Whole mitochondrial genomes reveal the relatedness of the browsing ant incursions in Australia.

Global trade and human movements outspread animal species, for example ants, from their native habitats to new areas. This causes biosecurity concerns because an exotic ant might have adverse impacts on agriculture, the environment, or health; thus, incurring economic losses. The browsing ant, Lepisiota frauenfeldi, was first detected in 2013 at the Perth Airport. Since then, more discrete browsing ant infestations have been found in Perth and at the Ports of Darwin and Brisbane. This exotic ant has been deemed a significant pest in Australia and eradication efforts are underway. However, tackling this invasion requires an understanding of how these infestations are related. Are they same or separate or a combination of both? Here, we carried out a phylogenetic analysis using high-throughput sequencing data to determine their relatedness. Our results showed that each interstate incursion was separate. Furthermore, the Western Australian incursions might have two introductions. These findings are critical in devising effective biosecurity measures. However, we discovered that this information could only be revealed by analysing the whole mitochondrial genome; not by a single mitochondrial gene as typically done for species identification. Here, we sequenced 51 whole mitogenomes including three of its congener L. incisa for the first time, for tracing future infestations.

Genetic diversity and SNP's from the chloroplast coding regions of virus-infected cassava.

Cassava is a staple food crop in sub-Saharan Africa; it is a rich source of carbohydrates and proteins which currently supports livelihoods of more than 800 million people worldwide. However, its continued production is at stake due to vector-transmitted diseases such as Cassava mosaic disease and Cassava brown streak disease. Currently, the management and control of viral diseases in cassava relies mainly on virus-resistant cultivars of cassava. Thus, the discovery of new target genes for plant virus resistance is essential for the development of more cassava varieties by conventional breeding or genetic engineering. The chloroplast is a common target for plant viruses propagation and is also a potential source for discovering new resistant genes for plant breeding. Non-infected and infected cassava leaf samples were obtained from different locations of East Africa in Tanzania, Kenya and Mozambique. RNA extraction followed by cDNA library preparation and Illumina sequencing was performed. Assembling and mapping of the reads were carried out and 33 partial chloroplast genomes were obtained. Bayesian phylogenetic analysis from 55 chloroplast protein-coding genes of a dataset with 39 taxa was performed and the single nucleotide polymorphisms for the chloroplast dataset were identified. Phylogenetic analysis revealed considerable genetic diversity present in chloroplast partial genome among cultivated cassava of East Africa. The results obtained may supplement data of previously selected resistant materials and aid breeding programs to find diversity and achieve resistance for new cassava varieties.

Tree Lab: Portable genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa.

In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer's field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

Comparative transcriptome analysis reveals genetic diversity in the endosymbiont Hamiltonella between native and exotic populations of Bemisia tabaci from Brazil.

The whitefly, Bemisia tabaci, is a species complex of more than 40 cryptic species and a major agricultural pest. It causes extensive damage to plants mainly by transmitting plant viruses. There is still a lack of genomic data available for the different whitefly species found in Brazil and their bacterial endosymbionts. Understanding the genetic and transcriptomic composition of these insect pests, the viruses they transmit and the microbiota is crucial to sustainable solutions for farmers to control whiteflies. Illumina RNA-Seq was used to obtain the transcriptome of individual whiteflies from 10 different populations from Brazil including Middle East-Asia Minor 1 (MEAM1), Mediterranean (MED) and New World 2 (NW2). Raw reads were assembled using CLC Genomics Workbench and subsequently mapped to reference genomes. We obtained whitefly complete mitochondrial genomes and draft genomes from the facultative bacterial endosymbiont Hamiltonella for further phylogenetic analyses. In addition, nucleotide sequences of the GroEL chaperonin gene from Hamiltonella from different populations were obtained and analysed. There was concordance in the species clustering using the whitefly complete mitogenome and the mtCOI gene tree. On the other hand, the phylogenetic analysis using the 12 ORF's of Hamiltonella clustered the native species NW2 apart from the exotics MEAM1 and MED. In addition, the amino acid analysis of GroEL chaperonin revealed a deletion only in Hamiltonella infecting NW2 among whiteflies populations analysed which was further confirmed by PCR and Sanger sequencing. The genomic data obtained in this study will aid understanding the functions that Hamiltonella may have in whitefly biology and serve as a reference for further studies regarding whiteflies in Brazil.

The first transcriptomes from field-collected individual whiteflies ( Bemisia tabaci, Hemiptera: Aleyrodidae): a case study of the endosymbiont composition.

Background:Bemisia tabaci species ( B. tabaci), or whiteflies, are the world's most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for B. tabaci are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations. Methods: In this study, we optimised a single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa 1 (SSA1) B. tabaci. Transcriptome sequencing resulted in 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 Contigs across B. tabaci transcriptomes. Results: Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont Portieraaleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. that were predominant across all four SSA1 B. tabaci samples with prevalence levels of between 54.1 to 75%. Amino acid alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Conclusions: The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies.