The cacao gene atlas: a transcriptome developmental atlas reveals highly tissue-specific and dynamically-regulated gene networks in Theobroma cacao L.

BACKGROUND: Theobroma cacao, the cocoa tree, is a tropical crop grown for its highly valuable cocoa solids and fat which are the basis of a 200-billion-dollar annual chocolate industry. However, the long generation time and difficulties associated with breeding a tropical tree crop have limited the progress of breeders to develop high-yielding disease-resistant varieties. Development of marker-assisted breeding methods for cacao requires discovery of genomic regions and specific alleles of genes encoding important traits of interest. To accelerate gene discovery, we developed a gene atlas composed of a large dataset of replicated transcriptomes with the long-term goal of progressing breeding towards developing high-yielding elite varieties of cacao. RESULTS: We describe the creation of the Cacao Transcriptome Atlas, its global characterization and define sets of genes co-regulated in highly organ- and temporally-specific manners. RNAs were extracted and transcriptomes sequenced from 123 different tissues and stages of development representing major organs and developmental stages of the cacao lifecycle. In addition, several experimental treatments and time courses were performed to measure gene expression in tissues responding to biotic and abiotic stressors. Samples were collected in replicates (3-5) to enable statistical analysis of gene expression levels for a total of 390 transcriptomes. To promote wide use of these data, all raw sequencing data, expression read mapping matrices, scripts, and other information used to create the resource are freely available online. We verified our atlas by analyzing the expression of genes with known functions and expression patterns in Arabidopsis (ACT7, LEA19, AGL16, TIP13, LHY, MYB2) and found their expression profiles to be generally similar between both species. We also successfully identified tissue-specific genes at two thresholds in many tissue types represented and a set of genes highly conserved across all tissues. CONCLUSION: The Cacao Gene Atlas consists of a gene expression browser with graphical user interface and open access to raw sequencing data files as well as the unnormalized and CPM normalized read count data mapped to several cacao genomes. The gene atlas is a publicly available resource to allow rapid mining of cacao gene expression profiles. We hope this resource will be used to help accelerate the discovery of important genes for key cacao traits such as disease resistance and contribute to the breeding of elite varieties to help farmers increase yields.

A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora.

BACKGROUND: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora. RESULTS: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp. CONCLUSIONS: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

Challenges and considerations for reproducibility of STARR-seq assays.

High-throughput methods such as RNA-seq, ChIP-seq, and ATAC-seq have well-established guidelines, commercial kits, and analysis pipelines that enable consistency and wider adoption for understanding genome function and regulation. STARR-seq, a popular assay for directly quantifying the activities of thousands of enhancer sequences simultaneously, has seen limited standardization across studies. The assay is long, with more than 250 steps, and frequent customization of the protocol and variations in bioinformatics methods raise concerns for reproducibility of STARR-seq studies. Here, we assess each step of the protocol and analysis pipelines from published sources and in-house assays, and identify critical steps and quality control (QC) checkpoints necessary for reproducibility of the assay. We also provide guidelines for experimental design, protocol scaling, customization, and analysis pipelines for better adoption of the assay. These resources will allow better optimization of STARR-seq for specific research needs, enable comparisons and integration across studies, and improve the reproducibility of results.

Complete Genome Sequence of Mycobacterium orygis Strain 51145.

We report the complete 4,352,172-bp genome sequence of Mycobacterium orygis strain 51145 assembled into a single circular chromosome. Comparative genomic analyses with other lineages of the Mycobacterium tuberculosis complex can provide insights into the biology, evolution, and epidemiology of this important group of pathogenic mycobacteria.

Metformin Affects the Transcriptomic Profile of Chicken Ovarian Cancer Cells.

Ovarian cancer is the most lethal gynecological malignancy in women. Metformin intake is associated with a reduced incidence of ovarian cancer and increased overall survival rate. We determined the effect of metformin on sphere formation, extracellular matrix invasion, and transcriptome profile of ovarian cancer cells (COVCAR) isolated from ascites of chickens that naturally developed ovarian cancer. We found that metformin treatment significantly decreased sphere formation and invasiveness of COVCAR cells. RNA-Seq data analysis revealed 0, 4, 365 differentially expressed genes in cells treated with 0.5, 1, 2 mM metformin, respectively compared to controls. Transcriptomic and ingenuity pathway analysis (IPA) revealed significant downregulation of MMP7, AICDA, GDPD2, APOC3, APOA1 and predicted inhibition of upstream regulators NFKB, STAT3, TP53 that are involved in epithelial-mesenchymal transition, DNA repair, and lipid metabolism. The analysis revealed significant upregulation of RASD2, IHH, CRABP-1 and predicted activation of upstream regulators VEGF and E2F1 that are associated with angiogenesis and cell cycle. Causal network analysis revealed novel pathways suggesting predicted inhibition of ovarian cancer through master regulator ASCL1 and dataset genes DCX, SEMA6B, HEY2, and KCNIP2. In summary, advanced pathway analysis in IPA revealed novel target genes, upstream regulators, and pathways affected by metformin treatment of COVCAR cells.

Transcriptome Analyses of Heart and Liver Reveal Novel Pathways for Regulating Songbird Migration.

Many birds undertake long biannual voyages during the night. During these times of the year birds drastically reduce their amount of sleep, yet curiously perform as well on tests of physical and cognitive performance than during non-migrating times of the year. This inherent physiological protection disappears when birds are forced to stay awake at other times of the year; thus these protective changes are only associated with the nocturnal migratory state. The goal of the current study was to identify the physiological mechanisms that confer protection against the consequences of sleep loss while simultaneously allowing for the increased physical performance required for migration. We performed RNA-seq analyses of heart and liver collected from birds at different times of day under different migratory states and analyzed these data using differential expression, pathway analysis and WGCNA. We identified changes in gene expression networks implicating multiple systems and pathways. These pathways regulate many aspects of metabolism, immune function, wound repair, and protection of multiple organ systems. Consequently, the circannual program controlling the appearance of the migratory phenotype involves the complex regulation of diverse gene networks associated with the physical demands of migration.

Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array.

Johne's disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis, is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. avium subsp. paratuberculosis and the human pathogen Mycobacterium tuberculosis, here, we applied a whole-proteome M. tuberculosis protein array to identify seroreactive and diagnostic M. avium subsp. paratuberculosis antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. avium subsp. paratuberculosis and M. tuberculosis showed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the M. tuberculosis protein array probed with sera from M. avium subsp. paratuberculosis-infected cattle showed antibody binding to 729 M. tuberculosis proteins, with 58% of them having ≥70% identity to M. avium subsp. paratuberculosis orthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosis antigens were orthologs of previously reported M. avium subsp. paratuberculosis antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. avium subsp. paratuberculosis recombinant proteins, representing reactive and nonreactive M. tuberculosis orthologs, further confirmed that the M. tuberculosis array has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. avium subsp. paratuberculosis proteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays.

Giraffe genome sequence reveals clues to its unique morphology and physiology.

The origins of giraffe's imposing stature and associated cardiovascular adaptations are unknown. Okapi, which lacks these unique features, is giraffe's closest relative and provides a useful comparison, to identify genetic variation underlying giraffe's long neck and cardiovascular system. The genomes of giraffe and okapi were sequenced, and through comparative analyses genes and pathways were identified that exhibit unique genetic changes and likely contribute to giraffe's unique features. Some of these genes are in the HOX, NOTCH and FGF signalling pathways, which regulate both skeletal and cardiovascular development, suggesting that giraffe's stature and cardiovascular adaptations evolved in parallel through changes in a small number of genes. Mitochondrial metabolism and volatile fatty acids transport genes are also evolutionarily diverged in giraffe and may be related to its unusual diet that includes toxic plants. Unexpectedly, substantial evolutionary changes have occurred in giraffe and okapi in double-strand break repair and centrosome functions.

Bone sialoprotein keratan sulfate proteoglycan (BSP-KSPG) and FGF-23 are important physiological components of medullary bone.

Medullary bone is a specialized bone found in the marrow cavity of laying birds. It provides a significant contribution to the calcium supply for egg shell formation. Medullary bone is distinguished from cortical bone by the presence of large amounts of a keratan sulfate proteoglycan (KSPG). The aims of the present experiment are to confirm the identity of the core protein of KSPG, identify a marker of medullary bone metabolism, and determine whether changes in keratan sulfate (KS) concentration in blood are associated with the egg-laying cycle. Using two different isolation techniques- one specific for bone and another for blood- we have identified bone sialoprotein (BSP) to be the core protein of this KSPG. We also determined that the amount of keratan sulfate (KS) in laying hen blood fluctuates in synchrony with the egg-laying cycle, and thus can serve as a specific marker for medullary bone metabolism. During the course of this investigation, we also found FGF-23 (phosphatonin) to be expressed in medullary bone, in synchrony with the egg-laying cycle. Western blotting was used to demonstrate the presence of this peptide in both laying hen blood and medullary bone extracts. The importance of FGF-23 (phosphatonin) and parathyroid hormone in normalizing the dramatic changes in plasma calcium and phosphorus during the 24h egg-laying cycle is discussed.

Genomic characterization of a turkey reovirus field strain by Next-Generation Sequencing.

The genome of a turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13), isolated from a turkey flock in Pennsylvania (PA) in 2013, has been sequenced using Next-Generation Sequencing (NGS) on the Illumina MiSeq platform. The genome of the PA TARV field strain was 23,496bp in length with 10 dsRNA segments encoding 12 viral proteins. The lengths of the genomic segments ranged from 1192bp (S4) to 3959bp (L1). The 5' and 3' conserved terminal sequences of the PA TARV field strain were similar to the two Minnesota (MN) TARVs (MN9 and MN10) published recently and avian orthoreovirus (ARV) reference strains. Phylogenetic analysis of the nucleotide sequences of all 10 genome segments revealed that there was a low to significant nucleotide sequence divergence between the PA TARV field strain and reference TARV and ARV strains. Analysis of the PA TARV sequence indicates that this PA TARV field strain is a unique strain and is different from the TARV MN9 or MN10 in M2 segment genes and ARV S1133 vaccine strain.

Mapping in an apple (Malus x domestica) F1 segregating population based on physical clustering of differentially expressed genes.

BACKGROUND: Apple tree breeding is slow and difficult due to long generation times, self-incompatibility, and complex genetics. The identification of molecular markers linked to traits of interest is a way to expedite the breeding process. In the present study, we aimed to identify genes whose steady-state transcript abundance was associated with inheritance of specific traits segregating in an apple (Malus × domestica) rootstock F1 breeding population, including resistance to powdery mildew (Podosphaera leucotricha) disease and woolly apple aphid (Eriosoma lanigerum). RESULTS: Transcription profiling was performed for 48 individual F1 apple trees from a cross of two highly heterozygous parents, using RNA isolated from healthy, actively-growing shoot tips and a custom apple DNA oligonucleotide microarray representing 26,000 unique transcripts. Genome-wide expression profiles were not clear indicators of powdery mildew or woolly apple aphid resistance phenotype. However, standard differential gene expression analysis between phenotypic groups of trees revealed relatively small sets of genes with trait-associated expression levels. For example, thirty genes were identified that were differentially expressed between trees resistant and susceptible to powdery mildew. Interestingly, the genes encoding twenty-four of these transcripts were physically clustered on chromosome 12. Similarly, seven genes were identified that were differentially expressed between trees resistant and susceptible to woolly apple aphid, and the genes encoding five of these transcripts were also clustered, this time on chromosome 17. In each case, the gene clusters were in the vicinity of previously identified major quantitative trait loci for the corresponding trait. Similar results were obtained for a series of molecular traits. Several of the differentially expressed genes were used to develop DNA polymorphism markers linked to powdery mildew disease and woolly apple aphid resistance. CONCLUSIONS: Gene expression profiling and trait-associated transcript analysis using an apple F1 population readily identified genes physically linked to powdery mildew disease resistance and woolly apple aphid resistance loci. This result was especially useful in apple, where extreme levels of heterozygosity make the development of reliable DNA markers quite difficult. The results suggest that this approach could prove effective in crops with complicated genetics, or for which few genomic information resources are available.

Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray.

An antibody microarray was developed to detect the "top six" non-O157 serogroups, O26, O45, O103, O111, O121, and O145 of Shiga toxin-producing Escherichia coli (STEC), that have been declared as adulterant in meat by the Food Safety and Inspection Service of the United States Department of Agriculture. The sensitivity of the array was 10(5)CFU and the limit of detection of each serogroup in artificially inoculated ground beef was 1-10 CFU following 12h of enrichment. Optimal concentrations of antibodies for printing and labeling and bacterial dilutions for binding to the antibodies were assessed. The array utilized a minimal amount of antibodies and other reagents and may be utilized for screening of multiple target O groups of STEC in parallel, directly from enriched samples in less than 3h. Furthermore, the antibody array provides the flexibility to include other O serogroups of E. coli and may be adopted for high throughput screening. The method is potentially applicable to detect the pathogenic STEC O groups of E. coli in meat and other food, thus improving food safety and public health.

Rootstock-regulated gene expression patterns associated with fire blight resistance in apple.

BACKGROUND: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. RESULTS: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. CONCLUSIONS: Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

GCAT-SEEKquence: genome consortium for active teaching of undergraduates through increased faculty access to next-generation sequencing data.

To transform undergraduate biology education, faculty need to provide opportunities for students to engage in the process of science. The rise of research approaches using next-generation (NextGen) sequencing has been impressive, but incorporation of such approaches into the undergraduate curriculum remains a major challenge. In this paper, we report proceedings of a National Science Foundation-funded workshop held July 11-14, 2011, at Juniata College. The purpose of the workshop was to develop a regional research coordination network for undergraduate biology education (RCN/UBE). The network is collaborating with a genome-sequencing core facility located at Pennsylvania State University (University Park) to enable undergraduate students and faculty at small colleges to access state-of-the-art sequencing technology. We aim to create a database of references, protocols, and raw data related to NextGen sequencing, and to find innovative ways to reduce costs related to sequencing and bioinformatics analysis. It was agreed that our regional network for NextGen sequencing could operate more effectively if it were partnered with the Genome Consortium for Active Teaching (GCAT) as a new arm of that consortium, entitled GCAT-SEEK(quence). This step would also permit the approach to be replicated elsewhere.

Characterization of the non-collagenous proteins in avian cortical and medullary bone.

Northern blotting, RT-PCR, and Western blotting techniques were used to characterize the matrix constituents of avian cortical and medullary bone. Extracts of bone tissue were found to contain multiple isoforms of bone sialoprotein (BSP), osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and dentin matrix protein-1 (DMP-1). Only single transcripts were observed with Northern blotting; therefore it was concluded that the isoforms were due to differences in post-translational modifications. Since medullary bone is rich in keratan sulfate (KS), RT-PCR was used to investigate the expression of known keratan sulfate-containing proteoglycans (KSPGs). Although this tissue was found to express lumican and osteoglycin/mimecan, there was little evidence to suggest that these proteoglycans were a major source of the keratan sulfate glycosaminoglycans. Treatment of medullary bone extracts with keratanase resulted in the appearance of a BSP immunoactive band of approximately 59 kDa. However, it was not possible to isolate and identify the intact keratan sulfate proteoglycan.

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.