicine was most efficient for chromosome doubling in 'Nova' and 'Orange Beauty,' respectively. For 'WUR 1553-7' the treatment with 20 ppm oryzalin was also effective. Cell numbers and first observations of the phenotype in the chromosome doubled plants show thicker leaves and bigger cells, as commonly observed after ploidy doubling. Due to the low number of chromosome doubled plants obtained more elaborate phenotyping will be performed on following generations cultivated under field conditions.

Population genomic structure of the gelatinous zooplankton species Mnemiopsis leidyi in its nonindigenous range in the North Sea.

Nonindigenous species pose a major threat for coastal and estuarine ecosystems. Risk management requires genetic information to establish appropriate management units and infer introduction and dispersal routes. We investigated one of the most successful marine invaders, the ctenophore Mnemiopsis leidyi, and used genotyping-by-sequencing (GBS) to explore the spatial population structure in its nonindigenous range in the North Sea. We analyzed 140 specimens collected in different environments, including coastal and estuarine areas, and ports along the coast. Single nucleotide polymorphisms (SNPs) were called in approximately 40 k GBS loci. Population structure based on the neutral SNP panel was significant (F ST .02; p < .01), and a distinct genetic cluster was identified in a port along the Belgian coast (Ostend port; pairwise F ST .02-.04; p < .01). Remarkably, no population structure was detected between geographically distant regions in the North Sea (the Southern part of the North Sea vs. the Kattegat/Skagerrak region), which indicates substantial gene flow at this geographical scale and recent population expansion of nonindigenous M. leidyi. Additionally, seven specimens collected at one location in the indigenous range (Chesapeake Bay, USA) were highly differentiated from the North Sea populations (pairwise F ST .36-.39; p < .01). This study demonstrates the utility of GBS to investigate fine-scale population structure of gelatinous zooplankton species and shows high population connectivity among nonindigenous populations of this recently introduced species in the North Sea. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at: The DNA sequences generated for this study are deposited in the NCBI sequence read archive under SRA accession numbers SRR6950721-SRR6950884, and will be made publically available upon publication of this manuscript.

High-Throughput Genome-Wide Genotyping To Optimize the Use of Natural Genetic Resources in the Grassland Species Perennial Ryegrass (Lolium perenne L.).

The natural genetic diversity of agricultural species is an essential genetic resource for breeding programs aiming to improve their ecosystem and production services. A large natural ecotype diversity is usually available for most grassland species. This could be used to recombine natural climatic adaptations and agronomic value to create improved populations of grassland species adapted to future regional climates. However describing natural genetic resources can be long and costly. Molecular markers may provide useful information to help this task. This opportunity was investigated for Lolium perenne L., using a set of 385 accessions from the natural diversity of this species collected right across Europe and provided by genebanks of several countries. For each of these populations, genotyping provided the allele frequencies of 189,781 SNP markers. GWAS were implemented for over 30 agronomic and/or putatively adaptive traits recorded in three climatically contrasted locations (France, Belgium, Germany). Significant associations were detected for hundreds of markers despite a strong confounding effect of the genetic background; most of them pertained to phenology traits. It is likely that genetic variability in these traits has had an important contribution to environmental adaptation and ecotype differentiation. Genomic prediction models calibrated using natural diversity were found to be highly effective to describe natural populations for almost all traits as well as commercial synthetic populations for some important traits such as disease resistance, spring growth or phenological traits. These results will certainly be valuable information to help the use of natural genetic resources of other species.

Environmental sustainability of conventional and organic farming: Accounting for ecosystem services in life cycle assessment.

Today, there is an ongoing debate about the environmental sustainability of the products of organic farming. To compare the performance of conventional and organic farming systems regarding environmental impact and productivity, the comprehensive environmental assessment tool 'life cycle assessment' can be used. The lower crop yields attained by organic systems compared to conventional farming systems might, however, outweigh the benefits of the use of more environmental-friendly practices when evaluating the environmental impact per product unit. Although these practices are beneficial for the environment, which is reflected in the delivery of a range of ecosystem services (ES), the focus is traditionally put only on the (harvested) product. Because the agricultural product involves actually a bundle of ES, the impact should be allocated among the whole output of an agricultural system. In this study, we propose an allocation procedure based on the capacity of agricultural systems to deliver ES to divide the environmental impact over all agricultural outputs (i.e. provisioning and other ES). Allocation factors are developed for conventional and organic arable farming systems. Applying these allocation factors, we demonstrate that for about half of the studied food products (including maize, potato), organic farming has clear environmental benefits in terms of resource consumption in comparison to conventional cultivation methods. This allocation approach allows a more complete comparison of the environmental sustainability of organically and conventionally produced food.