Chromosomal scale assembly reveals localized structural variants in avian caecal coccidian parasite Eimeria tenella.

Eimeria tenella is a major cause of caecal coccidiosis in commercial poultry chickens worldwide. Here, we report chromosomal scale assembly of Eimeria tenella strain APU2, a strain isolated from commercial broiler chickens in the U.S. We obtained 100× sequencing Oxford Nanopore Technology (ONT) and more than 800× Coverage of Illumina Next-Seq. We created the assembly using the hybrid approach implemented in MaSuRCA, achieving a contiguous 51.34 Mb chromosomal-scale scaffolding enabling identification of structural variations. The AUGUSTUS pipeline predicted 8060 genes, and BUSCO deemed the genomes 99% complete; 6278 (78%) genes were annotated with Pfam domains, and 1395 genes were assigned GO-terms. Comparing E. tenella strains (APU2, US isolate and Houghton, UK isolate) derived Houghton strain of E. tenella revealed 62,905 high stringency differences, of which 45,322 are single nucleotide polymorphisms (SNPs) (0.088%). The rate of transitions/transversions among the SNPs are 1.63 ts/tv. The strains possess conserved gene order but have profound sequence heterogeneity in a several chromosomal segments (chr 2, 11 and 15). Genic and intergenic variation in defined gene families was evaluated between the two strains to possibly identify sequences under selection. The average genic nucleotide diversity of 2.8 with average 2 kb gene length (0.145%) at genic level. We examined population structure using available E. tenella sequences in NCBI, revealing that the two E. tenella isolates from the U.S. (E. tenella APU2 and Wisconsin, "ERR296879") share a common maternal inheritance with the E. tenella Houghton. Our chromosomal level assembly promotes insight into Eimeria biology and evolution, hastening drug discovery and vaccine development.

Genome Resources for the Ex-Type of Phytophthora citricola, and Well-Authenticated Isolates of P. hibernalis, P. nicotianae, and P. syringae.

Phytophthora is one of the most important genera of plant pathogens, with many members causing high economic losses worldwide. To build robust molecular identification systems, it is very important to have information from well-authenticated specimens and, in preference, the ex-type specimens. The reference genomes of well-authenticated specimens form a critical foundation for genetics, biological research, and diagnostic applications. In this study, we describe four draft Phytophthora genome resources for the ex-type of Phytophthora citricola BL34 (P0716 WPC) (118 contigs for 50 Mb), and well-authenticated specimens of P. syringae BL57G (P10330 WPC) (591 contigs for 75 Mb), P. hibernalis BL41G (P3822 WPC) (404 contigs for 84 Mb), and P. nicotianae BL162 (P6303 WPC) (3,984 contigs for 108 Mb) generated with MinION long-read high-throughput sequencing technology (Oxford Nanopore Technologies). Using the quality reads, we assembled high-coverage genomes of P. citricola with 291× coverage and 16,662 annotated genes; P. nicotianae with 205× coverage and 29,271 annotated genes; P. syringae with 76× coverage and 23,331 annotated genes, and P. hibernalis with 42× coverage and 21,762 annotated genes. With the availability of genome sequences and their annotations, we predict that these draft genomes will be accommodating for various basic and applied research, including diagnostics to protect global agriculture.

Genome Resources of Four Distinct Pathogenic Races Within Fusarium oxysporum f. sp. vasinfectum that Cause Vascular Wilt Disease of Cotton.

Whole genome sequence (WGS) based identifications are being increasingly used by regulatory and public health agencies to facilitate the detection, investigation, and control of pathogens and pests. Fusarium oxysporum f. sp. vasinfectum is a significant vascular wilt pathogen of cultivated cotton and consists of several pathogenic races that are not each other's closest phylogenetic relatives. We have developed WGS assemblies for isolates of F. oxysporum f. sp. vasinfectum race 1 (FOV1), race 4 (FOV4), race 5 (FOV5), and race 8 (FOV8) using a combination of Nanopore (MinION) and Illumina sequencing technology (Mi-Seq). This resulted in assembled contigs with more than 100× coverage for each of the F. oxysporum f. sp. vasinfectum races and estimated genome sizes of FOV1 52 Mb, FOV4 68 Mb, FOV5 68 Mb, and FOV8 55 Mb. The AUGUSTUS gene prediction program predicted 16,263 genes in FOV1, 20,259 genes in FOV4, 20,375 genes in FOV5 and 16,615 genes in FOV8. We were able to identify 525 genes unique to FOV1, 570 unique to FOV4, 1,242 unique to FOV5, and 383 unique to FOV8. We expect that these findings will help in comparative genomics and in the identification of unique genes as candidate targets for diagnostic marker and methods development to permit rapid differentiation of F. oxysporum f. sp. vasinfectum subgroups.

Draft Genome Resource for the Ex-types of Phytophthora ramorum, P. kernoviae, and P. melonis, Species of Regulatory Concern, Using Ultra-Long Read MinION Nanopore Sequencing.

Phytophthora ramorum, P. kernoviae, and P. melonis are each species of current regulatory concern in the United States, the United Kingdom, and other areas of the world. Ex-type material are cultures and duplicates of the type that was used to describe each species and that are deposited in additional culture collections. Using these type specimens as references is essential to designing correct molecular identification and diagnostic systems. Here, we report a whole genome sequence for the Ex-type material of P. ramorum, P. kernoviae, and P. melonis generated using high-throughput sequencing via the MinION third generation platform from Oxford Nanopore Technology. We assembled the quality filtered reads into contigs for each species. We assembled the continuous contigs of P. ramorum, P. kernoviae, and P. melonis (1,322, 545, and 2,091 contigs, respectively). The ab initio prediction of genes from these species reveals that there are 16,838, 12,793, and 34,580 genes in P. ramorum, P. kernoviae, and P. melonis, respectively. Of the 34,580 P. melonis genes, 10,164 genes were conserved among all three of these Phytophthora species which may include pathogenicity genes. We compared the ex-type of P. ramorum EU1 lineage assembly with another selected isolate of EU1 available at the National Center for Biotechnology Information and found 251,859 single nucleotide polymorphisms (SNPs) genome-wide; the comparison with the EU2 lineage genome isolate revealed 441,859 SNPs genome-wide. This genome resource of the ex-types of P. ramorum, and P. kernoviae is a significant contribution as these species are among the most important pathogens of regulatory concern in different regions of the world.

RNA-Seq transcriptome analysis of Amaranthus palmeri with differential tolerance to glufosinate herbicide.

Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in some cases seriously threatens the sustainability of crop production in North America. Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record of glufosinate use at the collection site, survival of plants within the population are likely due to genetic expression that pre-dates selection; in the formal parlance of weed science this is described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was measured using Illumina DNA sequence reads from non-treated and treated surviving (presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282 sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were differentially expressed between treated T and S plants. Of the upregulated genes in treated T plants, 210 were more highly induced than were the upregulated genes in the treated S plants. Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase (GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription factor, NADH-ubiquinone oxidoreductase, ABA 8'-hydroxylase, and cytochrome P450 genes (CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real time-PCR. While GST was upregulated in treated tolerant plants in at least one population, CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are candidates for contributing tolerance to glufosinate. Taken together, these results show that differential induction of stress-protection genes in a population can enable some individuals to survive herbicide application. Elevated expression of detoxification-related genes can get fixed in a population with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained selection pressure could select for mutation(s) in the GS2 gene with the same consequence.

Arabidopsis Novel Glycine-Rich Plasma Membrane PSS1 Protein Enhances Disease Resistance in Transgenic Soybean Plants.

Nonhost resistance is defined as the immunity of a plant species to all nonadapted pathogen species. Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 is nonhost to the oomycete plant pathogen Phytophthora sojae and the fungal plant pathogen Fusarium virguliforme that are pathogenic to soybean (Glycine max). Previously, we reported generating the pss1 mutation in the pen1-1 genetic background as well as genetic mapping and characterization of the Arabidopsis nonhost resistance Phytophthora sojae-susceptible gene locus, PSS1 In this study, we identified six candidate PSS1 genes by comparing single-nucleotide polymorphisms of (1) the bulked DNA sample of seven F2:3 families homozygous for the pss1 allele and (2) the pen1-1 mutant with Columbia-0. Analyses of T-DNA insertion mutants for each of these candidate PSS1 genes identified the At3g59640 gene encoding a glycine-rich protein as the putative PSS1 gene. Later, complementation analysis confirmed the identity of At3g59640 as the PSS1 gene. PSS1 is induced following P. sojae infection as well as expressed in an organ-specific manner. Coexpression analysis of the available transcriptomic data followed by reverse transcriptase-polymerase chain reaction suggested that PSS1 is coregulated with ATG8a (At4g21980), a core gene in autophagy. PSS1 contains a predicted single membrane-spanning domain. Subcellular localization study indicated that it is an integral plasma membrane protein. Sequence analysis suggested that soybean is unlikely to contain a PSS1-like defense function. Following the introduction of PSS1 into the soybean cultivar Williams 82, the transgenic plants exhibited enhanced resistance to F. virguliforme, the pathogen that causes sudden death syndrome.

Investigation of the Fusarium virguliforme Transcriptomes Induced during Infection of Soybean Roots Suggests that Enzymes with Hydrolytic Activities Could Play a Major Role in Root Necrosis.

Sudden death syndrome (SDS) is caused by the fungal pathogen, Fusarium virguliforme, and is a major threat to soybean production in North America. There are two major components of this disease: (i) root necrosis and (ii) foliar SDS. Root symptoms consist of root necrosis with vascular discoloration. Foliar SDS is characterized by interveinal chlorosis and leaf necrosis, and in severe cases by flower and pod abscission. A major toxin involved in initiating foliar SDS has been identified. Nothing is known about how root necrosis develops. In order to unravel the mechanisms used by the pathogen to cause root necrosis, the transcriptome of the pathogen in infected soybean root tissues of a susceptible cultivar, 'Essex', was investigated. The transcriptomes of the germinating conidia and mycelia were also examined. Of the 14,845 predicted F. virguliforme genes, we observed that 12,017 (81%) were expressed in germinating conidia and 12,208 (82%) in mycelia and 10,626 (72%) in infected soybean roots. Of the 10,626 genes induced in infected roots, 224 were transcribed only following infection. Expression of several infection-induced genes encoding enzymes with oxidation-reduction properties suggests that degradation of antimicrobial compounds such as the phytoalexin, glyceollin, could be important in early stages of the root tissue infection. Enzymes with hydrolytic and catalytic activities could play an important role in establishing the necrotrophic phase. The expression of a large number of genes encoding enzymes with catalytic and hydrolytic activities during the late infection stages suggests that cell wall degradation could be involved in root necrosis and the establishment of the necrotrophic phase in this pathogen.

Tanscriptomic Study of the Soybean-Fusarium virguliforme Interaction Revealed a Novel Ankyrin-Repeat Containing Defense Gene, Expression of Whose during Infection Led to Enhanced Resistance to the Fungal Pathogen in Transgenic Soybean Plants.

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliforme infection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction.

Identification of Highly Variable Supernumerary Chromosome Segments in an Asexual Pathogen.

Supernumerary chromosome segments are known to harbor different transposons from their essential counterparts. The aim of this study was to investigate the role of transposons in the origin and evolution of supernumerary segments in the asexual fungal pathogen Fusarium virguliforme. We compared the genomes of 11 isolates comprising six Fusarium species that cause soybean sudden death syndrome (SDS) or bean root rot (BRR), and identified significant levels of genetic variation in A+T-rich repeat blocks of the essential chromosomes and in A+T-neutral regions of the supernumerary segments. The A+T-rich repeat blocks in the essential chromosomes were highly variable between F. virguliforme and non-F. virguliforme isolates, but were scarcely variable between F. virguliforme isolates. The A+T-neutral regions in the supernumerary segments, however, were highly variable between F. virguliforme isolates, with a statistically significant number (21 standard deviations above the mean) of single nucleotide polymorphisms (SNPs). And supernumerary sequence types and rearrangement patterns of some F. virguliforme isolates were present in an isolate of F. cuneirostrum but not in the other F. virguliforme isolates. The most variable and highly expressed region in the supernumerary segments contained an active DNA transposon that was a most conserved match between F. virguliforme and the unrelated fungus Tolypocladium inflatum. This transposon was absent from two of the F. virguliforme isolates. Furthermore, transposons in the supernumerary segments of some F. virguliforme isolates were present in non-F. virguliforme isolates, but were absent from the other F. virguliforme isolates. Two supernumerary P450 enzymes were 43% and 57% identical to their essential counterparts. This study has raised the possibility that transposons generate genetic variation in supernumerary chromosome segments by frequent horizontal transfer within and between closely related species.

A strategy for genome-wide identification of gene based polymorphisms in rice reveals non-synonymous variation and functional genotypic markers.

The genetic diversity of plants has traditionally been employed to improve crop plants to suit human needs, and in the future feed the increasing population and protect crops from environmental stresses and climate change. Genome-wide sequencing is a reality and can be used to make association to crop traits to be utilized by high-throughput marker based selection methods. This study describes a strategy of using next generation sequencing (NGS) data from the rice genome to make comparisons to the high-quality reference genome, identify functional polymorphisms within genes that might result in function changes and be used to study correlations to traits and employed in genetic mapping. We analyzed the NGS data of Oryza sativa ssp indica cv. G4 covering 241 Mb with ∼20X coverage and compared to the reference genome of Oryza sativa ssp. japonica to describe the genome-wide distribution of gene-based single nucleotide polymorphisms (SNPs). The analysis shows that the 63% covered genome consists of 1.6 million SNPs with 6.9 SNPs/Kb, and including 80,146 insertions and 92,655 deletions (INDELs) genome-wide. There are a total of 1,139,801 intergenic SNPs, 295,136 SNPs in intronic/non-coding regions, 195,098 in coding regions, 23,242 SNPs at the five-prime (5') UTR regions and 22,686 SNPs at the three-prime (3') UTR region. SNP variation was found in 40,761 gene loci, which include 75,262 synonymous and 119,836 non-synonymous changes, and functional reading frame changes through 3,886 inducing STOP-codon (isSNP) and 729 preventing STOP-codon (psSNP) variation. There are quickly evolving 194 high SNP hotspot genes (>100 SNPs/gene), and 1,513 out of 2,458 transcription factors displaying 2,294 non-synonymous SNPs that can be a major source of phenotypic diversity within the species. All data is searchable at https://plantstress-pereira.uark.edu/oryza2. We envision that this strategy will be useful for the identification of genes for crop traits and molecular breeding of rice cultivars.

The genome sequence of the fungal pathogen Fusarium virguliforme that causes sudden death syndrome in soybean.

UNLABELLED: Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease. METHODOLOGY/PRINCIPAL FINDINGS: We have generated the draft genome sequence of F. virguliforme by conducting whole-genome shotgun sequencing on a 454 GS-FLX Titanium sequencer. Initially, single-end reads of a 400-bp shotgun library were assembled using the PCAP program. Paired end sequences from 3 and 20 Kb DNA fragments and approximately 100 Kb inserts of 1,400 BAC clones were used to generate the assembled genome. The assembled genome sequence was 51 Mb. The N50 scaffold number was 11 with an N50 Scaffold length of 1,263 Kb. The AUGUSTUS gene prediction program predicted 14,845 putative genes, which were annotated with Pfam and GO databases. Gene distributions were uniform in all but one of the major scaffolds. Phylogenic analyses revealed that F. virguliforme was closely related to the pea pathogen, Nectria haematococca. Of the 14,845 F. virguliforme genes, 11,043 were conserved among five Fusarium species: F. virguliforme, F. graminearum, F. verticillioides, F. oxysporum and N. haematococca; and 1,332 F. virguliforme-specific genes, which may include pathogenicity genes. Additionally, searches for candidate F. virguliforme pathogenicity genes using gene sequences of the pathogen-host interaction database identified 358 genes. CONCLUSIONS: The F. virguliforme genome sequence and putative pathogenicity genes presented here will facilitate identification of pathogenicity mechanisms involved in SDS development. Together, these resources will expedite our efforts towards discovering pathogenicity mechanisms in F. virguliforme. This will ultimately lead to improvement of SDS resistance in soybean.

Sequence based polymorphic (SBP) marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome.

BACKGROUND: Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs) for any genomic regions. Here a sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described. RESULTS: A ~3X genome coverage sequence of the Arabidopsis thaliana ecotype, Niederzenz (Nd-0) was obtained by applying Illumina's sequencing by synthesis (Solexa) technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0) genome sequence identified putative single nucleotide polymorphisms (SNPs) throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated. CONCLUSIONS: The SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for cloning genes based on their genetic map positions and identifying tightly linked molecular markers for selecting desirable genotypes in animal and plant breeding experiments.

Sequence analysis of the long arm of rice chromosome 11 for rice-wheat synteny.

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10x redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score >/= 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.