Gene expression during the early stages of host perception and attachment in adult female Rhipicephalus microplus ticks.

The cattle tick, Rhipicephalus microplus, is a serious pest of cattle, with significant economic consequences to the livestock industries of tropical and semitropical countries. Rhipicephalus microplus belongs to the Metastriata group of the Ixodidae family known as hard ticks. When adult hard ticks feed, mating has not yet occurred and an initial host attachment phase of 1-2 days is followed by a slow feeding phase that can last several days. Once mating occurs, feeding concludes with a rapid engorgement phase that is completed in 12-36 h. Our group's interest in mining the genome and transcriptome of R. microplus for novel targets for development of tick control technologies led us to investigate the early transcriptional events occurring upon tick attachment and subsequent feeding. We placed newly molted unfed adult R. microplus females upon a bovine host and harvested the attached ticks after 3, 6, 12, and 24 h. We also placed a group of these ticks in a gas-permeable tube taped onto the side of the bovine host. These ticks were able to sense the host but unable to penetrate the tube to begin attachment and were ultimately harvested after 3 h. This study produced a comprehensive transcriptome from newly molted adult ticks and will provide a useful resource for studies of tick feeding and host perception and also assist genome annotation refinements.

Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

Doing Genetic and Genomic Biology Using the Legume Information System and Associated Resources.

In this chapter, we introduce the main components of the Legume Information System ( https://legumeinfo.org ) and several associated resources. Additionally, we provide an example of their use by exploring a biological question: is there a common molecular basis, across legume species, that underlies the photoperiod-mediated transition from vegetative to reproductive development, that is, days to flowering? The Legume Information System (LIS) holds genetic and genomic data for a large number of crop and model legumes and provides a set of online bioinformatic tools designed to help biologists address questions and tasks related to legume biology. Such tasks include identifying the molecular basis of agronomic traits; identifying orthologs/syntelogs for known genes; determining gene expression patterns; accessing genomic datasets; identifying markers for breeding work; and identifying genetic similarities and differences among selected accessions. LIS integrates with other legume-focused informatics resources such as SoyBase ( https://soybase.org ), PeanutBase ( https://peanutbase.org ), and projects of the Legume Federation ( https://legumefederation.org ).

The adult horn fly transcriptome and its complement of transcripts encoding cytochrome P450s, glutathione S-transferases, and esterases.

The horn fly, Haematobia irritans, is a blood-feeding parasitic fly with a global distribution that includes Europe, Africa, Asia, and the Americas. The fly has a major detrimental economic impact upon cattle production, with losses estimated at over $800 million annually in the United States and $2.5 billion in Brazil alone. Insecticide resistance in specific horn fly populations has been a problem for many years and there are several mechanisms whereby resistance develops. Little is known about the complement of metabolic enzymes encoded by the horn fly's genome that might provide the fly with detoxification or sequestration pathways to survive insecticide treatments. The cytochrome P450, glutathione S-transferase, and esterase enzyme families contain members that are capable of sequestering and/or detoxifying xenobiotic molecules such as insecticides. We sought to develop a comprehensive dataset of metabolic enzyme-encoding transcript sequences from the adult horn fly, as this is the life stage whose actions directly impose the economic costs to cattle producers. We used an Illumina paired-end read RNA-Seq approach to determine the adult horn fly transcriptomes from laboratory and field populations of horn flies with varying levels of pesticide resistance, including untreated and pyrethroid-treated newly eclosed adult flies. We followed with bioinformatic analyses to discern sequences putatively encoding cytochrome P450, esterase, and GST enzymes. We utilized read-mapping of RNA-Seq data and quantitative real-time polymerase chain reaction (qRT-PCR) to examine gene expression levels of specific P450 transcripts in several fly populations with varying degrees of pesticide resistance.

The genome sequence of segmental allotetraploid peanut Arachis hypogaea.

Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.

The assembled transcriptome of the adult horn fly, Haematobia irritans.

The horn fly, Haematobia irritans irritans (Linnaeus, 1758; Diptera: Muscidae), a hematophagous external parasite of cattle, causes considerable economic losses to the livestock industry worldwide. This pest is mainly controlled with insecticides; however, horn fly populations from several countries have developed resistance to many of the products available for their control. In an attempt to better understand the adult horn fly and the development of resistance in natural populations, we used an Illumina paired-end read HiSeq and GAII approach to determine the transcriptomes of untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males from a Louisiana population of horn flies with a moderate level of pyrethroid resistance. A total of 128,769,829, 127,276,458, 67,653,920, and 64,270,124 quality-filtered Illumina reads were obtained for untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males, respectively. The de novo assemblies using CLC Genomics Workbench 8.0.1 yielded 15,699, 11,961, 2672, 7278 contigs (≥ 200 nt) for untreated control adult females, untreated control adult males, permethrin-treated surviving adult males and permethrin + piperonyl butoxide-treated killed adult males, respectively. More than 56% of the assembled contigs of each data set had significant hits in the BlastX (UniProtKB/Swiss-Prot database) (E <0.001). The number of contigs in each data set with InterProScan, GO mapping, Enzyme codes and KEGG pathway annotations were: Untreated Control Adult Females - 10,331, 8770, 2963, 2183; Untreated control adult males - 8392, 7056, 2449, 1765; Permethrin-treated surviving adult males - 1992, 1609, 641, 495; Permethrin + PBO-treated killed adult males - 5561, 4463, 1628, 1211.

The testes transcriptome of the New World Screwworm, Cochliomyia hominivorax.

The New World Screwworm (NWS), Cochliomyia hominivorax, is a pest insect that is endemic to subtropical and tropical regions of the Western Hemisphere. The female lays eggs in open wounds or orifices of warm-blooded animals. Upon hatching, the resulting larvae feed upon the host׳s living tissues, which can become infected and death can occur. The sterile insect technique was developed to eradicate this pest from North America and new female conditional-lethal strains that generate only male individuals are being developed for use in the eradication program. To facilitate the identification of useful transcripts and gene promoters for these new strains, we used an Illumina Hi-Seq protocol to sequence the testes transcriptome of NWS. We report the assembly of 4149 transcripts (≥200 nt) from testes dissected from NWS males obtained from the J06 strain used in the screwworm production plant in Pacora, Panama. Functional annotation resulted in 2060, 2031, 558, and 325 transcripts with assigned BlastX, Gene Ontology, Enzyme Codes, and KEGG pathway information, respectively. In the Gene Ontology annotations, 6% and 3% of the transcripts in the Biological Process Ontology were noted as Developmental Process and Reproduction, respectively. This data set will serve as a resource to facilitate studies of sex determination in the NWS and the development of recombinant vectors that can be used to create new male-only strains of NWS.

Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens.

The MinION is a portable single-molecule DNA sequencing instrument that was released by Oxford Nanopore Technologies in 2014, producing long sequencing reads by measuring changes in ionic flow when single-stranded DNA molecules translocate through the pores. While MinION long reads have an error rate substantially higher than the ones produced by short-read sequencing technologies, they can generate de novo assemblies of microbial genomes, after an initial correction step that includes alignment of Illumina sequencing data or detection of overlaps between Oxford Nanopore reads to improve accuracy. In this study, MinION reads were generated from the multi-chromosome genome of Agrobacterium tumefaciens strain LBA4404. Errors in the consensus two-directional (sense and antisense) "2D" sequences were first characterized by way of comparison with an internal reference assembly. Both Illumina-based correction and self-correction were performed and the resulting corrected reads assembled into high-quality hybrid and non-hybrid assemblies. Corrected read datasets and assemblies were subsequently compared. The results shown here indicate that both hybrid and non-hybrid methods can be used to assemble Oxford Nanopore reads into informative multi-chromosome assemblies, each with slightly different outcomes in terms of contiguity and accuracy.

Comprehensive transcriptome assembly of Chickpea (Cicer arietinum L.) using sanger and next generation sequencing platforms: development and applications.

A comprehensive transcriptome assembly of chickpea has been developed using 134.95 million Illumina single-end reads, 7.12 million single-end FLX/454 reads and 139,214 Sanger expressed sequence tags (ESTs) from >17 genotypes. This hybrid transcriptome assembly, referred to as Cicer arietinumTranscriptome Assembly version 2 (CaTA v2, available at http://data.comparative-legumes.org/transcriptomes/cicar/lista_cicar-201201), comprising 46,369 transcript assembly contigs (TACs) has an N50 length of 1,726 bp and a maximum contig size of 15,644 bp. Putative functions were determined for 32,869 (70.8%) of the TACs and gene ontology assignments were determined for 21,471 (46.3%). The new transcriptome assembly was compared with the previously available chickpea transcriptome assemblies as well as to the chickpea genome. Comparative analysis of CaTA v2 against transcriptomes of three legumes - Medicago, soybean and common bean, resulted in 27,771 TACs common to all three legumes indicating strong conservation of genes across legumes. CaTA v2 was also used for identification of simple sequence repeats (SSRs) and intron spanning regions (ISRs) for developing molecular markers. ISRs were identified by aligning TACs to the Medicago genome, and their putative mapping positions at chromosomal level were identified using transcript map of chickpea. Primer pairs were designed for 4,990 ISRs, each representing a single contig for which predicted positions are inferred and distributed across eight linkage groups. A subset of randomly selected ISRs representing all eight chickpea linkage groups were validated on five chickpea genotypes and showed 20% polymorphism with average polymorphic information content (PIC) of 0.27. In summary, the hybrid transcriptome assembly developed and novel markers identified can be used for a variety of applications such as gene discovery, marker-trait association, diversity analysis etc., to advance genetics research and breeding applications in chickpea and other related legumes.

Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships.

Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.

Transcriptome analysis of cytokinin response in tomato leaves.

Tomato is one of the most economically and agriculturally important Solanaceous species and vegetable crops, serving as a model for examination of fruit biology and compound leaf development. Cytokinin is a plant hormone linked to the control of leaf development and is known to regulate a wide range of genes including many transcription factors. Currently there is little known of the leaf transcriptome in tomato and how it might be regulated by cytokinin. We employ high throughput mRNA sequencing technology and bioinformatic methodologies to robustly analyze cytokinin regulated tomato leaf transcriptomes. Leaf samples of two ages, 13d and 35d were treated with cytokinin or the solvent vehicle control dimethyl sulfoxide (DMSO) for 2 h or 24 h, after which RNA was extracted for sequencing. To confirm the accuracy of RNA sequencing results, we performed qPCR analysis of select transcripts identified as cytokinin regulated by the RNA sequencing approach. The resulting data provide the first hormone transcriptome analysis of leaves in tomato. Specifically we identified several previously untested tomato orthologs of cytokinin-related genes as well as numerous novel cytokinin-regulated transcripts in tomato leaves. Principal component analysis of the data indicates that length of cytokinin treatment and plant age are the major factors responsible for changes in transcripts observed in this study. Two hour cytokinin treatment showed a more robust transcript response indicated by both greater fold change of induced transcripts and the induction of twice as many cytokinin-related genes involved in signaling, metabolism, and transport in young vs. older leaves. This difference in transcriptome response in younger vs. older leaves was also found to a lesser extent with an extended (24 h) cytokinin treatment. Overall data presented here provides a solid foundation for future study of cytokinin and cytokinin regulated genes involved in compound leaf development or other developmental processes in tomato.