The green ash transcriptome and identification of genes responding to abiotic and biotic stresses.

BACKGROUND: To develop a set of transcriptome sequences to support research on environmental stress responses in green ash (Fraxinus pennsylvanica), we undertook deep RNA sequencing of green ash tissues under various stress treatments. The treatments, including emerald ash borer (EAB) feeding, heat, drought, cold and ozone, were selected to mimic the increasing threats of climate change and invasive pests faced by green ash across its native habitat. RESULTS: We report the generation and assembly of RNA sequences from 55 green ash samples into 107,611 putative unique transcripts (PUTs). 52,899 open reading frames were identified. Functional annotation of the PUTs by comparison to the Uniprot protein database identified matches for 63 % of transcripts and for 98 % of transcripts with ORFs. Further functional annotation identified conserved protein domains and assigned gene ontology terms to the PUTs. Examination of transcript expression across different RNA libraries revealed that expression patterns clustered based on tissues regardless of stress treatment. The transcripts from stress treatments were further examined to identify differential expression. Tens to hundreds of differentially expressed PUTs were identified for each stress treatment. A set of 109 PUTs were found to be consistently up or down regulated across three or more different stress treatments, representing basal stress response candidate genes in green ash. In addition, 1956 simple sequence repeats were identified in the PUTs, of which we identified 465 high quality DNA markers and designed flanking PCR primers. CONCLUSIONS: North American native ash trees have suffered extensive mortality due to EAB infestation, creating a need to breed or select for resistant green ash genotypes. Stress from climate change is an additional concern for longevity of native ash populations. The use of genomics could accelerate management efforts. The green ash transcriptome we have developed provides important sequence information, genetic markers and stress-response candidate genes.

A high-quality reference genome for Fraxinus pennsylvanica for ash species restoration and research.

Green ash (Fraxinus pennsylvanica) is the most widely distributed ash tree in North America. Once common, it has experienced high mortality from the non-native invasive emerald ash borer (EAB; Agrilus planipennis). A small percentage of native green ash trees that remain healthy in long-infested areas, termed "lingering ash," display partial resistance to the insect, indicating that breeding and propagating populations with higher resistance to EAB may be possible. To assist in ash breeding, ecology and evolution studies, we report the first chromosome-level assembly from the genus Fraxinus for F. pennsylvanica with over 99% of bases anchored to 23 haploid chromosomes, spanning 757 Mb in total, composed of 49.43% repetitive DNA, and containing 35,470 high-confidence gene models assigned to 22,976 Asterid orthogroups. We also present results of range-wide genetic variation studies, the identification of candidate genes for important traits including potential EAB-resistance genes, and an investigation of comparative genome organization among Asterids based on this reference genome platform. Residual duplicated regions within the genome probably resulting from a recent whole genome duplication event in Oleaceae were visualized in relation to wild olive (Olea europaea var. sylvestris). We used our F. pennsylvanica chromosome assembly to construct reference-guided assemblies of 27 previously sequenced Fraxinus taxa, including F. excelsior. Thus, we present a significant step forward in genomic resources for research and protection of Fraxinus species.

Development of novel genic microsatellite markers from transcriptome sequencing in sugar maple (Acer saccharum Marsh.).

BACKGROUND: Sugar maple (Acer saccharum Marsh.) is a hardwood tree species native to northeastern North America and economically valued for its wood and sap. Yet, few molecular genetic resources have been developed for this species to date. Microsatellite markers have been a useful tool in population genetics, e.g., to monitor genetic variation and to analyze gene flow patterns. The objective of this study is to develop a reference transcriptome and microsatellite markers in sugar maple. FINDINGS: A set of 117,861 putative unique transcripts were assembled using 29.2 Gb of RNA sequencing data derived from different tissues and stress treatments. From this set of sequences a total of 1068 microsatellite motifs were identified. Out of 58 genic microsatellite markers tested on a population of 47 sugar maple trees in upper Michigan, 22 amplified well, of which 16 were polymorphic and 6 were monomorphic. Values for expected heterozygosity varied from 0.224 to 0.726 for individual loci. Of the 16 polymorphic markers, 15 exhibited transferability to other Acer L. species. CONCLUSIONS: Genic microsatellite markers can be applied to analyze genetic variation in potentially adaptive genes relative to genomic reference markers as a basis for the management of sugar maple genetic resources in the face of climate change.

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing.

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.