John A. Crow

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote–eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes.

BackgroundMeiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.ResultsA high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.ConclusionThese findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.

A Comprehensive Transcriptome Assembly of Pigeonpea (Cajanus cajan L.) using Sanger and Second-Generation Sequencing Platforms

A comprehensive transcriptome assembly for pigeonpea has been developed by analyzing 128.9 million short Illumina GA IIx single end reads, 2.19 million single end FLX/454 reads, and 18 353 Sanger expressed sequenced tags from more than 16 genotypes. The resultant transcriptome assembly, referred to as CcTA v2, comprised 21 434 transcript assembly contigs (TACs) with an N50 of 1510 bp, the largest one being ∼8 kb. Of the 21 434 TACs, 16 622 (77.5%) could be mapped on to the soybean genome build 1.0.9 under fairly stringent alignment parameters. Based on knowledge of intron junctions, 10 009 primer pairs were designed from 5033 TACs for amplifying intron spanning regions (ISRs). By using in silico mapping of BAC-end-derived SSR loci of pigeonpea on the soybean genome as a reference, putative mapping positions at the chromosome level were predicted for 6284 ISR markers, covering all 11 pigeonpea chromosomes. A subset of 128 ISR markers were analyzed on a set of eight genotypes. While 116 markers were validated, 70 markers showed one to three alleles, with an average of 0.16 polymorphism information content (PIC) value. In summary, the CcTA v2 transcript assembly and ISR markers will serve as a useful resource to accelerate genetic research and breeding applications in pigeonpea.

Prevalence of single nucleotide polymorphism among 27 diverse alfalfa genotypes as assessed by transcriptome sequencing

BackgroundAlfalfa, a perennial, outcrossing species, is a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, particularly if many genome-wide markers are available. Transcriptome sequencing enables efficient high-throughput discovery of single nucleotide polymorphism (SNP) markers for a complex polyploid species.ResultThe transcriptomes of 27 alfalfa genotypes, including elite breeding genotypes, parents of mapping populations, and unimproved wild genotypes, were sequenced using an Illumina Genome Analyzer IIx. De novo assembly of quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of individual reads to the contigs enabled the detection of 872,384 SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants at about 95% of SNP sites identified among five wild, unimproved genotypes are still present in cultivated alfalfa, and all four US breeding programs also contain a high proportion of these SNPs. Thus, little evidence exists among this dataset for loss of significant DNA sequence diversity from either domestication or breeding of alfalfa. Structure analysis indicated that individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa) were clearly separated.ConclusionWe used transcriptome sequencing to discover large numbers of SNPs segregating in elite breeding populations of alfalfa. Little loss of SNP diversity was evident between unimproved and elite alfalfa germplasm. The EST and SNP markers generated from this study are publicly available at the Legume Information System (http://medsa.comparative-legumes.org/) and can contribute to future alfalfa research and breeding applications.

Next-generation sequencing-based transcriptomic and proteomic analysis of the common reed, Phragmites australis (Poaceae), reveals genes involved in invasiveness and rhizome specificity

PREMISE OF THE STUDY The common reed (Phragmites australis), one of the most widely distributed of all angiosperms, uses its rhizomes (underground stems) to invade new territory, making it one of the most successful weedy species worldwide. Characterization of the rhizome transcriptome and proteome is needed to identify candidate genes and proteins involved in rhizome growth, development, metabolism, and invasiveness. METHODS We employed next-generation sequencing technologies including 454 and Illumina platforms to characterize the reed rhizome transcriptome and used quantitative proteomics techniques to identify the rhizome proteome. KEY RESULTS Combining 336514 Roche 454 Titanium reads and 103350802 Illumina paired-end reads in a de novo hybrid assembly yielded 124450 unique transcripts with an average length of 549 bp, of which 54317 were annotated. Rhizome-specific and differentially expressed transcripts were identified between rhizome apical tips (apical meristematic region) and rhizome elongation zones. A total of 1280 nonredundant proteins were identified and quantified using GeLC-MS/MS based label-free proteomics, where 174 and 77 proteins were preferentially expressed in the rhizome elongation zone and apical tip tissues, respectively. Genes involved in allelopathy and in controlling development and potentially invasiveness were identified. CONCLUSIONS In addition to being a valuable sequence and protein data resource for studying plant rhizome species, our results provide useful insights into identifying specific genes and proteins with potential roles in rhizome differentiation, development, and function.

Asian citrus psyllid expression profiles suggest Candidatus Liberibacter asiaticus-mediated alteration of adult nutrition and metabolism, and of nymphal development and immunity.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the insect vector of the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease, or Huanglongbing (HLB). The widespread invasiveness of the psyllid vector and HLB in citrus trees worldwide has underscored the need for non-traditional approaches to manage the disease. One tenable solution is through the deployment of RNA interference technology to silence protein-protein interactions essential for ACP-mediated CLas invasion and transmission. To identify psyllid interactor-bacterial effector combinations associated with psyllid-CLas interactions, cDNA libraries were constructed from CLas-infected and CLas-free ACP adults and nymphs, and analyzed for differential expression. Library assemblies comprised 24,039,255 reads and yielded 45,976 consensus contigs. They were annotated (UniProt), classified using Gene Ontology, and subjected to in silico expression analyses using the Transcriptome Computational Workbench (TCW) (http://www.sohomoptera.org/ACPPoP/). Functional-biological pathway interpretations were carried out using the Kyoto Encyclopedia of Genes and Genomes databases. Differentially expressed contigs in adults and/or nymphs represented genes and/or metabolic/pathogenesis pathways involved in adhesion, biofilm formation, development-related, immunity, nutrition, stress, and virulence. Notably, contigs involved in gene silencing and transposon-related responses were documented in a psyllid for the first time. This is the first comparative transcriptomic analysis of ACP adults and nymphs infected and uninfected with CLas. The results provide key initial insights into host-parasite interactions involving CLas effectors that contribute to invasion-virulence, and to host nutritional exploitation and immune-related responses that appear to be essential for successful ACP-mediated circulative, propagative CLas transmission.

Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

A systems-wide comparison of red rice (Oryza longistaminata) tissues identifies rhizome specific genes and proteins that are targets for cultivated rice improvement

BackgroundThe rhizome, the original stem of land plants, enables species to invade new territory and is a critical component of perenniality, especially in grasses. Red rice (Oryza longistaminata) is a perennial wild rice species with many valuable traits that could be used to improve cultivated rice cultivars, including rhizomatousness, disease resistance and drought tolerance. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development and function in this plant.ResultsWe used an integrated approach to compare the transcriptome, proteome and metabolome of the rhizome to other tissues of red rice. 116 Gb of transcriptome sequence was obtained from various tissues and used to identify rhizome-specific and preferentially expressed genes, including transcription factors and hormone metabolism and stress response-related genes. Proteomics and metabolomics approaches identified 41 proteins and more than 100 primary metabolites and plant hormones with rhizome preferential accumulation. Of particular interest was the identification of a large number of gene transcripts from Magnaportha oryzae, the fungus that causes rice blast disease in cultivated rice, even though the red rice plants showed no sign of disease.ConclusionsA significant set of genes, proteins and metabolites appear to be specifically or preferentially expressed in the rhizome of O. longistaminata. The presence of M. oryzae gene transcripts at a high level in apparently healthy plants suggests that red rice is resistant to this pathogen, and may be able to provide genes to cultivated rice that will enable resistance to rice blast disease.

Comparison of Potato and Asian Citrus Psyllid Adult and Nymph Transcriptomes Identified Vector Transcripts with Potential Involvement in Circulative, Propagative Liberibacter Transmission

The potato psyllid (PoP) Bactericera cockerelli (Sulc) and Asian citrus psyllid (ACP) Diaphorina citri Kuwayama are the insect vectors of the fastidious plant pathogen, Candidatus Liberibacter solanacearum (CLso) and Ca. L. asiaticus (CLas), respectively. CLso causes Zebra chip disease of potato and vein-greening in solanaceous species, whereas, CLas causes citrus greening disease. The reliance on insecticides for vector management to reduce pathogen transmission has increased interest in alternative approaches, including RNA interference to abate expression of genes essential for psyllid-mediated Ca. Liberibacter transmission. To identify genes with significantly altered expression at different life stages and conditions of CLso/CLas infection, cDNA libraries were constructed for CLso-infected and -uninfected PoP adults and nymphal instars. Illumina sequencing produced 199,081,451 reads that were assembled into 82,224 unique transcripts. PoP and the analogous transcripts from ACP adult and nymphs reported elsewhere were annotated, organized into functional gene groups using the Gene Ontology classification system, and analyzed for differential in silico expression. Expression profiles revealed vector life stage differences and differential gene expression associated with Liberibacter infection of the psyllid host, including invasion, immune system modulation, nutrition, and development.

Molecular and Biological Characterization of a New Isolate of Guinea Pig Cytomegalovirus

Development of a vaccine against congenital infection with human cytomegalovirus is complicated by the issue of re-infection, with subsequent vertical transmission, in women with pre-conception immunity to the virus. The study of experimental therapeutic prevention of re-infection would ideally be undertaken in a small animal model, such as the guinea pig cytomegalovirus (GPCMV) model, prior to human clinical trials. However, the ability to model re-infection in the GPCMV model has been limited by availability of only one strain of virus, the 22122 strain, isolated in 1957. In this report, we describe the isolation of a new GPCMV strain, the CIDMTR strain. This strain demonstrated morphological characteristics of a typical Herpesvirinae by electron microscopy. Illumina and PacBio sequencing demonstrated a genome of 232,778 nt. Novel open reading frames ORFs not found in reference strain 22122 included an additional MHC Class I homolog near the right genome terminus. The CIDMTR strain was capable of dissemination in immune compromised guinea pigs, and was found to be capable of congenital transmission in GPCMV-immune dams previously infected with salivary gland‑adapted strain 22122 virus. The availability of a new GPCMV strain should facilitate study of re-infection in this small animal model.