Ann-Christin Rönnberg-Wästljung

Willow genotype, but not drought treatment, affects foliar phenolic concentrations and leaf-beetle resistance

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 between Salix viminalis (L.) (Salicaceae) (high in condensed tannins) and Salix 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 in P. vulgatissima larval 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 vulgatissima preferred 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.

QTL analyses of drought tolerance and growth for a Salix dasyclados × Salix viminalis hybrid in contrasting water regimes

Quantitative trait loci (QTL) for growth traits and water-use efficiency have been identified in two water regimes (normal and drought-treated) and for a treatment index. A tetraploid hybrid F2 population originating from a cross between a Salix dasyclados clone (SW901290) and a Salix viminalis clone (‘Jorunn’) was used in the study. The growth response of each individual including both above and below ground dry-matter production (i.e. shoot length, shoot diameter, aboveground and root dry weight, internode length, root dry weight/total dry weight, relative growth rate and leaf nitrogen content) was analysed in a replicated block experiment with two water treatments. A composite interval mapping approach was used to estimate number of QTL, the magnitude of the QTL and their position on genetic linkage maps. QTL specific for each treatment and for the treatment index were found, but QTL common across the treatments and the treatment index were also detected. Each QTL explained from 8% to 29% of the phenotypic variation, depending on trait and treatment. Clusters of QTL for different traits were mapped close to each other at several linkage groups, indicating either a common genetic base or tightly linked QTL. Common QTL identified between treatments and treatment index in the complex trait dry weight can be useful tools in the breeding and selection for drought stress tolerance in Salix.

QTL mapping of resistance to leaf rust in Salix

Genetic mapping of quantitative trait loci (QTL) for resistance to Melampsora leaf rust was performed in two willow families: the progeny from a backcross between Salix viminalis and a hybrid S. viminalisu2009×u2009Salix schwerinii (population S1), and the F1 progeny of a cross between two S. viminalis (population S3). Disease levels were scored in the field for three consecutive years. In the laboratory, five different rust strains were sprayed onto leaf disks and the following resistance components were scored: latent period, diameter and number of uredinia, and flecking. One major QTL and 14 smaller were identified in the S1 host population. One rust strain, that represents a Melampsora form with limited incidence on S. viminalis, showed lower aggressiveness to the S1 host population together with a different pattern in QTLs. In the S3 host population, we detected 13 QTLs for rust resistance, of which two were located to the same genomic regions as those found for the S1 population. We showed that the strongest QTL co-segregated with a gene homologous to a known Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat resistance gene in poplar. The importance of the identified QTLs is discussed in relation to breeding for durable resistance.

Quantitative trait loci for resistance to herbivores in willow: field experiments with varying soils and climates

As a basis for genetic improvement of willow (Salix spp.) for use in wood biomass production, quantitative trait loci (QTLs) responsible for resistance to herbivores have been identified in a tetraploid hybrid F2 population originating from a cross between Salix dasyclados (Wimm.) and Salix viminalis (L.) (Salicaceae). Symptoms of herbivory, caused by various insects and game, and, in addition, leaf rust, were assessed in three field locations with varying soils and climates. Eleven damage traits (lost leaf area, leaf discoloration, leaf blisters, leaf‐mite symptoms, leaf‐margin cuts, and various estimates of shoot‐tip damage by a gall midge, game, and lepidopterans) were submitted to QTL analysis. A composite interval mapping approach was used to estimate the number of QTLs, the magnitude of the QTLs, and their position on genetic linkage maps. Most of the identified QTLs were specific for each trait and location, but a few QTLs common across the locations were also detected. Each QTL explained between 8 and 24% of the phenotypic variation, depending on damage trait and field location. Clusters of QTLs for different traits were found at several linkage groups, indicating either a common genetic base or tightly linked QTL. Our results emphasize the need for verification of QTL studies over different environments.

Resistance to Melampsora larici-epitea leaf rust in Salix: analyses of quantitative trait loci.

Quantitative resistance ofSalix toMelampsora larici-epitea leaf rust was studied in 2Salix mapping populations. One population was a backcross between aS. schwerinii ×S. viminalis hybrid andS. viminalis, and the other was an F2 population betweenS. viminalis andS. dasyclados. A leaf disc bioassay was used to study the components of quantitative resistance (latent period, uredinia number, and uredinia size) to 3 isolates of the leaf rust. The analysis of quantitative trait loci (QTLs) revealed 9 genomic regions in the backcross population and 7 genomic regions in the F2 population that were important for rust resistance, with QTLs explaining 8–26% of the phenotypic variation. An important genomic region was identified for the backcross population in linkage group 2, where QTLs were identified for all resistance components for 2 of the rust isolates. Four of the QTLs had overlapping mapping intervals, demonstrating a common genetic background for latent period, uredinia diameter, and uredinia number. QTLs specific to some rust isolates and to some resistance components were also found, indicating a combination of common and specific mechanisms involved in the various resistance components. Breeding implications in relation to these findings are discussed.

Association mapping in Salix viminalis L. (Salicaceae) – identification of candidate genes associated with growth and phenology

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 ( Radj2 ) explained by these associations ranged from 0.3% to 4.4%, suggesting that the studied traits are controlled by many loci of limited individual impact. Despite this, a SNP in the EARLY FLOWERING 3 gene was repeatedly associated (FDR < 0.2) with bud burst. The rare homozygous genotype exhibited 0.4–1.0 lower bud burst scores than the other genotype classes on a five‐grade scale. Consequently, this marker could be promising for use in MAS and the gene deserves further study. Otherwise, associations were less consistent across sites, likely due to their small Radj2 estimates and to considerable G × E interactions indicated by multivariate association analyses and modest trait accession correlations across sites (0.32–0.61).

International transfer of Alnus, Populus, and Salix germplasm: Early test results

Abstract The interin results of a genetic testing program established within the International Energy Agency (IEA) Forestry Energy Agreement are reported. The ‘Joint Evaluation Activity’ was initiated to evaluate Alnus, Populus and Salix germplasm collected by IEA projects and national programs. The project was designed to compare the growth rates of selected genetic materials under a variety of growing environments, and identify germplasm with potential for biomass cultivation. The project was also designed to examine the limitations of long-distance international transfer of alder, poplar, and willow clones/provenances/species among national biomass programs. Four field tests were established, each containing selected clones and provenances of alder poplar and willow. Three-year growth data are reported here that identify genetic materials with superior growth potential in one or more of the test environments. They also identify the maximum climatic tolerance of several previously untested species/provenances. The results can be used to guide the selection of materials for future exchanges among the national programs participating in the study.

Genetics of phenotypic plasticity and biomass traits in hybrid willows across contrasting environments and years

Abstract Background and Aims Phenotypic plasticity can affect the geographical distribution of taxa and greatly impact the productivity of crops across contrasting and variable environments. The main objectives of this study were to identify genotype–phenotype associations in key biomass and phenology traits and the strength of phenotypic plasticity of these traits in a short-rotation coppice willow population across multiple years and contrasting environments to facilitate marker-assisted selection for these traits. Methods A hybrid Salix viminalisu2009×u2009(S. viminalis × Salix schwerinii) population with 463 individuals was clonally propagated and planted in three common garden experiments comprising one climatic contrast between Sweden and Italy and one water availability contrast in Italy. Several key phenotypic traits were measured and phenotypic plasticity was estimated as the trait value difference between experiments. Quantitative trait locus (QTL) mapping analyses were conducted using a dense linkage map and phenotypic effects of S. schwerinii haplotypes derived from detected QTL were assessed. Key Results Across the climatic contrast, clone predictor correlations for biomass traits were low and few common biomass QTL were detected. This indicates that the genetic regulation of biomass traits was sensitive to environmental variation. Biomass QTL were, however, frequently shared across years and across the water availability contrast. Phenology QTL were generally shared between all experiments. Substantial phenotypic plasticity was found among the hybrid offspring, that to a large extent had a genetic origin. Individuals carrying influential S. schwerinii haplotypes generally performed well in Sweden but less well in Italy in terms of biomass production. Conclusions The results indicate that specific genetic elements of S. schwerinii are more suited to Swedish conditions than to those of Italy. Therefore, selection should preferably be conducted separately for such environments in order to maximize biomass production in admixed S. viminalis × S. schwerinii populations.

Identification of a Differentially Expressed TIR-NBS-LRR Gene in a Major QTL Associated to Leaf Rust Resistance in Salix.

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.