Alison M. Morse

RNA-seq: technical variability and sampling.

BackgroundRNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript.ResultsIn this study three independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage.ConclusionsTechnical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.

Evolution of Genome Size and Complexity in Pinus

Background Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood. Methodology/Principal Findings Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA. Conclusions/Significance Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes.

Ago HITS-CLIP Expands Understanding of Kaposi's Sarcoma-associated Herpesvirus miRNA Function in Primary Effusion Lymphomas

KSHV is the etiological agent of Kaposis sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentricCastlemans disease (MCD). The fact that KSHV-encoded miRNAs are readily detectable in all KSHV-associated tumors suggests a potential role in viral pathogenesis and tumorigenesis. MiRNA-mediated regulation of gene expression is a complex network with each miRNA having many potential targets, and to date only few KSHV miRNA targets have been experimentally determined. A detailed understanding of KSHV miRNA functions requires high-through putribonomics to globally analyze putative miRNA targets in a cell type-specific manner. We performed Ago HITS-CLIP to identify viral and cellular miRNAs and their cognate targets in two latently KSHV-infected PEL cell lines. Ago HITS-CLIP recovered 1170 and 950 cellular KSHVmiRNA targets from BCBL-1 and BC-3, respectively. Importantly, enriched clusters contained KSHV miRNA seed matches in the 3′UTRs of numerous well characterized targets, among them THBS1, BACH1, and C/EBPβ. KSHV miRNA targets were strongly enriched for genes involved in multiple pathways central for KSHV biology, such as apoptosis, cell cycle regulation, lymphocyte proliferation, and immune evasion, thus further supporting a role in KSHV pathogenesis and potentially tumorigenesis. A limited number of viral transcripts were also enriched by HITS-CLIP including vIL-6 expressed only in a subset of PEL cells during latency. Interestingly, Ago HITS-CLIP revealed extremely high levels of Ago-associated KSHV miRNAs especially in BC-3 cells where more than 70% of all miRNAs are of viral origin. This suggests that in addition to seed match-specific targeting of cellular genes, KSHV miRNAs may also function by hijacking RISCs, thereby contributing to a global de-repression of cellular gene expression due to the loss of regulation by human miRNAs. In summary, we provide an extensive list of cellular and viral miRNA targets representing an important resource to decipher KSHV miRNA function.

The Cytokinin Type-B Response Regulator PtRR13 Is a Negative Regulator of Adventitious Root Development in Populus

Adventitious root formation at the base of plant cuttings is an innate de novo organogenesis process that allows massive vegetative propagation of many economically and ecologically important species. The early molecular events following shoot excision are not well understood. Using whole-genome microarrays, we detected significant transcriptome remodeling during 48 h following shoot removal in Populus tremula × Populus alba softwood cuttings in the absence of exogenous auxin, with 27% and 36% of the gene models showing differential abundance between 0 and 6 h and between 6 and 24 h, respectively. During these two time intervals, gene networks involved in protein turnover, protein phosphorylation, molecular transport, and translation were among the most significantly regulated. Transgenic lines expressing a constitutively active form of the Populus type-B cytokinin response regulator PtRR13 (ΔDDKPtRR13) have a delayed rooting phenotype and cause misregulation of CONTINUOUS VASCULAR RING1, a negative regulator of vascularization; PLEIOTROPIC DRUG RESISTANCE TRANSPORTER9, an auxin efflux transporter; and two APETALA2/ETHYLENE RESPONSE FACTOR genes with sequence similarity to TINY. Inappropriate cytokinin action via ΔDDKPtRR13 expression appeared to disrupt adventitious root development 24 h after shoot excision, when root founder cells are hypothesized to be sensitive to the negative effects of cytokinin. Our results are consistent with PtRR13 acting downstream of cytokinin to repress adventitious root formation in intact plants, and that reduced cytokinin signaling after shoot excision enables coordinated expression of ethylene, auxin, and vascularization pathways leading to adventitious root development.

Evolution and diversity of invertase genes in Populus trichocarpa

Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyzes the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families thought to have distinct evolutionary origins and can be distinguished by their pH optima for activity: acid invertases and neutral/alkaline invertases. The acid invertases apparently originated in eubacteria and are targeted to the cell wall and vacuole, while neutral/alkaline invertases apparently originated in cyanobacteria and function in the cytosol. The recently sequenced genome of Populus trichocharpa (Torr. and Gray) allowed us to identify the genes encoding invertase in this woody perennial. Here we describe the identification of eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1–3), and five of which belong to the cell wall targeted group (PtCIN1–5). Similarly, we report the identification of 16 neutral/alkaline invertase genes (PtNIN1–16). Expression analyses using whole genome microarrays and RT-PCR reveal evidence for expression of all invertase family members. An examination of the micro-syntenic regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis invertases. We also find evidence for expression of a novel intronless vacuolar invertase (PtVIN1), which apparently arose from a processed PtVIN2 transcript that re-inserted into the genome. To our knowledge, this is the first intronless invertase found in plants. This work increases the understanding of the role this family plays in carbon allocation and partitioning in forest trees as well as its evolutionary development.

Exploring Diversification and Genome Size Evolution in Extant Gymnosperms through Phylogenetic Synthesis

Gymnosperms, comprising cycads, Ginkgo, Gnetales, and conifers, represent one of the major groups of extant seed plants. Yet compared to angiosperms, little is known about the patterns of diversification and genome evolution in gymnosperms. We assembled a phylogenetic supermatrix containing over 4.5 million nucleotides from 739 gymnosperm taxa. Although 93.6% of the cells in the supermatrix are empty, the data reveal many strongly supported nodes that are generally consistent with previous phylogenetic analyses, including weak support for Gnetales sister to Pinaceae. A lineage through time plot suggests elevated rates of diversification within the last 100 million years, and there is evidence of shifts in diversification rates in several clades within cycads and conifers. A likelihood-based analysis of the evolution of genome size in 165 gymnosperms finds evidence for heterogeneous rates of genome size evolution due to an elevated rate in Pinus.

Genetic dissection of fusiform rust and pitch canker disease traits in loblolly pine

Loblolly pine (Pinus taeda L.) exhibits genetic resistance to fusiform rust disease (incited by the biotrophic fungus, Cronartium quercuum f. sp. fusiforme) and pitch canker disease (incited by the necrotrophic fungus, Fusarium circinatum). In this study, a total of 14,015 loblolly pine cuttings from 1,065 clones were screened in controlled greenhouse conditions to identify phenotypes of clones, families, and parents that guide a genetic dissection of disease traits associated with pitch canker and fusiform rust. A total of 23,373 phenotypic data points were collected for lesion length (pitch canker) and gall score, gall length, and gall width (fusiform rust). We verified heritable fusiform rust and pitch canker traits and calculated parental, clonal, and full-sib family rankings for both diseases. Genetic correlations revealed that traits associated with fusiform rust are genetically distinct from one another, and that the genetic mechanisms underlying pitch canker and fusiform rust resistance are independent. The disease phenotyping described here is a critical step towards identifying specific loci and alleles associated with fusiform rust and pitch canker resistance.

Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts

This work identified aromatic amino acid hydroxylase (AAH)-like sequences from nonflowering plants. The encoded proteins were shown to be plastid targeted, to have Phe hydroxylase activity, and, unlike animal and bacterial AAHs, to use a folate as cofactor rather than a pterin. Ablating AAH in moss led to accumulation of Phe and caffeate esters. Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism.

Pine genes regulated by the necrotrophic pathogen Fusarium circinatum.

A targeted genomics approach was used to construct a cDNA array of potential pathogen-regulated genes for investigating host–pathogen interactions in pine trees (Pinus species). This array, containing a nonredundant set of 311 cDNAs, was assembled by combining smaller sets of cDNAs generated by differential display or suppression subtraction hybridization using a variety of pathogen treatments and elicitors. The array was probed to identify host genes regulated by Fusarium circinatum, a necrotrophic fungus that incites pitch canker disease on pine stems. A set of 29 cDNAs were induced during the disease state. Notably, cDNAs on the array that were derived from experiments with fusiform rust, incited by Cronartium quercuum f. sp. fusiforme (a biotrophic fungus) were unregulated by Fusarium. The results imply distinct genetic responses in pine to diseases incited by necrotrophs and biotrophs. This cDNA collection expands the genomics toolkit for understanding interactions between conifers and their microbial associates in forest ecosystems.

High-Throughput Phenotyping of Maize Leaf Physiological and Biochemical Traits Using Hyperspectral Reflectance

Partial least-squares regression modeling of leaf reflectance spectra provides a high-throughput and accurate approach to phenotyping photosynthesis and leaf biochemistry in maize. High-throughput, noninvasive field phenotyping has revealed genetic variation in crop morphological, developmental, and agronomic traits, but rapid measurements of the underlying physiological and biochemical traits are needed to fully understand genetic variation in plant-environment interactions. This study tested the application of leaf hyperspectral reflectance (λ = 500–2,400 nm) as a high-throughput phenotyping approach for rapid and accurate assessment of leaf photosynthetic and biochemical traits in maize (Zea mays). Leaf traits were measured with standard wet-laboratory and gas-exchange approaches alongside measurements of leaf reflectance. Partial least-squares regression was used to develop a measure of leaf chlorophyll content, nitrogen content, sucrose content, specific leaf area, maximum rate of phosphoenolpyruvate carboxylation, [CO2]-saturated rate of photosynthesis, and leaf oxygen radical absorbance capacity from leaf reflectance spectra. Partial least-squares regression models accurately predicted five out of seven traits and were more accurate than previously used simple spectral indices for leaf chlorophyll, nitrogen content, and specific leaf area. Correlations among leaf traits and statistical inferences about differences among genotypes and treatments were similar for measured and modeled data. The hyperspectral reflectance approach to phenotyping was dramatically faster than traditional measurements, enabling over 1,000 rows to be phenotyped during midday hours over just 2 to 4 d, and offers a nondestructive method to accurately assess physiological and biochemical trait responses to environmental stress.