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Bioenergy will be one of the most important renewable energy sources in the conversion from fossil fuels to bio‐based products. Short rotation coppice Salix could be a key player in this conversion since Salix has rapid growth, positive energy balance, easy to manage cultivation system with vegetative propagation of plant material and multiple harvests from the same plantation. The aim of the present paper is to provide an overview of the main challenges and key issues in willow genetic improvement toward sustainable biofuel value chains. Primarily based on results from the research project "Optimized Utilization of Salix" (OPTUS), the influence of Salix wood quality on the potential for biofuel use is discussed, followed by issues related to the conversion of Salix biomass into liquid and gaseous transportation fuels. Thereafter, the studies address genotypic influence on soil carbon sequestration in Salix plantations, as well as on soil carbon dynamics and climate change impacts. Finally, the opportunities for plant breeding are discussed using willow as a resource for sustainable biofuel production. Substantial phenotypic and genotypic variation was reported for different wood quality traits important in biological (i.e., enzymatic and anaerobic) and thermochemical conversion processes, which is a prerequisite for plant breeding. Furthermore, different Salix genotypes can affect soil carbon sequestration variably, and life cycle assessment illustrates that these differences can result in different climate mitigation potential depending on genotype. Thus, the potential of Salix plantations for sustainable biomass production and its conversion into biofuels is shown. Large genetic variation in various wood and biomass traits, important for different conversion processes and carbon sequestration, provides opportunities to enhance the sustainability of the production system via plant breeding. This includes new breeding targets in addition to traditional targets for high yield to improve biomass quality and carbon sequestration potential. [ABSTRACT FROM AUTHOR]

Transition towards a bio-based society requires large amounts of woody biomass to be converted into biofuels and biomaterials. Populus species are good candidates for growth in short rotations, but there is a lack of climate-adapted plant material suitable for growth at the high latitudes of the Nordic–Baltic region. Here we studied the growth and phenology traits in 63 Populus trichocarpa clones earlier preliminary selected for growth at northern latitudes, in three different field sites; i.e. in central Sweden, eastern and western Latvia. The material showed moderate broad sense heritabilities, with high values for phenology traits, indicating opportunities for selection. Genotype × environment (g × e) interaction was identified for all traits, but the phenotypic correlation between pairs of sites provided more detailed information indicating the strength of the g × e interaction. The between-clone variation in plasticity was high, and we identified some clones showing a high and stable performance across the three sites. These clones are of particular interest for the commercial deployment and future breeding of Populus material for the Nordic–Baltic region. [ABSTRACT FROM AUTHOR]

An earlier identified major quantitative trait locus for resistance towards the willow leaf rust fungus Melampsora larici-epitea in a Salix viminalis x (S. viminalis × S. schwerinii) population was used to identify potential resistance genes to the rust pathogen. Screening a genomic bacterial artificial chromosome library with markers from the peak position of the QTL region revealed one gene with TIR-NBS-LRR (Toll Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat) domain structure indicative of a resistance gene. The resistance gene analog was denoted RGA1 and further analysis revealed a number of non-synonymous single nucleotide polymorphisms in the LRR domain between the resistant and susceptible Salix genotypes. Gene expression levels under controlled conditions showed a significantly lower constitutive expression of RGA1 in the susceptible genotype. In addition, the susceptible genotype showed a significantly reduced expression level of the RGA1 gene at 24 hours post inoculation with M. larici-epitea. This indicates that the pathogen may actively suppress RGA1 gene expression allowing a compatible plant-pathogen interaction and causing infection. [ABSTRACT FROM AUTHOR]

Plant canopy optimization models predict that leaf nitrogen (N) distribution in the canopy will parallel the vertical light gradient, and numerous studies with many species have confirmed this prediction. Further, it is predicted that for a given canopy leaf area, a low vertical light extinction coefficient will promote rapid growth. Therefore, the ideal canopy of fast-growing plants should combine high leaf area index with a lowlight extinction coefficient; the latter being reflected in a flat vertical leaf N gradient throughout the canopy. Based on data from an experimental Salix stand (six varieties) grown on agricultural land in central Sweden, we tested the hypothesis that shoot growth is correlated with vertical leaf N gradient in canopies of hybrid willows bred for biomass production, which could have implications for Salix breeding. Tree improvement research requires screening of growth-related traits in large numbers of plants, but assessment of canopy leaf N gradients by chemical analysis is expensive, time-consuming and destructive. An alternative to analytical methods is to estimate leaf N gradients nondestructively with an optical chlorophyll meter (SPA method). Here we provide a specific calibration for interpreting SPAD data measured in hybrid willows grown in biomass plantations on fertile agricultural land. Based on SPAD measurements, a significant and inverse relationship (r² = 0.88) was found between shoot biomass growth and vertical leaf N gradient across canopies of six Salix varieties. [ABSTRACT FROM AUTHOR]

Cassava ( Manihot esculenta Crantz) is a starchy root crop grown in the tropics mainly by small-scale farmers even though agro-industrial processing is rapidly increasing. For this processing market improved varieties with high dry matter root content (DMC) is required. Potentially toxic cyanogenic glucosides are synthesized in the leaves and translocated to the roots. Selection for varieties with low cyanogenic glucoside potential (CNP) and high DMC is among the principal objectives in cassava breeding programs. However, these traits are highly influenced by the environmental conditions and the genetic control of these traits is not well understood. An S1 population derived from a cross between two bred cassava varieties (MCOL 1684 and Rayong 1) that differ in CNP and DMC was used to study the heritability and genetic basis of these traits. A broad-sense heritability of 0.43 and 0.42 was found for CNP and DMC, respectively. The moderate heritabilities for DMC and CNP indicate that the phenotypic variation of these traits is explained by a genetic component. We found two quantitative trait loci (QTL) on two different linkage groups controlling CNP and six QTL on four different linkage groups controlling DMC. One QTL for CNP and one QTL for DMC mapped near each other, suggesting pleiotrophy and/or linkage of QTL. The two QTL for CNP showed additive effects while the six QTL for DMC showed additive effect, dominance or overdominance. This study is a first step towards developing molecular marker tools for efficient breeding of CNP and DMC in cassava. [ABSTRACT FROM AUTHOR]

• Phenotypic correlations and quantitative trait loci (QTL) for important growth traits and a surrogate of intrinsic water-use efficiency (leaf δ13C) were analysed in a willow pedigree of 92 full-sibling clones grown under two water regimes. The major objective was to examine the genetic basis of the phenotypic correlations. • Cuttings of Salix were glasshouse-grown during one growing season. The relative growth rate (RGR) and underlying traits were assessed. QTL analysis was conducted based on an available linkage map for Salix. • Leaf area productivity and leaf nitrogen productivity were more important in determining RGR than leaf area ratio and specific leaf area. However, phenotypic correlations among growth traits partly varied between the two environments. QTL were detected for most growth traits, among them many common QTL for different traits. The QTL pattern reflected the phenotypic correlation pattern. None of the QTL for the complex traits was consistent across the different environments. • The results demonstrate a genetic basis for phenotypic correlations among growth traits in Salix, and provide evidence for the existence of ‘master switches’ regulating some of the traits. [ABSTRACT FROM AUTHOR]

In a greenhouse experiment we examined the effect of willow genotype and irrigation regime (moderate drought and well-watered) on plant growth parameters, foliar nitrogen, and phenolic concentrations, as well as on the preference and performance of the blue leaf beetle,Phratora vulgatissima(L.) (Coleoptera: Chrysomelidae). The 10 vegetatively propagated willow genotypes in the experiments were F2 full-sibling hybrids, originated from a cross betweenSalix viminalis(L.) (Salicaceae) (high in condensed tannins) andSalix dasyclados(L.) (Salicaceae) (rich in phenolic glycosides). Insect bioassays were conducted on detached leaves in Petri dishes as well as with free-living insects on intact potted plants. The 10-week long irrigation treatments caused statistically significant phenotypic differences in the potted willow saplings. Total biomass was somewhat higher in the well-watered treatment. The root to total biomass ratio was higher in the drought-treatment plants. There was significant genotypic variation in foliar nitrogen concentrations, and they were higher in the drought-treatment plants. There was also a strong genotypic variation in each of the phenolic substances analyzed. Condensed tannins, which accounted for the greatest proportion of total phenolic mass, were higher in the well-watered treatment. There was, however, no difference in levels of the other phenolics (salicylates, cinnamic acid, flavonoids, and chlorogenic acid) between irrigation treatments. The sum of these phenolics was higher in the well-watered treatment. There was a strong variation inP. vulgatissimalarval development on different willow genotypes, and larval performance was negatively correlated with levels of salicylates and cinnamic acid. There was, however, no effect of irrigation treatment on larval performance.Phratora vulgatissimapreferred to feed on well-watered plants, and we found a preference for oviposition there, but neither feeding nor oviposition site preference was affected by willow genotype. Adult feeding and oviposition preferences were not correlated with larval performance. [ABSTRACT FROM AUTHOR]

Describes the genetic structure of Salix viminalis. Influence of geographic origin and growth traits on genetic structure; Biological causes of high levels of dominance genetic variance; Process of vegetative propagation.

Animal manure represents a major source of renewable energy that can be converted into biogas using anaerobic digestion. In order to most efficiently utilize this resource, it can be co-digested with energy dense, high biomethanation potential feedstocks such as energy crops. However, such feedstocks typically require pretreatments which are not feasible for small-scale facilities. We investigated the use of single-stage and the sequential co-digestion of comminuted but otherwise non-pretreated Salix with animal manure, and further investigated the effects of coppicing frequency and clone choice on biomethanation potential and the area requirements for a typical Swedish farm-scale anaerobic digester using Salix and manure as feedstock. In comparison with conventional single-stage digestion, sequential digestion increased the volumetric and specific methane production by 57% to 577 NmL L−1 d−1 and 192 NmL (g volatile solids (VS))−1, respectively. Biomethanation potential was the highest for the two-year-old shoots, although gains in biomass productivity suggest that every-third-year coppicing may be a better strategy for supplying Salix feedstock for anaerobic digestion. The biomethane production performance of the sequential digestion of minimally pretreated Salix mirrors that of hydrothermally pretreated hardwoods and may provide an option where such pretreatments are not feasible. [ABSTRACT FROM AUTHOR]

Background: Sex chromosomes are in some species largely undifferentiated (homomorphic) with restricted sex determination regions. Homomorphic but different sex chromosomes are found in the closely related genera Populus and Salix indicating flexible sex determination systems, ideal for studies of processes involved in sex chromosome evolution. We have performed genome-wide association studies of sex and analysed sex chromosomes in a population of 265 wild collected Salix viminalis accessions and studied the sex determining locus. Results: A total of 19,592 markers were used in association analyses using both Fisher's exact tests and a single-marker mixed linear model, which resulted in 48 and 41 sex-associated (SA) markers respectively. Across all 48 SA markers, females were much more often heterozygous than males, which is expected if females were the heterogametic sex. The majority of the SA markers were, based on positions in the S. purpurea genome, located on chromosome 15, previously demonstrated to be the sex chromosome. Interestingly, when mapping the genotyping-by-sequencing sequence tag harbouring the two SA markers with the highest significance to the S. viminalis genomic scaffolds, five regions of very high similarity were found: three on a scaffold that represents a part of chromosome 15, one on a scaffold that represents a part of chromosome 9 and one on a scaffold not anchored to the genome. Based on segregation differences of the alleles at the two marker positions and on differences in PCR amplification between females and males we conclude that females had multiple copies of this DNA fragment (chromosome 9 and 15), whereas males only had one (chromosome 9). We therefore postulate that the female specific sequences have been copied from chromosome 9 and inserted on chromosome 15, subsequently developing into a hemizygous W chromosome linked region. Conclusions: Our results support that sex determination in S. viminalis is controlled by one locus on chromosome 15. The segregation patterns observed at the SA markers furthermore confirm that S. viminalis females are the heterogametic sex. We also identified a translocation from chromosome 9 to the W chromosome. [ABSTRACT FROM AUTHOR]

Background: Entering and exiting winter dormancy present important trade-offs between growth and survival at northern latitudes. Many forest trees display local adaptation across latitude in traits associated with these phenology transitions. Transfers of a species outside its native range introduce the species to novel combinations of environmental conditions potentially requiring different combinations of alleles to optimize growth and survival. In this study, we performed genome wide association analyses and a selection scan in a P. trichocarpa mapping population derived from crossings between clones collected across the native range and introduced into Sweden. GWAS analyses were performed using phenotypic data collected across two field seasons and in a controlled phytotron experiment. Results: We uncovered 584 putative candidate genes associated with spring and autumn phenology traits as well as with growth. Many regions harboring variation significantly associated with the initiation of leaf shed and leaf autumn coloring appeared to have been evolving under positive selection in the native environments of P. trichocarpa. A comparison between the candidate genes identified with results from earlier GWAS analyses performed in the native environment found a smaller overlap for spring phenology traits than for autumn phenology traits, aligning well with earlier observations that spring phenology transitions have a more complex genetic basis than autumn phenology transitions. Conclusions: In a small and structured introduced population of P. trichocarpa, we find complex genetic architectures underlying all phenology and growth traits, and identify multiple putative candidate genes despite the limitations of the study population. [ABSTRACT FROM AUTHOR]

Background: Sex chromosomes have evolved independently multiple times in eukaryotes and are therefore considered a prime example of convergent genome evolution. Sex chromosomes are known to emerge after recombination is halted between a homologous pair of chromosomes, and this leads to a range of non-adaptive modifications causing gradual degeneration and gene loss on the sex-limited chromosome. However, the proximal causes of recombination suppression and the pace at which degeneration subsequently occurs remain unclear. Results: Here, we use long- and short-read single-molecule sequencing approaches to assemble and annotate a draft genome of the basket willow, Salix viminalis, a species with a female heterogametic system at the earliest stages of sex chromosome emergence. Our single-molecule approach allowed us to phase the emerging Z and W haplotypes in a female, and we detected very low levels of Z/W single-nucleotide divergence in the non-recombining region. Linked-read sequencing of the same female and an additional male (ZZ) revealed the presence of two evolutionary strata supported by both divergence between the Z and W haplotypes and by haplotype phylogenetic trees. Gene order is still largely conserved between the Z and W homologs, although the W-linked region contains genes involved in cytokinin signaling regulation that are not syntenic with the Z homolog. Furthermore, we find no support across multiple lines of evidence for inversions, which have long been assumed to halt recombination between the sex chromosomes. Conclusions: Our data suggest that selection against recombination is a more gradual process at the earliest stages of sex chromosome formation than would be expected from an inversion and may result instead from the accumulation of transposable elements. Our results present a cohesive understanding of the earliest genomic consequences of recombination suppression as well as valuable insights into the initial stages of sex chromosome formation and regulation of sex differentiation. [ABSTRACT FROM AUTHOR]

An increasing interest to convert lignocellulosic biomass into biofuels has highlighted the potential of using willows for this purpose, due to its fast growth in short rotation coppice systems. Here, we use a mapping population of 463 individuals of a cross between Salix viminalis and S. viminalis × S. schwerinii to investigate the genetic background of different wood chemical traits, information of importance for breeding towards different uses of wood. Furthermore, using a subset of the mapping population, the correlation between biogas production and chemical traits was investigated. The phenotyping of wood was carried by Furrier-transformed-Infrared spectrometry (FT-IR) and water content analysis. Quantitative trait loci (QTLs) analysis was used to identify regions in the genome of importance for the phenotypic variation of these chemical traits. We found 27 QTLs for various traits. On linkage group (LG) VI-1, QTLs for signals assigned to G-lignin, lignin, and the S/G ratio were collocated and on LG XIV we found a cluster of QTLs representing signals assigned to lignin, cellulose, hemicellulose, and water. The QTLs explained from 3.4 to 6.9% of the phenotypic variation indicating a quantitative genetic background where many genes influence the traits. For the biogas production, a positive and negative correlation was seen with the signals assigned to acetyl and lignin, respectively. This study represents a first step in the understanding of the genetic background of wood chemical traits for willows, information needed for complementary studies, mapping of important genes, and for breeding of varieties for biofuel production purposes. [ABSTRACT FROM AUTHOR]

Willow species ( Salix) are important as short-rotation biomass crops for bioenergy, which creates a demand for faster genetic improvement and breeding through deployment of molecular marker-assisted selection ( MAS). To find markers associated with important adaptive traits, such as growth and phenology, for use in MAS, we genetically dissected the trait variation of a Salix viminalis (L.) population of 323 accessions. The accessions were sampled throughout northern Europe and were established at two field sites in Pustnäs, Sweden, and at Woburn, UK, offering the opportunity to assess the impact of genotype-by-environment interactions (G × E) on trait-marker associations. Field measurements were recorded for growth and phenology traits. The accessions were genotyped using 1536 SNP markers developed from phenology candidate genes and from genes previously observed to be differentially expressed in contrasting environments. Association mapping between 1233 of these SNPs and the measured traits was performed taking into account population structure and threshold selection bias. At a false discovery rate ( FDR) of 0.2, 29 SNPs were associated with bud burst, leaf senescence, number of shoots or shoot diameter. The percentage of accession variation ( [ABSTRACT FROM AUTHOR]

Background: Drought is a major environmental stress that can have severe impacts on plant productivity and survival. Understanding molecular mechanisms of drought responses is crucial in order to breed for drought adapted plant cultivars. The aim of the present study was to investigate phenotypic and transcriptional drought responses in two willow genotypes (520 and 592) originating from an experimental cross between S. viminalis × (S. viminalis × S. schwerinii). Willows are woody perennials in the Salicaceae plant family that are grown as bioenergy crops worldwide. Methods: An experiment was conducted where plants were exposed to drought and different eco-physiological parameters were assessed. RNA-seq data was furthermore generated with the Illumina technology from root tips and leaves from plants grown in drought and well-watered (WW) conditions. The RNA-seq data was assembled de novo with the Trinity assembler to create a reference gene set to which the reads were mapped in order to obtain differentially expressed genes (DEGs) between the drought and WW conditions. To investigate molecular mechanisms involved in the drought response, GO enrichment analyses were conducted. Candidate genes with a putative function in the drought response were also identified. Results: A total of 52,599 gene models were obtained and after filtering on gene expression (FPKM ≥ 1), 35,733 gene models remained, of which 24,421 contained open reading frames. A total of 5,112 unique DEGs were identified between drought and WW conditions, of which the majority were found in the root tips. Phenotypically, genotype 592 displayed less growth reduction in response to drought compared to genotype 520. At the transcriptional level, genotype 520 displayed a greater response in the leaves as more DEGs were found in genotype 520 compared to genotype 592. In contrast, the transcriptional responses in the root tips were rather similar between the two genotypes. A core set of candidate genes encoding proteins with a putative function in drought response was identified, for example MYBs and bZIPs as well as chlorophyll a/b binding proteins. Discussion: We found substantial differences in drought responses between the genotypes, both at the phenotypic and transcriptional levels. In addition to the genotypic variation in several traits, we also found indications for genotypic variation in trait plasticity, which could play a role in drought adaptation. Furthermore, the two genotypes displayed overall similar transcriptional responses in the root tips, but more variation in the leaves. It is thus possible that the observed phenotypic differences could be a result of transcriptional differences mostly at the leaf level. Conclusions: This study has contributed to a better general understanding of drought responses in woody plants, specifically in willows, and has implications for breeding research towards more drought adapted plants. [ABSTRACT FROM AUTHOR]

Background and Aims The major objective was to identify plant traits functionally important for optimization of shoot growth and nitrogen (N) economy under drought. Although increased leaf N content (area basis) has been observed in dry environments and theory predicts increased leaf N to be an acclimation to drought, experimental evidence for the prediction is rare. Methods A pedigree of 200 full-sibling hybrid willows was pot-grown in a glasshouse in three replicate blocks and exposed to two water regimes for 3 weeks. Drought conditions were simulated as repeated periods of water shortage. The total leaf mass and area, leaf area efficiency (shoot growth per unit leaf area, EA), area-based leaf N content (NA), total leaf N pool (NL) and leaf N efficiency (shoot growth per unit leaf N, EN) were assessed. Key Results In the water-stress treatment, shoot biomass growth was N limited in the genotypes with low NL, but increasingly limited by other factors in the genotypes with greatest NL. The NA was increased by drought, and drought-induced shift in NA varied between genotypes (significant G × E). Judged from the EA–NA relationship, optimal NA was 16 % higher in the water-stress compared with the well-watered treatment. Biomass allocation to leaves and shoots varied between treatments, but the treatment response of the leaf : shoot ratio was similar across all genotypes. Conclusions It is concluded that N-uptake efficiency and leaf N efficiency are important traits to improve growth under drought. Increased leaf N content (area basis) is an acclimation to optimize N economy under drought. The leaf N content is an interesting trait for breeding of willow bioenergy crops in a climate change future. In contrast, leaf biomass allocation is a less interesting breeding target to improve yield under drought. [ABSTRACT FROM AUTHOR]

Background: Salix (willow) and Populus (poplar) are members of the Salicaceae family and they share many ecological as well as genetic and genomic characteristics. The interest of using willow for biomass production is growing, which has resulted in increased pressure on breeding of high yielding and resistant clones adapted to different environments. The main purpose of this work was to develop dense genetic linkage maps for mapping of traits related to yield and resistance in willow. We used the Populus trichocarpa genome to extract evenly spaced markers and mapped the orthologous loci in the willow genome. The marker positions in the two genomes were used to study genome evolution since the divergence of the two lineages some 45 mya. Results: We constructed two linkage maps covering the 19 linkage groups in willow. The most detailed consensus map, S1, contains 495 markers with a total genetic distance of 2477 cM and an average distance of 5.0 cM between the markers. The S3 consensus map contains 221 markers and has a total genetic distance of 1793 cM and an average distance of 8.1 cM between the markers. We found high degree of synteny and gene order conservation between willow and poplar. There is however evidence for two major interchromosomal rearrangements involving poplar LG I and XVI and willow LG Ib, suggesting a fission or a fusion in one of the lineages, as well as five intrachromosomal inversions. The number of silent substitutions were three times lower (median: 0.12) between orthologs than between paralogs (median: 0.37 - 0.41). Conclusions: The relatively slow rates of genomic change between willow and poplar mean that the genomic resources in poplar will be most useful in genomic research in willow, such as identifying genes underlying QTLs of important traits. Our data suggest that the whole-genome duplication occurred long before the divergence of the two genera, events which have until now been regarded as contemporary. Estimated silent substitution rates were 1.28 × 10-9 and 1.68 × 10-9 per site and year, which are close to rates found in other perennials but much lower than rates in annuals. [ABSTRACT FROM AUTHOR]

Background: Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefinery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. Results: Sugar release (glucose and xylose) after pretreatment and enzymatic saccharification proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h2) of 0.23–0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h2 = 0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker–trait associations below a suggestive significance threshold, including one marker associated with glucose release. Conclusions: We identified lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identified for sugar release, meriting further investigation while also highlighting the difficulties in employing genomewide association mapping for complex traits. [ABSTRACT FROM AUTHOR]