[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Background:
Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., an endangered ancient legume species, endemic to the Gobi desert in north-western China. As the only evergreen broadleaf shrub in this area, A. mongolicus plays an important role in the region's ecological-environmental stability. Despite the strong potential of A. mongolicus in providing new insights on drought tolerance, sequence information on the species in public databases remains scarce. To both learn about the role of gene expression in drought stress tolerance in A. mongolicus and to expand genomic resources for the species, transcriptome sequencing of stress-treated A. mongolicus plants was performed.
Using 454 pyrosequencing technology, 8,480 and 7,474 contigs were generated after de novo assembly of RNA sequences from leaves of untreated and drought-treated plants, respectively. After clustering using TGICL and CAP3 programs, a combined assembly of all reads produced a total of 11,357 putative unique transcripts (PUTs). Functional annotation and classification of the transcripts were conducted by aligning the 11,357 PUTs against the public protein databases and nucleotide database (Nt). Between control and drought-treated plants, 1,620 differentially expressed genes (DEGs) were identified, of which 1,106 were up-regulated and 514 were down-regulated. The differential expression of twenty candidate genes in metabolic pathways and transcription factors families related to stress-response were confirmed by quantitative real-time PCR. Representatives of several large gene families, such as WRKY and P5CS, were identified and verified in A. mongolicus for the first time.
The additional transcriptome resources, gene expression profiles, functional annotations, and candidate genes provide a more comprehensive understanding of the stress response pathways in xeric-adapted plant species such as A. mongolicus.
[Show abstract][Hide abstract] ABSTRACT: A set of seven new nuclear microsatellite markers (nSSRs) was developed for sugar maple (Acer saccharum Marsh.) using paired-end Illumina sequencing. Out of 96 primers screened in a panel of six unrelated individuals, seven markers amplified polymorphic products. The utility of these markers, in addition to six already published microsatellites, for genetic variation and gene flow studies was assessed. Out of the seven newly developed markers, three amplified multiple fragments and were interpreted as dominant (absence/presence) markers, while four markers amplified a maximum of two amplification products per sample. The six published microsatellites and three of the four newly developed markers showed regular segregation in an open-pollinated single tree progeny. Observed heterozygosity (Ho) and expected heterozygosity (He) in 48 individuals from one population ranged from 0.436 to 0.917 and from 0.726 to 0.894, respectively. Dominant markers revealed 64 variable positions and moderate genetic variation with- in the population (He = 0.102, Shannon’s I = 0.193). Paternity analyses in the program CERVUS at co-dominant markers showed effective dispersal of pollen in the sugar maple population both at 95% and 80% confidence levels. Dependent on the confidence level, the mean pollen dispersal distance within the population ranged from 33.25 m to 38.75 m and gene flow from outside the stand from 78% to 82%. The absence of fine-scale Spatial Genetic Structure (SGS) suggested effective dispersal of both seeds and pollen.
Preview · Article · Jan 2015 · Annals of Forest Research
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Aquaporin (AQP) proteins are involved in water homeostasis in cells at all taxonomic levels of life. Phosphorylation of some AQPs has been proposed to regulate water permeability via gating of the channel itself. We analyzed plasma membrane intrinsic proteins (PIP) from Camelina and characterized their biological functions under both stressful and favorable conditions. A three-dimensional theoretical model of the Camelina AQP proteins was built by homology modeling which could prove useful in further functional characterization of AQPs. CsPIP2;1 was strongly and constitutively expressed in roots and leaves of Camelina, suggesting that this gene is related to maintenance of homeostasis during salt and drought stresses. CsPIP2s exhibited water channel activity in Xenopus oocytes. We then examined the roles of CsPIP2;1 phosphorylation at Ser273 and Ser277 in the regulation of water permeability using phosphorylation mutants. A single deletion strain of CsPIP2;1 was generated to serve as the primary host for testing AQP expression constructs. A Ser277 to alanine mutation (to prevent phosphorylation) did not change CsPIP2;1 water permeability while a Ser273 mutation to alanine did affect water permeability. Furthermore, a CsPIP2;1 point mutation when ectopically expressed in yeast resulted in lower growth in salt and drought conditions compared with controls, and confirmation of Ser273 as the phosphorylation site. Our results support the idea that post-translational modifications in the Ser273 regulatory domains of the C-terminus fine tune water flux through CsPIP2;1.
No preview · Article · Sep 2014 · Journal of Plant Physiology
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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, beta-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 beta-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.
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.
[Show abstract][Hide abstract] ABSTRACT: • Premise of the study: Fourteen genomic microsatellite markers were developed and characterized in honey locust, Gleditsia triacanthos, using Illumina sequencing. Due to their high variability, these markers can be applied in analyses of genetic diversity and structure, and in mating system and gene flow studies.
• Methods and Results: Thirty-six individuals from across the species range were included in a genetic diversity analysis and yielded three to 20 alleles per locus. Observed heterozygosity and expected heterozygosity ranged from 0.214 to 0.944 and from 0.400 to 0.934, respectively, with minimal occurrence of null alleles. Regular segregation of maternal alleles was observed at seven loci and moderate segregation distortion at four of 11 loci that were heterozygous in the seed parent.
• Conclusions: Honey locust is an important agroforestry tree capable of very fast growth and tolerance of poor site conditions. This is the first report of genomic microsatellites for this species.
Full-text · Article · Dec 2013 · APPLICATIONS IN PLANT SCIENCES
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The Asian longhorned beetle (Anoplophoraglabripennis) is an invasive, wood-boring pest that thrives in the heartwood of deciduous tree species. A large impediment faced by A. glabripennis as it feeds on woody tissue is lignin, a highly recalcitrant biopolymer that reduces access to sugars and other nutrients locked in cellulose and hemicellulose. We previously demonstrated that lignin, cellulose, and hemicellulose are actively deconstructed in the beetle gut and that the gut harbors an assemblage of microbes hypothesized to make significant contributions to these processes. While lignin degrading mechanisms have been well characterized in pure cultures of white rot basidiomycetes, little is known about such processes in microbial communities associated with wood-feeding insects. The goals of this study were to develop a taxonomic and functional profile of a gut community derived from an invasive population of larval A. glabripennis collected from infested host trees and to identify genes that could be relevant for the digestion of woody tissue and nutrient acquisition. To accomplish this goal, we taxonomically and functionally characterized the A. glabripennis midgut microbiota through amplicon and shotgun metagenome sequencing and conducted a large-scale comparison with the metagenomes from a variety of other herbivore-associated communities. This analysis distinguished the A. glabripennis larval gut metagenome from the gut communities of other herbivores, including previously sequenced termite hindgut metagenomes. Genes encoding enzymes were identified in the A. glabripennis gut metagenome that could have key roles in woody tissue digestion including candidate lignin degrading genes (laccases, dye-decolorizing peroxidases, novel peroxidases and β-etherases), 36 families of glycoside hydrolases (such as cellulases and xylanases), and genes that could facilitate nutrient recovery, essential nutrient synthesis, and detoxification. This community could serve as a reservoir of novel enzymes to enhance industrial cellulosic biofuels production or targets for novel control methods for this invasive and highly destructive insect.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Jatropha has potential to be an important bio-fuel crop due to such advantages as high seed oil content and the ability to grow well on marginal lands less suited for food crops. Despite its ability to grow on marginal land, Jatropha is still susceptible to high salt and drought stresses, which can significantly reduce plant growth, stomatal conductance, sap-flow rate, and plant sap volume. This study was undertaken to collect basic knowledge of the physiological and molecular aspects of Jatropha response to salt and drought stresses, and to elucidate how Jatropha recovers from stress. From these studies we identified candidate genes that may be useful for the development of Jatropha cultivars that will grow efficiently in arid and barren lands. Of particular interest, two plasma membrane intrinsic proteins were identified: Jatropha plasma membrane intrinsic protein 1 (JcPIP1) and Jatropha plasma membrane intrinsic protein 2 (JcPIP2). The expression levels of JcPIP1 were dramatically increased in roots, stems, and leaves during the recovery from stress, whereas the JcPIP2 gene transcripts levels were induced in roots and stems during the water deficit stress. The protein levels of JcPIP1 and JcPIP2 were consistent with the gene expression patterns. Based on these results, we hypothesized that JcPIP1 plays a role in the recovery events from water stresses, while JcPIP2 is important in early responses to water stress. Virus induced gene silencing technology revealed that both JcPIP1 and JcPIP2 have positive roles in response to water deficit stresses, but have antagonistic functions at the recovery stage. We suggest that both JcPIP1 and JcPIP2 may play important roles in responses to water deficit conditions and both have potential as targets for genetic engineering.
No preview · Article · Mar 2013 · Journal of plant physiology