In silico prediction of candidate gene targets for the management of African cassava whitefly (Bemisia tabaci, SSA1-SG1), a key vector of viruses causing cassava brown streak disease.

Whiteflies (Bemisia tabaci sensu lato) have a wide host range and are globally important agricultural pests. In Sub-Saharan Africa, they vector viruses that cause two ongoing disease epidemics: cassava brown streak disease and cassava mosaic virus disease. These two diseases threaten food security for more than 800 million people in Sub-Saharan Africa. Efforts are ongoing to identify target genes for the development of novel management options against the whitefly populations that vector these devastating viral diseases affecting cassava production in Sub-Saharan Africa. This study aimed to identify genes that mediate osmoregulation and symbiosis functions within cassava whitefly gut and bacteriocytes and evaluate their potential as key gene targets for novel whitefly control strategies. The gene expression profiles of dissected guts, bacteriocytes and whole bodies were compared by RNAseq analysis to identify genes with significantly enriched expression in the gut and bacteriocytes. Phylogenetic analyses identified three candidate osmoregulation gene targets: two α-glucosidases, SUC 1 and SUC 2 with predicted function in sugar transformations that reduce osmotic pressure in the gut; and a water-specific aquaporin (AQP1) mediating water cycling from the distal to the proximal end of the gut. Expression of the genes in the gut was enriched 23.67-, 26.54- and 22.30-fold, respectively. Genome-wide metabolic reconstruction coupled with constraint-based modeling revealed four genes (argH, lysA, BCAT & dapB) within the bacteriocytes as potential targets for the management of cassava whiteflies. These genes were selected based on their role and essentiality within the different essential amino acid biosynthesis pathways. A demonstration of candidate osmoregulation and symbiosis gene targets in other species of the Bemisia tabaci species complex that are orthologs of the empirically validated osmoregulation genes highlights the latter as promising gene targets for the control of cassava whitefly pests by in planta RNA interference.

Near-infrared spectroscopy and machine learning algorithms for rapid and non-invasive detection of Trichuris.

BACKGROUND: Trichuris trichiura (whipworm) is one of the most prevalent soil transmitted helminths (STH) affecting 604-795 million people worldwide. Diagnostic tools that are affordable and rapid are required for detecting STH. Here, we assessed the performance of the near-infrared spectroscopy (NIRS) technique coupled with machine learning algorithms to detect Trichuris muris in faecal, blood, serum samples and non-invasively through the skin of mice. METHODOLOGY: We orally infected 10 mice with 30 T. muris eggs (low dose group), 10 mice with 200 eggs (high dose group) and 10 mice were used as the control group. Using the NIRS technique, we scanned faecal, serum, whole blood samples and mice non-invasively through their skin over a period of 6 weeks post infection. Using artificial neural networks (ANN) and spectra of faecal, serum, blood and non-invasive scans from one experiment, we developed 4 algorithms to differentiate infected from uninfected mice. These models were validated on mice from a second independent experiment. PRINCIPAL FINDINGS: NIRS and ANN differentiated mice into the three groups as early as 2 weeks post infection regardless of the sample used. These results correlated with those from concomitant serological and parasitological investigations. SIGNIFICANCE: To our knowledge, this is the first study to demonstrate the potential of NIRS as a diagnostic tool for human STH infections. The technique could be further developed for large scale surveillance of soil transmitted helminths in human populations.

First Report of the Detection of DENV1 in Human Blood Plasma with Near-Infrared Spectroscopy.

Dengue virus (DENV) is the world's most common arboviral infection, with an estimated 3.9 million people at risk of the infection, 100 million symptomatic cases and 10,000 deaths per year. Current diagnosis for DENV includes the use of molecular methods, such as polymerase chain reaction, which can be costly for routine use. The near-infrared spectroscopy (NIR) technique is a high throughput technique that involves shining a beam of infrared light on a biological sample, collecting a reflectance spectrum, and using machine learning algorithms to develop predictive algorithms. Here, we used NIR to detect DENV1 artificially introduced into whole blood, plasma, and serum collected from human donors. Machine learning algorithms were developed using artificial neural networks (ANN) and the resultant models were used to predict independent samples. DENV in plasma samples was detected with an overall accuracy, sensitivity, and specificity of 90% (N = 56), 88.5% (N = 28) and 92.3% (N = 28), respectively. However, a predictive sensitivity of 33.3% (N = 16) and 80% (N = 10) and specificity of 46.7% (N = 16) and 32% (N = 10) was achieved for detecting DENV1 in whole blood and serum samples, respectively. DENV1 peaks observed at 812 nm and 819 nm represent C-H stretch, peaks at 1130-1142 nm are related to methyl group and peaks at 2127 nm are related to saturated fatty groups. Our findings indicate the potential of NIR as a diagnostic tool for DENV, however, further work is recommended to assess its sensitivity for detecting DENV in people naturally infected with the virus and to determine its capacity to differentiate DENV serotypes and other arboviruses.

Characterization of transposable elements within the Bemisia tabaci species complex.

BACKGROUND: Whiteflies are agricultural pests that cause negative impacts globally to crop yields resulting at times in severe economic losses and food insecurity. The Bemisia tabaci whitefly species complex is the most damaging in terms of its broad crop host range and its ability to serve as vector for over 400 plant viruses. Genomes of whiteflies belonging to this species complex have provided valuable genomic data; however, transposable elements (TEs) within these genomes remain unexplored. This study provides the first accurate characterization of TE content within the B. tabaci species complex. RESULTS: This study identified that an average of 40.61% of the genomes of three whitefly species (MEAM1, MEDQ, and SSA-ECA) consists of TEs. The majority of the TEs identified were DNA transposons (22.85% average) while SINEs (0.14% average) were the least represented. This study also compared the TE content of the three whitefly genomes with three other hemipteran genomes and found significantly more DNA transposons and less LINEs in the whitefly genomes. A total of 63 TE superfamilies were identified to be present across the three whitefly species (39 DNA transposons, six LTR, 16 LINE, and two SINE). The sequences of the identified TEs were clustered which generated 5766 TE clusters. A total of 2707 clusters were identified as uniquely found within the whitefly genomes while none of the generated clusters were from both whitefly and non-whitefly TE sequences. This study is the first to characterize TEs found within different B. tabaci species and has created a standardized annotation workflow that could be used to analyze future whitefly genomes. CONCLUSION: This study is the first to characterize the landscape of TEs within the B. tabaci whitefly species complex. The characterization of these elements within the three whitefly genomes shows that TEs occupy significant portions of B. tabaci genomes, with DNA transposons representing the vast majority. This study also identified TE superfamilies and clusters of TE sequences of potential interest, providing essential information, and a framework for future TE studies within this species complex.

Trimming and Validation of Illumina Short Reads Using Trimmomatic, Trinity Assembly, and Assessment of RNA-Seq Data.

Next-generation sequencing (NGS) technologies can generate billions of reads in a single sequencing run. However, with such high-throughput comes quality issues which have to be addressed before undertaking downstream analysis. Quality control on short reads is usually performed at default settings due to a lack of in-depth understanding of a particular software's parameters and their effect if changed on the output. Here we demonstrate how to optimize read trimming using Trimmomatic. We highlight the benefits of trimming by comparing the quality of transcripts assembled using trimmed and untrimmed reads.

De Novo Assembly of Linked Reads Using Supernova 2.0.

The recent emergence of "third-generation" sequencing platforms which address shortcomings of standard short reads has allowed the resolution of complex genomic regions during genome assembly. However, sequencing costs for third-generation platforms continue to be high. Novel approaches that leverage the low cost of short-read sequencing while capturing long-range information have been developed. In this chapter, we focus on one such approach, the 10x Genomics' Chromium system. We demonstrate the assembly of the B73 maize reference genome using the Supernova assembler. We also offer suggestions on how one might improve the resulting assembly through analysis of assembly metrics.

Lysine provisioning by horizontally acquired genes promotes mutual dependence between whitefly and two intracellular symbionts.

Horizontal gene transfer is widespread in insects bearing intracellular symbionts. Horizontally transferred genes (HTGs) are presumably involved in amino acid synthesis in sternorrhynchan insects. However, their role in insect-symbiont interactions remains largely unknown. We found symbionts Portiera, Hamiltonella and Rickettsia possess most genes involved in lysine synthesis in the whitefly Bemisia tabaci MEAM1 although their genomes are reduced. Hamiltonella maintains a nearly complete lysine synthesis pathway. In contrast, Portiera and Rickettsia require the complementation of whitefly HTGs for lysine synthesis and have lysE, encoding a lysine exporter. Furthermore, each horizontally transferred lysine gene of ten B. tabaci cryptic species shares an evolutionary origin. We demonstrated that Hamiltonella did not alter the titers of Portiera and Rickettsia or lysine gene expression of Portiera, Rickettsia and whiteflies. Hamiltonella also did not impact on lysine levels or protein localization in bacteriocytes harboring Portiera and ovaries infected with Rickettsia. Complementation with whitefly lysine synthesis HTGs rescued E. coli lysine gene knockout mutants. Silencing whitefly lysA in whiteflies harboring Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia without influencing the expression of Hamiltonella lysA. Furthermore, silencing whitefly lysA in whiteflies lacking Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia in ovarioles. Therefore, we, for the first time, demonstrated an essential amino acid lysine synthesized through HTGs is important for whitefly reproduction and fitness of both obligate and facultative symbionts, and it illustrates the mutual dependence between whitefly and its two symbionts. Collectively, this study reveals that acquisition of horizontally transferred lysine genes contributes to coadaptation and coevolution between B. tabaci and its symbionts.

Genetic diversity of whitefly (Bemisia spp.) on crop and uncultivated plants in Uganda: implications for the control of this devastating pest species complex in Africa.

Over the past three decades, highly increased whitefly (Bemisia tabaci) populations have been observed on the staple food crop cassava in eastern Africa and associated with ensuing viral disease pandemics and food insecurity. Increased whitefly numbers have also been observed in other key agricultural crops and weeds. Factors behind the population surges on different crops and their interrelationships are unclear, although in cassava they have been associated with specific populations within the Bemisia tabaci species complex known to infest cassava crops in Africa. This study carried out an in-depth survey to understand the distribution of B. tabaci populations infesting crops and uncultivated plant hosts in Uganda, a centre of origin for this pest complex. Whitefly samples were collected from 59 identified plant species and 25 unidentified weeds in a countrywide survey. Identities of 870 individual adult whiteflies were determined through mitochondrial cytochrome oxidase 1 sequences (651 bp) in the 3' barcode region used for B. tabaci systematics. Sixteen B. tabaci and five related whitefly putative species were identified based on > 4.0% nucleotide divergence, of which three are proposed as novel B. tabaci putative species and four as novel closely related whitefly species. The most prevalent whiteflies were classified as B. tabaci MED-ASL (30.5% of samples), sub-Saharan Africa 1 (SSA1, 22.7%) and Bemisia Uganda1 (12.1%). These species were also indicated to be the most polyphagous occurring on 33, 40 and 25 identified plant species, respectively. Multiple (≥ 3) whitefly species occurred on specific crops (bean, eggplant, pumpkin and tomato) and weeds (Sida acuta and Ocimum gratissimum). These plants may have increased potential to act as reservoirs for mixed infections of whitefly-vectored viruses. Management of whitefly pest populations in eastern Africa will require an integration of approaches that consider their degree of polyphagy and a climate that enables the continuous presence of crop and uncultivated plant hosts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10340-021-01355-6.

Molecular Evolution of the Glutathione S-Transferase Family in the Bemisia tabaci Species Complex.

The glutathione S-transferase (GST) family plays an important role in the adaptation of herbivorous insects to new host plants and other environmental constrains. The family codes for enzymes that neutralize reactive oxygen species and phytotoxins through the conjugation of reduced glutathione. Here, we studied the molecular evolution of the GST family in Bemisia tabaci, a complex of >35 sibling species, differing in their geographic and host ranges. We tested if some enzymes evolved different functionality, by comparing their sequences in six species, representing five of the six major genetic clades in the complex. Comparisons of the nonsynonymous to synonymous substitution ratios detected positive selection events in 11 codons of 5 cytosolic GSTs. Ten of them are located in the periphery of the GST dimer, suggesting a putative involvement in interactions with other proteins. Modeling the tertiary structure of orthologous enzymes, identified additional 19 mutations in 9 GSTs, likely affecting the enzymes' functionality. Most of the mutation events were found in the environmentally responsive classes Delta and Sigma, indicating a slightly different delta/sigma tool box in each species. At a broader genomic perspective, our analyses indicated a significant expansion of the Delta GST class in B. tabaci and a general association between the diet breadth of hemipteran species and their total number of GST genes. We raise the possibility that at least some of the identified changes improve the fitness of the B. tabaci species carrying them, leading to their better adaptation to specific environments.

Molecular Characterization of a New Virus Species Identified in Yam (Dioscorea spp.) by High-Throughput Sequencing.

To date, several viruses of different genera have been reported to infect yam (Dioscorea spp.). The full diversity of viruses infecting yam, however, remains to be explored. High-throughput sequencing (HTS) methods are increasingly being used in the discovery of new plant viral genomes. In this study, we employed HTS on yam to determine whether any undiscovered viruses were present that would restrict the international distribution of yam germplasm. We discovered a new virus sequence present in 31 yam samples tested and have tentatively named this virus "yam virus Y" (YVY). Twenty-three of the samples in which YVY was detected showed mosaic and chlorotic leaf symptoms, but Yam mosaic virus was also detected in these samples. Complete genome sequences of two YVY viral isolates were assembled and found to contain five open reading frames (ORFs). ORF1 encodes a large replication-associated protein, ORF2, ORF3 and ORF4 constitute the putative triple gene block proteins, and ORF5 encodes a putative coat protein. Considering the species demarcation criteria of the family Betaflexiviridae, YVY should be considered as a novel virus species in the family Betaflexiviridae. Further work is needed to understand the association of this new virus with any symptoms and yield loss and its implication on virus-free seed yam production.

Tissue culture and next-generation sequencing: A combined approach for detecting yam (Dioscorea spp.) viruses.

In vitro culture offers many advantages for yam germplasm conservation, propagation and international distribution. However, low virus titres in the generated tissues pose a challenge for reliable virus detection, which makes it difficult to ensure that planting material is virus-free. In this study, we evaluated next-generation sequencing (NGS) for virus detection following yam propagation using a robust tissue culture methodology. We detected and assembled the genomes of novel isolates of already characterised viral species of the genera Badnavirus and Potyvirus, confirming the utility of NGS in diagnosing yam viruses and contributing towards the safe distribution of germplasm.

Integrated physical map of bread wheat chromosome arm 7DS to facilitate gene cloning and comparative studies.

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere.

A differential k-mer analysis pipeline for comparing RNA-Seq transcriptome and meta-transcriptome datasets without a reference.

Next-generation DNA sequencing technologies, such as RNA-Seq, currently dominate genome-wide gene expression studies. A standard approach to analyse this data requires mapping sequence reads to a reference and counting the number of reads which map to each gene. However, for many transcriptome studies, a suitable reference genome is unavailable, especially for meta-transcriptome studies which assay gene expression from mixed populations of organisms. Where a reference is unavailable, it is possible to generate a reference by the de novo assembly of the sequence reads. However, the high cost of generating high-coverage data for de novo assembly hinders this approach and more importantly the accurate assembly of such data is challenging, especially for meta-transcriptome data, and resulting assemblies frequently suffer from collapsed regions or chimeric sequences. As an alternative to the standard reference mapping approach, we have developed a k-mer-based analysis pipeline (DiffKAP) to identify differentially expressed reads between RNA-Seq datasets without the requirement for a reference. We compared the DiffKAP approach with the traditional Tophat/Cuffdiff method using RNA-Seq data from soybean, which has a suitable reference genome. We subsequently examined differential gene expression for a coral meta-transcriptome where no reference is available, and validated the results using qRT-PCR. We conclude that DiffKAP is an accurate method to study differential gene expression in complex meta-transcriptomes without the requirement of a reference genome.

Species-complex diversification and host-plant associations in Bemisia tabaci: A plant-defence, detoxification perspective revealed by RNA-Seq analyses.

Insect-plant associations and their role in diversification are mostly studied in specialists. Here, we aimed to identify macroevolution patterns in the relationships between generalists and their host plants that have the potential to promote diversification. We focused on the Bemisia tabaci species complex containing more than 35 cryptic species. Mechanisms for explaining this impressive diversification have focused so far on allopatric forces that assume a common, broad, host range. We conducted a literature survey which indicated that species in the complex differ in their host range, with only few showing a truly broad one. We then selected six species, representing different phylogenetic groups and documented host ranges. We tested whether differences in the species expression profiles of detoxification genes are shaped more by their phylogenetic relationships or by their ability to successfully utilize multiple hosts, including novel ones. Performance assays divided the six species into two groups of three, one showing higher performance on various hosts than the other (the lower performance group). The same grouping pattern appeared when the species were clustered according to their expression profiles. Only species placed in the lower performance group showed a tendency to lower the expression of multiple genes. Taken together, these findings bring evidence for the existence of a common detoxification "machinery," shared between species that can perform well on multiple hosts. We raise the possibility that this "machinery" might have played a passive role in the diversification of the complex, by allowing successful migration to new/novel environments, leading, in some cases, to fragmentation and speciation.

Complete genome sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT virus 3, reveals the first evidence of recombination in yam badnaviruses.

Yams (Dioscorea spp.) host a diverse range of badnaviruses (genus Badnavirus, family Caulimoviridae). The first complete genome sequence of Dioscorea bacilliform RT virus 3 (DBRTV3), which belongs to the monophyletic species group K5, is described. This virus is most closely related to Dioscorea bacilliform SN virus (DBSNV, group K4) based on a comparison of genome sequences. Recombination analysis identified a unique recombination event in DBRTV3, with DBSNV likely to be the major parent and Dioscorea bacilliform AL virus (DBALV) the minor parent, providing the first evidence for recombination in yam badnaviruses. This has important implications for yam breeding programmes globally.

The pangenome of hexaploid bread wheat.

There is an increasing understanding that variation in gene presence-absence plays an important role in the heritability of agronomic traits; however, there have been relatively few studies on variation in gene presence-absence in crop species. Hexaploid wheat is one of the most important food crops in the world and intensive breeding has reduced the genetic diversity of elite cultivars. Major efforts have produced draft genome assemblies for the cultivar Chinese Spring, but it is unknown how well this represents the genome diversity found in current modern elite cultivars. In this study we build an improved reference for Chinese Spring and explore gene diversity across 18 wheat cultivars. We predict a pangenome size of 140 500 ± 102 genes, a core genome of 81 070 ± 1631 genes and an average of 128 656 genes in each cultivar. Functional annotation of the variable gene set suggests that it is enriched for genes that may be associated with important agronomic traits. In addition to variation in gene presence, more than 36 million intervarietal single nucleotide polymorphisms were identified across the pangenome. This study of the wheat pangenome provides insight into genome diversity in elite wheat as a basis for genomics-based improvement of this important crop. A wheat pangenome, GBrowse, is available at http://appliedbioinformatics.com.au/cgi-bin/gb2/gbrowse/WheatPan/, and data are available to download from http://wheatgenome.info/wheat_genome_databases.php.

An efficient approach to BAC based assembly of complex genomes.

BACKGROUND: There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. RESULTS: We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. CONCLUSIONS: We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.

High-resolution skim genotyping by sequencing reveals the distribution of crossovers and gene conversions in Cicer arietinum and Brassica napus.

KEY MESSAGE: We characterise the distribution of crossover and non-crossover recombination in Brassica napus and Cicer arietinum using a low-coverage genotyping by sequencing pipeline SkimGBS. The growth of next-generation DNA sequencing technologies has led to a rapid increase in sequence-based genotyping for applications including diversity assessment, genome structure validation and gene-trait association. We have established a skim-based genotyping by sequencing method for crop plants and applied this approach to genotype-segregating populations of Brassica napus and Cicer arietinum. Comparison of progeny genotypes with those of the parental individuals allowed the identification of crossover and non-crossover (gene conversion) events. Our results identify the positions of recombination events with high resolution, permitting the mapping and frequency assessment of recombination in segregating populations.