Host genomic influence on bacterial composition in the switchgrass rhizosphere.

Host genetic variation can shape the diversity and composition of associated microbiomes, which may reciprocally influence host traits and performance. While the genetic basis of phenotypic diversity of plant populations in nature has been studied, comparatively little research has investigated the genetics of host effects on their associated microbiomes. Switchgrass (Panicum virgatum) is a highly outcrossing, perennial, grass species with substantial locally adaptive diversity across its native North American range. Here, we compared 383 switchgrass accessions in a common garden to determine the host genotypic influence on rhizosphere bacterial composition. We hypothesized that the composition and diversity of rhizosphere bacterial assemblages would differentiate due to genotypic differences between hosts (potentially due to root phenotypes and associated life history variation). We observed higher alpha diversity of bacteria associated with upland ecotypes and tetraploids, compared to lowland ecotypes and octoploids, respectively. Alpha diversity correlated negatively with flowering time and plant height, indicating that bacterial composition varies along switchgrass life history axes. Narrow-sense heritability (h2 ) of the relative abundance of 21 core bacterial families was observed. Overall compositional differences among tetraploids, due to genetic variation, supports widespread genotypic influence on the rhizosphere microbiome. Tetraploids were only considered due to complexities associated with the octoploid genomes. Lastly, a genome-wide association study identified 1861 single-nucleotide polymorphisms associated with 110 families and genes containing them related to potential regulatory functions. Our findings suggest that switchgrass genomic and life-history variation influences bacterial composition in the rhizosphere, potentially due to host adaptation to local environments.

NaCl-induced CsRCI2E and CsRCI2F interact with aquaporin CsPIP2;1 to reduce water transport in Camelina sativa L.

Rare cold-inducible 2 (RCI2) proteins are small hydrophobic proteins that are known to be localized in cellular membranes. The function of RCI2 proteins has been reported to be associated with low-temperature, salt, and drought stress tolerances as a membrane potential regulator; however, the specific functions are still unknown. The PIP2 (plasma membrane intrinsic protein 2) aquaporins are proteins that transport water and small solutes into the cell. The expression and activity of PIP2 proteins, like RCI2, are also related to salt- and drought-stress tolerance. In this study, we identified novel protein interactions between RCI2 and PIP2; 1, including protein accumulation changes in the bioenergy crop Camelina sativa L. under various NaCl stress conditions. Accumulation of both CsRCI2E and CsRCI2F proteins increased with NaCl stress; however, to differing levels depending on the NaCl stress intensity. A co-immunoprecipitation test revealed interaction between CsRCI2E-CsPIP2 and CsRCI2F-CsPIP2. Moreover, co-expression of the four CsRCI2 proteins with CsPIP2; 1 in Xenopus laevis oocytes reduced water transport activity. Furthermore, the abundance of CsPIP2; 1 protein was decreased under CsRCI2E and CsRCI2F co-expression. These results suggest that NaCl-induced expression of CsRCI2E and CsRCI2F contributes to the regulation of CsPIP2; 1.

Genome-wide association study identifies a major gene for beech bark disease resistance in American beech (Fagus grandifolia Ehrh.).

BACKGROUND: The American Beech tree (Fagus grandifolia Ehrh.), native to eastern North America, is ecologically important and provides high quality wood products. This species is susceptible to beech bark disease (BBD) and is facing high rates of mortality in North America. The disease occurs from an interaction between the woolly beech scale insect (Cryptococcus fagisuga), one of two species of the fungus Neonectria (N. faginata or N. ditissima), and American Beech trees. METHODS: In this case-control genome-wide association study (GWAS), we tested 16 K high quality SNPs using the Affymetrix Axiom 1.5 K - 50 K assay to genotype an association population of 514 individuals. We also conducted linkage analysis in a full-sib family of 115 individuals. Fisher's exact test and logistic regression tests were performed to test associations between SNPs and phenotypes. RESULTS: Association tests revealed four highly significant SNPs on chromosome (Chr) 5 for a single gene (Mt), which encodes a mRNA for metallothionein-like protein (metal ion binding) in Fagus sylvatica. Metallothioneins represent Cys-rich metal chelators able to coordinate metal atoms and may play an important role in the resistance mechanisms against beech scale insect. CONCLUSION: The GWAS study has identified a single locus of major effect contributing to beech bark disease resistance. Knowledge of this genetic locus contributing to resistance might be used in applied breeding, conservation and restoration programs.

High-quality genetic mapping with ddRADseq in the non-model tree Quercus rubra.

BACKGROUND: Restriction site associated DNA sequencing (RADseq) has the potential to be a broadly applicable, low-cost approach for high-quality genetic linkage mapping in forest trees lacking a reference genome. The statistical inference of linear order must be as accurate as possible for the correct ordering of sequence scaffolds and contigs to chromosomal locations. Accurate maps also facilitate the discovery of chromosome segments containing allelic variants conferring resistance to the biotic and abiotic stresses that threaten forest trees worldwide. We used ddRADseq for genetic mapping in the tree Quercus rubra, with an approach optimized to produce a high-quality map. Our study design also enabled us to model the results we would have obtained with less depth of coverage. RESULTS: Our sequencing design produced a high sequencing depth in the parents (248×) and a moderate sequencing depth (15×) in the progeny. The digital normalization method of generating a de novo reference and the SAMtools SNP variant caller yielded the most SNP calls (78,725). The major drivers of map inflation were multiple SNPs located within the same sequence (77% of SNPs called). The highest quality map was generated with a low level of missing data (5%) and a genome-wide threshold of 0.025 for deviation from Mendelian expectation. The final map included 849 SNP markers (1.8% of the 78,725 SNPs called). Downsampling the individual FASTQ files to model lower depth of coverage revealed that sequencing the progeny using 96 samples per lane would have yielded too few SNP markers to generate a map, even if we had sequenced the parents at depth 248×. CONCLUSIONS: The ddRADseq technology produced enough high-quality SNP markers to make a moderately dense, high-quality map. The success of this project was due to high depth of coverage of the parents, moderate depth of coverage of the progeny, a good framework map, an optimized bioinformatics pipeline, and rigorous premapping filters. The ddRADseq approach is useful for the construction of high-quality genetic maps in organisms lacking a reference genome if the parents and progeny are sequenced at sufficient depth. Technical improvements in reduced representation sequencing (RRS) approaches are needed to reduce the amount of missing data.

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.

Substantial genome synteny preservation among woody angiosperm species: comparative genomics of Chinese chestnut (Castanea mollissima) and plant reference genomes.

BACKGROUND: Chinese chestnut (Castanea mollissima) has emerged as a model species for the Fagaceae family with extensive genomic resources including a physical map, a dense genetic map and quantitative trait loci (QTLs) for chestnut blight resistance. These resources enable comparative genomics analyses relative to model plants. We assessed the degree of conservation between the chestnut genome and other well annotated and assembled plant genomic sequences, focusing on the QTL regions of most interest to the chestnut breeding community. RESULTS: The integrated physical and genetic map of Chinese chestnut has been improved to now include 858 shared sequence-based markers. The utility of the integrated map has also been improved through the addition of 42,970 BAC (bacterial artificial chromosome) end sequences spanning over 26 million bases of the estimated 800 Mb chestnut genome. Synteny between chestnut and ten model plant species was conducted on a macro-syntenic scale using sequences from both individual probes and BAC end sequences across the chestnut physical map. Blocks of synteny with chestnut were found in all ten reference species, with the percent of the chestnut physical map that could be aligned ranging from 10 to 39 %. The integrated genetic and physical map was utilized to identify BACs that spanned the three previously identified QTL regions conferring blight resistance. The clones were pooled and sequenced, yielding 396 sequence scaffolds covering 13.9 Mbp. Comparative genomic analysis on a microsytenic scale, using the QTL-associated genomic sequence, identified synteny from chestnut to other plant genomes ranging from 5.4 to 12.9 % of the genome sequences aligning. CONCLUSIONS: On both the macro- and micro-synteny levels, the peach, grape and poplar genomes were found to be the most structurally conserved with chestnut. Interestingly, these results did not strictly follow the expectation that decreased phylogenetic distance would correspond to increased levels of genome preservation, but rather suggest the additional influence of life-history traits on preservation of synteny. The regions of synteny that were detected provide an important tool for defining and cataloging genes in the QTL regions for advancing chestnut blight resistance research.

Functional genomics and microbiome profiling of the Asian longhorned beetle (Anoplophora glabripennis) reveal insights into the digestive physiology and nutritional ecology of wood feeding beetles.

BACKGROUND: Wood-feeding beetles harbor an ecologically rich and taxonomically diverse assemblage of gut microbes that appear to promote survival in woody tissue, which is devoid of nitrogen and essential nutrients. Nevertheless, the contributions of these apparent symbionts to digestive physiology and nutritional ecology remain uncharacterized in most beetle lineages. RESULTS: Through parallel transcriptome profiling of beetle- and microbial- derived mRNAs, we demonstrate that the midgut microbiome of the Asian longhorned beetle (Anoplophora glabripennis), a member of the beetle family Cerambycidae, is enriched in biosynthetic pathways for the synthesis of essential amino acids, vitamins, and sterols. Consequently, the midgut microbiome of A. glabripennis can provide essential nutrients that the beetle cannot obtain from its woody diet or synthesize itself. The beetle gut microbiota also produce their own suite of transcripts that can enhance lignin degradation, degrade hemicellulose, and ferment xylose and wood sugars. An abundance of cellulases from several glycoside hydrolase families are expressed endogenously by A. glabripennis, as well as transcripts that allow the beetle to convert microbe-synthesized essential amino acids into non-essential amino acids. A. glabripennis and its gut microbes likely collaborate to digest carbohydrates and convert released sugars and amino acid intermediates into essential nutrients otherwise lacking from their woody host plants. CONCLUSIONS: The nutritional provisioning capabilities of the A. glabripennis gut microbiome may contribute to the beetles' unusually broad host range. The presence of some of the same microbes in the guts of other Cerambycidae and other wood-feeding beetles suggests that partnerships with microbes may be a facilitator of evolutionary radiations in beetles, as in certain other groups of insects, allowing access to novel food sources through enhanced nutritional provisioning.

Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases.

BACKGROUND: The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp. RESULTS: We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase. CONCLUSIONS: Genome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the upregulated secreted proteins in the necrotrophic phase are hypothesized to be actively attacking the plant cell walls and plant cellular components resulting in necrosis. These genes are being used to develop a new understanding of how this disease interaction progresses and to identify potential targets to reduce the impact of this devastating disease.

FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar.

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles of vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.

Midgut transcriptome profiling of Anoplophora glabripennis, a lignocellulose degrading cerambycid beetle.

BACKGROUND: Wood-feeding insects often work in collaboration with microbial symbionts to degrade lignin biopolymers and release glucose and other fermentable sugars from recalcitrant plant cell wall carbohydrates, including cellulose and hemicellulose. Here, we present the midgut transcriptome of larval Anoplophora glabripennis, a wood-boring beetle with documented lignin-, cellulose-, and hemicellulose- degrading capabilities, which provides valuable insights into how this insect overcomes challenges associated with feeding in woody tissue. RESULTS: Transcripts from putative protein coding regions of over 9,000 insect-derived genes were identified in the A. glabripennis midgut transcriptome using a combination of 454 shotgun and Illumina paired-end reads. The most highly-expressed genes predicted to encode digestive-related enzymes were trypsins, carboxylesterases, ß-glucosidases, and cytochrome P450s. Furthermore, 180 unigenes predicted to encode glycoside hydrolases (GHs) were identified and included several GH 5, 45, and 48 cellulases, GH 1 xylanases, and GH 1 ß-glucosidases. In addition, transcripts predicted to encode enzymes involved in detoxification were detected, including a substantial number of unigenes classified as cytochrome P450s (CYP6B) and carboxylesterases, which are hypothesized to play pivotal roles in detoxifying host tree defensive chemicals and could make important contributions to A. glabripennis' expansive host range. While a large diversity of insect-derived transcripts predicted to encode digestive and detoxification enzymes were detected, few transcripts predicted to encode enzymes required for lignin degradation or synthesis of essential nutrients were identified, suggesting that collaboration with microbial enzymes may be required for survival in woody tissue. CONCLUSIONS: A. glabripennis produces a number of enzymes with putative roles in cell wall digestion, detoxification, and nutrient extraction, which likely contribute to its ability to thrive in a broad range of host trees. This system is quite different from the previously characterized termite fermentation system and provides new opportunities to discover enzymes that could be exploited for cellulosic ethanol biofuel production or the development of novel methods to control wood-boring pests.

Populus euphratica: the transcriptomic response to drought stress.

Populus euphratica Olivier is widely established in arid and semiarid regions but lags in the availability of transcriptomic resources in response to water deficiency. To investigate the mechanisms that allow P. euphratica to maintain growth in arid regions, the responses of the plant to soil water deficit were analyzed at a systems level using physiological and pyrosequencing approaches. We generated 218,601 and 287,120 reads from non-stressed control and drought-stressed P. euphratica leaves respectively, totaling over 200 million base pairs. After assembly, 24,013 transcripts were yielded with an average length of 1,128 bp. We determined 2,279 simple sequence repeats, which may have possible information for understanding drought adaption of woody plants. Stomatal closure was inhibited under moderate drought to maintain a relatively high rate of CO2 assimilation and water transportation, which was supposed to be important for P. euphratica to maintain normal growth and develop vigorous root systems in an adverse environment. This was accompanied by strong transcriptional remodeling of stress-perception, signaling and transcription regulation, photoprotective system, oxidative stress detoxification, and other stress responsive genes. In addition, genes involved in stomatal closure inhibition, ascorbate-glutathione pathway and ubiquitin-proteasome system that may specially modulate the drought stress responses of P. euphratica are highlighted. Our analysis provides a comprehensive picture of how P. euphratica responds to drought stress at physiological and transcriptome levels which may help to understand molecular mechanisms associated with drought response and could be useful for genetic engineering of woody plants.

Wood chemistry analysis and expression profiling of a poplar clone expressing a tyrosine-rich peptide.

KEY MESSAGE: Our study has identified pathways and gene candidates that may be associated with the greater flexibility and digestibility of the poplar cell walls. With the goal of facilitating lignin removal during the utilization of woody biomass as a biofuel feedstock, we previously transformed a hybrid poplar clone with a partial cDNA sequence encoding a tyrosine- and hydroxyproline-rich glycoprotein from parsley. A number of the transgenic lines released more polysaccharides following protease digestion and were more flexible than wild-type plants, but otherwise normal in phenotype. Here, we report that overexpression of the tyrosine-rich peptide encoding sequence in these transgenic poplar plants did not significantly alter total lignin quantity or quality (S/G lignin ratio), five- and six-carbon sugar contents, growth rate, or susceptibility to a major poplar fungal pathogen, Septoria musiva. Whole-genome microarray analysis revealed a total of 411 differentially expressed transcripts in transgenic lines, all with decreased transcript abundance relative to wild-type plants. Their corresponding genes were overrepresented in functional categories such as secondary metabolism, amino acid metabolism, and energy metabolism. Transcript abundance was decreased primarily for five types of genes encoding proteins involved in cell-wall organization and in lignin biosynthesis. The expression of a subset of 19 of the differentially regulated genes by qRT-PCR validated the microarray results. Our study has identified pathways and gene candidates that may be the underlying cause for the enhanced flexibility and digestibility of the stems of poplar plants expressing the TYR transgene.

Genome-wide association study of 23 flowering phenology traits and 4 floral agronomic traits in tree peony (Paeonia section Moutan DC.) reveals five genes known to regulate flowering time.

Tree peony is a unique traditional flower in China, with large, fragrant, and colorful flowers. However, a relatively short and concentrated flowering period limits the applications and production of tree peony. A genome-wide association study (GWAS) was conducted to accelerate molecular breeding for the improvement of flowering phenology traits and ornamental phenotypes in tree peony. A diverse panel of 451 tree peony accessions was phenotyped for 23 flowering phenology traits and 4 floral agronomic traits over 3 years. Genotyping by sequencing (GBS) was used to obtain a large number of genome-wide single-nucleotide polymorphisms (SNPs) (107 050) for the panel genotypes, and 1047 candidate genes were identified by association mapping. Eighty-two related genes were observed during at least 2 years for flowering, and seven SNPs repeatedly identified for multiple flowering phenology traits over multiple years were highly significantly associated with five genes known to regulate flowering time. We validated the temporal expression profiles of these candidate genes and highlighted their possible roles in the regulation of flower bud differentiation and flowering time in tree peony. This study shows that GWAS based on GBS can be used to identify the genetic determinants of complex traits in tree peony. The results expand our understanding of flowering time control in perennial woody plants. Identification of markers closely related to these flowering phenology traits can be used in tree peony breeding programs for important agronomic traits.

Chestnut resistance to the blight disease: insights from transcriptome analysis.

BACKGROUND: A century ago, Chestnut Blight Disease (CBD) devastated the American chestnut. Backcross breeding has been underway to introgress resistance from Chinese chestnut into surviving American chestnut genotypes. Development of genomic resources for the family Fagaceae, has focused in this project on Castanea mollissima Blume (Chinese chestnut) and Castanea dentata (Marsh.) Borkh (American chestnut) to aid in the backcross breeding effort and in the eventual identification of blight resistance genes through genomic sequencing and map based cloning. A previous study reported partial characterization of the transcriptomes from these two species. Here, further analyses of a larger dataset and assemblies including both 454 and capillary sequences were performed and defense related genes with differential transcript abundance (GDTA) in canker versus healthy stem tissues were identified. RESULTS: Over one and a half million cDNA reads were assembled into 34,800 transcript contigs from American chestnut and 48,335 transcript contigs from Chinese chestnut. Chestnut cDNA showed higher coding sequence similarity to genes in other woody plants than in herbaceous species. The number of genes tagged, the length of coding sequences, and the numbers of tagged members within gene families showed that the cDNA dataset provides a good resource for studying the American and Chinese chestnut transcriptomes. In silico analysis of transcript abundance identified hundreds of GDTA in canker versus healthy stem tissues. A significant number of additional DTA genes involved in the defense-response not reported in a previous study were identified here. These DTA genes belong to various pathways involving cell wall biosynthesis, reactive oxygen species (ROS), salicylic acid (SA), ethylene, jasmonic acid (JA), abscissic acid (ABA), and hormone signalling. DTA genes were also identified in the hypersensitive response and programmed cell death (PCD) pathways. These DTA genes are candidates for host resistance to the chestnut blight fungus, Cryphonectria parasitica. CONCLUSIONS: Our data allowed the identification of many genes and gene network candidates for host resistance to the chestnut blight fungus, Cryphonectria parasitica. The similar set of GDTAs in American chestnut and Chinese chestnut suggests that the variation in sensitivity to this pathogen between these species may be the result of different timing and amplitude of the response of the two to the pathogen infection. Resources developed in this study are useful for functional genomics, comparative genomics, resistance breeding and phylogenetics in the Fagaceae.

Integrated analysis of miRNAome transcriptome and degradome reveals miRNA-target modules governing floral florescence development and senescence across early- and late-flowering genotypes in tree peony.

As a candidate national flower of China, tree peony has extremely high ornamental, medicinal and oil value. However, the short florescence and rarity of early-flowering and late-flowering varieties restrict further improvement of the economic value of tree peony. Specific miRNAs and their target genes engaged in tree peony floral florescence, development and senescence remain unknown. This report presents the integrated analysis of the miRNAome, transcriptome and degradome of tree peony petals collected from blooming, initial flowering, full blooming and decay stages in early-flowering variety Paeonia ostii 'Fengdan', an early-flowering mutant line of Paeonia ostii 'Fengdan' and late-flowering variety Paeonia suffruticosa 'Lianhe'. Transcriptome analysis revealed a transcript ('psu.G.00014095') which was annotated as a xyloglucan endotransglycosylase/hydrolase precursor XTH-25 and found to be differentially expressed across flower developmental stages in Paeonia ostii 'Fengdan' and Paeonia suffruticosa 'Lianhe'. The miRNA-mRNA modules were presented significant enrichment in various pathways such as plant hormone signal transduction, indole alkaloid biosynthesis, arachidonic acid metabolism, folate biosynthesis, fatty acid elongation, and the MAPK signaling pathway. Multiple miRNA-mRNA-TF modules demonstrated the potential functions of MYB-related, bHLH, Trihelix, NAC, GRAS and HD-ZIP TF families in floral florescence, development, and senescence of tree peony. Comparative spatio-temporal expression investigation of eight floral-favored miRNA-target modules suggested that transcript 'psu.T.00024044' and microRNA mtr-miR166g-5p are involved in the floral florescence, development and senescence associated agronomic traits of tree peony. The results might accelerate the understanding of the potential regulation mechanism in regards to floral florescence, development and abscission, and supply guidance for tree peony breeding of varieties with later and longer florescence characteristics.

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.

Lignin degradation in wood-feeding insects.

The aromatic polymer lignin protects plants from most forms of microbial attack. Despite the fact that a significant fraction of all lignocellulose degraded passes through arthropod guts, the fate of lignin in these systems is not known. Using tetramethylammonium hydroxide thermochemolysis, we show lignin degradation by two insect species, the Asian longhorned beetle (Anoplophora glabripennis) and the Pacific dampwood termite (Zootermopsis angusticollis). In both the beetle and termite, significant levels of propyl side-chain oxidation (depolymerization) and demethylation of ring methoxyl groups is detected; for the termite, ring hydroxylation is also observed. In addition, culture-independent fungal gut community analysis of A. glabripennis identified a single species of fungus in the Fusarium solani/Nectria haematococca species complex. This is a soft-rot fungus that may be contributing to wood degradation. These results transform our understanding of lignin degradation by wood-feeding insects.

Shotgun proteomic analysis of yeast-elicited California poppy (Eschscholzia californica) suspension cultures producing enhanced levels of benzophenanthridine alkaloids.

The California poppy, Eschscholzia californica, produces benzophenanthridine alkaloids (BPAs), an important class of biologically active compounds. Cell cultures of E. californica were investigated as an alternative and scalable method for producing these valuable compounds; treatment with yeast extract increased production from low levels to 23 mg/g dry weight (DW) of BPAs. A shotgun proteomic analysis of E. californica cell cultures was undertaken to explore changes in metabolism associated with enhanced BPA production. We implemented differential centrifugation and then shotgun proteomics based on nanoliquid chromatography/mass spectrometry (nano-LC-MS/MS) for peptide separation and analysis. A unigene database available for E. californica was translated and utilized for protein identification. Approximately 646 proteins (3% false discovery rate at the protein level) were identified. Differentially abundant proteins observed with elicitation included enzymes involved in (S)-adenosyl methionine (SAM) biosynthesis and BPA biosynthesis. These results demonstrate (1) the identification of proteins from a medicinal plant using shotgun proteomics combined with a well-annotated, translated unigene database and (2) the potential utility of proteomics for exploring changes in metabolism associated with enhanced secondary metabolite production.

Genes acquired by horizontal transfer are potentially involved in the evolution of phytopathogenicity in Moniliophthora perniciosa and Moniliophthora roreri, two of the major pathogens of cacao.

Moniliophthora perniciosa and Moniliophthora roreri are phytopathogenic basidiomycete species that infect cacao causing two important diseases in this crop: "Witches' Broom" and "Frosty Pod Rot", respectively. The ability of species from this genus (Moniliophthora) to cause disease is exceptional in the family Marasmiaceae. Species in closely related genera including, Marasmius, Crinipellis, and Chaetocalathus, are mainly saprotrophs and are not known to cause disease. In this study, the possibility that this phytopathogenic lifestyle has been acquired by horizontal gene transfer (HGT) was investigated. A stringent genome comparison pipeline was used to identify potential genes that have been obtained by Moniliophthora through HGT. This search led to the identification of three genes: a metallo-dependent hydrolase (MDH), a mannitol phosphate dehydrogenase (MPDH), and a family of necrosis-inducing proteins (NEPs). Phylogenetic analysis of these genes suggests that Moniliophthora acquired NEPs from oomycetes, MDH from actinobacteria and MPDH from firmicutes. Based on the known gene functions and on previous studies of M. perniciosa infection and development, a correlation between gene acquisition and the evolution of the phytopathogenic genus Moniliophthora can be postulated.

Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides x P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence.

BACKGROUND: Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR. RESULTS: CAD family genes were distributed in three classes based on sequence conservation. All the three classes are represented by seedless as well as seed plants, including the class of bona fide lignin pathway genes. The expression of some CAD/CAD-like genes that are not associated with xylem development were induced following herbivore damage in leaves, while other genes were induced in only bark or xylem tissues. Five of the CAD/CAD-like genes, however, showed a shift in expression from one tissue to another between non-treated and herbivore-treated plants. Systemic expression of the CAD/CAD-like genes was generally suppressed. CONCLUSIONS: Our results indicated a correlation between the evolution of the CAD gene family and lignin and that the three classes of genes may have evolved in the ancestor of land plants. Our results also suggest that the CAD/CAD-like genes have evolved a diversity of expression profiles and potentially different functions, but that they are nonetheless co-regulated under stress conditions.