Kelly J. Vining

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Epigenetic regulation of adaptive responses of forest tree species to the environment

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular “memory”. Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

Genome resequencing reveals multiscale geographic structure and extensive linkage disequilibrium in the forest tree Populus trichocarpa

• Plant population genomics informs evolutionary biology, breeding, conservation and bioenergy feedstock development. For example, the detection of reliable phenotype-genotype associations and molecular signatures of selection requires a detailed knowledge about genome-wide patterns of allele frequency variation, linkage disequilibrium and recombination. • We resequenced 16 genomes of the model tree Populus trichocarpa and genotyped 120 trees from 10 subpopulations using 29,213 single-nucleotide polymorphisms. • Significant geographic differentiation was present at multiple spatial scales, and range-wide latitudinal allele frequency gradients were strikingly common across the genome. The decay of linkage disequilibrium with physical distance was slower than expected from previous studies in Populus, with r(2) dropping below 0.2 within 3-6 kb. Consistent with this, estimates of recent effective population size from linkage disequilibrium (N(e) ≈ 4000-6000) were remarkably low relative to the large census sizes of P. trichocarpa stands. Fine-scale rates of recombination varied widely across the genome, but were largely predictable on the basis of DNA sequence and methylation features. • Our results suggest that genetic drift has played a significant role in the recent evolutionary history of P. trichocarpa. Most importantly, the extensive linkage disequilibrium detected suggests that genome-wide association studies and genomic selection in undomesticated populations may be more feasible in Populus than previously assumed.

Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa

BackgroundCytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation.ResultsWe investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue, then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes.ConclusionsDNA methylation varies in a highly gene- and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements.

A genetic linkage map of black raspberry (Rubus occidentalis) and the mapping of Ag 4 conferring resistance to the aphid Amphorophora agathonica

Key messageWe have constructed a densely populated, saturated genetic linkage map of black raspberry and successfully placed a locus for aphid resistance.AbstractBlack raspberry (Rubus occidentalis L.) is a high-value crop in the Pacific Northwest of North America with an international marketplace. Few genetic resources are readily available and little improvement has been achieved through breeding efforts to address production challenges involved in growing this crop. Contributing to its lack of improvement is low genetic diversity in elite cultivars and an untapped reservoir of genetic diversity from wild germplasm. In the Pacific Northwest, where most production is centered, the current standard commercial cultivar is highly susceptible to the aphid Amphorophora agathonica Hottes, which is a vector for the Raspberry mosaic virus complex. Infection with the virus complex leads to a rapid decline in plant health resulting in field replacement after only 3–4 growing seasons. Sources of aphid resistance have been identified in wild germplasm and are used to develop mapping populations to study the inheritance of these valuable traits. We have constructed a genetic linkage map using single-nucleotide polymorphism and transferable (primarily simple sequence repeat) markers for F1 population ORUS 4305 consisting of 115 progeny that segregate for aphid resistance. Our linkage map of seven linkage groups representing the seven haploid chromosomes of black raspberry consists of 274 markers on the maternal map and 292 markers on the paternal map including a morphological locus for aphid resistance. This is the first linkage map of black raspberry and will aid in developing markers for marker-assisted breeding, comparative mapping with other Rubus species, and enhancing the black raspberry genome assembly.

Genotyping-by-sequencing enables linkage mapping in three octoploid cultivated strawberry families

Genotyping-by-sequencing (GBS) was used to survey genome-wide single-nucleotide polymorphisms (SNPs) in three biparental strawberry (Fragaria × ananassa) populations with the goal of evaluating this technique in a species with a complex octoploid genome. GBS sequence data were aligned to the F. vesca ‘Fvb’ reference genome in order to call SNPs. Numbers of polymorphic SNPs per population ranged from 1,163 to 3,190. Linkage maps consisting of 30–65 linkage groups were produced from the SNP sets derived from each parent. The linkage groups covered 99% of the Fvb reference genome, with three to seven linkage groups from a given parent aligned to any particular chromosome. A phylogenetic analysis performed using the POLiMAPS pipeline revealed linkage groups that were most similar to ancestral species F. vesca for each chromosome. Linkage groups that were most similar to a second ancestral species, F. iinumae, were only resolved for Fvb 4. The quantity of missing data and heterogeneity in genome coverage inherent in GBS complicated the analysis, but POLiMAPS resolved F. × ananassa chromosomal regions derived from diploid ancestor F. vesca.

Germplasm resources for verticillium wilt resistance breeding and genetics in strawberry (Fragaria)

BACKGROUND: The fungal disease verticillium wilt has been recognized as an obstacle to strawberry production since its initial description in 1931. The full potential of genetic resistance as a solution to this problem has yet to be determined or realized. OBJECTIVE: Our investigations are concerned with defining new sources of resistance to verticillium wilt disease in cultivated and wild strawberry germplasm, and with advancing genetic studies on the basis of resistance/susceptibility. METHODS: We screened 23 diploid, 1 decaploid, and 26 octoploid Fragaria (strawberry) germplasm accessions and cultigens for response to root-dip inoculation with Verticillium dahliae isolate V1. Pedigree relationships of 10 studied cultigens were examined. Crosses were performed between resistant and susceptible accessions. RESULTS: Variability in inoculation response existed within and between species at diploid and octoploid levels. Very or moderately resistant accessions were found within each of three diploid and three octoploid species. Moderately or very susceptible accessions were documented within F. vesca and each octoploid species. Segregation for resistance/susceptibility was evident in progeny populations. CONCLUSIONS: The verticillium wilt resistance ratings reported here and discussed in relation to prior studies adds to the body of publically available knowledge about sources of wilt resistance and susceptibility in Fragaria germplasm.

Evaluation of genetic diversity among Russet potato clones and varieties from breeding programs across the United States

DNA fingerprinting is a powerful tool for plant diversity studies, cultivar identification, and germplasm conservation and management. In breeding programs, fingerprinting and diversity analysis provide an insight into the extent of genetic variability available in the breeding material, which in turn helps breeders to maintain a pool of highly diverse genotypes by avoiding the selection of closely related parents. Oblong-long tubers with russeting skin characterize Russet potato, a primary potato market class in the United States, and especially in the western production regions. The aim of this study was to estimate the level of genetic diversity within this market class potato, utilizing clones and varieties from various breeding programs across the United States. A collection of 264 Russet and non-Russet breeding clones and varieties was fingerprinted using 23 highly polymorphic genome-wide simple sequence repeat (SSR) markers, resulting in 142 polymorphic alleles. The number of alleles produced per SSR varied from 2 to 10, with an average of 6.2 alleles per marker. The polymorphic information content and expected heterozygosity of SSRs ranged from 0.37 to 0.89 and 0.50 to 0.89 with an average of 0.77 and 0.81, respectively. Out of these 23 markers, we propose nine SSR markers best suited for fingerprinting Russet potatoes based on polymorphic information content, heterozygosity and ease of scoring. Diversity analysis of these clones suggest that there is significant diversity across the breeding material and the diversity has been evenly distributed among all the regional breeding programs.

Sequence and Analysis of the Black Raspberry ( Rubus occidentalis ) Genome

The US Pacific Northwest is the primary production region of black raspberry, and this high-value specialty crop has been underutilized for several decades. Black raspberries contain high levels of anthocyanins and other bioactive compounds, which has sparked a renewed interest in breeding programs and cultivation. Despite this potential, black raspberry stands have seen a marked decline that many attribute to disease pressures and only three new cultivars have been released over the last 20 years. Here we discuss the available genomic resources for black raspberry, including the recently released draft genome. These resources will expedite marker-assisted improvement of raspberry with applications across the Rosaceae family. The 243 Mb black raspberry genome was sequenced using an Illumina-based whole genome shotgun sequencing approach, and a chromosome-scale assembly was generated using a high-density genetic map. Black raspberry is the sixth genome to be sequenced in the Rosaceae facilitating in-depth comparative genomics across the family. Black raspberry and the diploid wild strawberry (Fragaria vesca) are largely collinear with some lineage-specific structural rearrangements. The genome has 28,005 genes which is comparable to other Rosaceae species and includes a number of recently duplicated genes which may be related to domestication. Gene expression atlases during fruit ripening and Verticillium inoculation provide insights into ripening and disease resistance, respectively. Together the resources discussed here will provide tools for the improved understanding and breeding of Rosaceae crops.

Characterization of aphid resistance loci in black raspberry (Rubus occidentalis L.)

Viruses vectored by the aphid Amphorophora agathonica cause decline in black raspberry plant health resulting in a shortened life and poor fruit quality of the infected plantings. New aphid resistant cultivars could increase the longevity of plantings providing growers and processors with consistent fruit production. Recent exploration of the native range of black raspberry identified three sources of aphid resistance: Ag4 from Ontario (ON), Canada, Ag5 from Maine (ME), and a third source from Michigan (MI) with no formal designation. The objectives of this study were to assess segregation of these three sources of aphid resistance in populations with single and combined sources and develop markers that can identify each source of resistance. A genetic linkage map constructed for ORUS 4305 placed the ON aphid resistance locus on Rubus linkage group (RLG) 6. Segregation ratios in populations with single and combined sources, and linkage mapping in two populations (ORUS 4304 and ORUS 4812) segregating for the Ag5 and MI sources, respectively, indicated that these three sources of resistance are each conferred by single dominant genes/alleles that are linked on RLG6. Confirmation of marker association in 16 validation populations identified four markers that could be used to predict resistance; however, none could distinguish between the ON and MI sources. These four markers may be useful for screening populations to enrich the field-planted progeny for aphid resistance. Fine mapping of the resistance loci is needed to develop functional markers at each of the resistance loci to enable pyramiding and durable aphid resistance.