Fractionation and subfunctionalization following genome duplications : mechanisms that drive gene content and their consequences

A gene's duplication relaxes selection. Loss of duplicate, low-function DNA (fractionation) sometimes follows, mostly by deletion in plants, but mostly via the pseudogene pathway in fish and other clades with smaller population sizes. Subfunctionalization — the founding term of the Xfunctionalization lexicon — while not the general cause of differences in duplicate gene retention, becomes primary as the number of a gene's cis — regulatory sites increases. Balanced gene drive explains retention for the average gene. Both maintenance-of-balance and subfunctionalization drive gene content nonrandomly, and currently fall outside of our accepted Theory of Evolution. The ‘typical’ mutation encountered by a gene duplicate is not a neutral loss-of-function; dominant mutations (Muller's lexicon; these are not neutral) abound, and confound X functionalization terms like ‘neofunctionalization’. Confusion of words may cause confusion of thought. As with many plants, fish tetraploidies provide a higher throughput surrogate-genetic method to infer function from human and other vertebrate ENCODE-like regulatory sites. ‘Not only have studies on polyploid fractionation led to reconsiderations of fundamental evolutionary theory, but fractionation in polyploids permits higher-throughput comparative genomic experiments using ENCODE-like data yielding the logical precision expected of genetic analyses.’Keywords: The Theory of Evolution, fractionation, Gene Balance Hypothesis, genome dominance, Whole genome duplication, subfunctionalization, dominant mutatio

The best of both worlds: combining lineage specific and universal bait sets in target-enrichment hybridization reactions

PREMISE: Researchers adopting target-enrichment approaches often struggle with the decision of whether to use universal or lineage-specific probe sets. To circumvent this quandary, we investigate the efficacy of a simultaneous enrichment by combining universal probes and lineage-specific probes in a single hybridization reaction, to benefit from the qualities of both probe sets with little added cost or effort.METHODS AND RESULTS: Using 26 Brassicaceae libraries and standard enrichment protocols, we compare results from three independent data sets. A large average fraction of reads mapping to the Angiosperms353 (24–31%) and Brassicaceae (35–59%) targets resulted in a sizable reconstruction of loci for each target set (x̄ ≥ 70%).CONCLUSIONS: High levels of enrichment and locus reconstruction for the two target sets demonstrate that the sampling of genomic regions can be easily extended through the combination of probe sets in single enrichment reactions. We hope that these findings will facilitate the production of expanded data sets that answer individual research questions and simultaneously allow wider applications by the research community as a whole.NaturalisPlant science

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples

Bioinformatics and Next Generation Sequencing: Applications of Arthropod Genomes

Over the past decade, the Next Generation Sequencing (NGS) technology has been broadly applied in many areas such as genomics, medical diagnosis, biotechnology, virology, biological systematics, forensic biology, and anthropology. Taken together, it has offered us brilliant insights into life sciences. Most of the work presented in this thesis describes NGS applications on genome assembly, genome annotation, and comparative genomics, using arthropods as case studies: (1) by sequencing and analyzing the genomes of three Tetranychus spider mites with three completely different feeding behaviors, we uncovered genomic signature variations and indicative of pest adaptations; (2) we sequenced, assembled and annotated five Brevipalpus flat mite genomes and their corresponding endosymbiont Cardinium genomes. Comparative genomics reveals herbivorous pest adaptations and parthenogenesis; (3) the complete genomic analysis of parasitoid wasp Copidosoma floridanum indicates the mechanism of polyembryony of such primary parasite of moths. By bioinformatics and genomics approaches, my study provides the genomic basis and establishes the hypotheses for the future biology in pest and arthropod researches. These NGS applications of arthropod genomes will offer new insights into arthropod evolution and plant-herbivore interactions, open unique opportunities to develop novel plant protection strategies, and additionally, provide arthropod genomic resources as well

Restoring Bee Diversity and Pollination Services through Revegetation

Habitat loss is causing declines in native bees and reducing associated pollination services. Revegetation can be used to reverse these declines, and is a restoration technique attracting growing effort and resources. However, a lack of understanding around the quality of revegetation needed to support native bees and their ecological roles remains, limiting opportunities to improve revegetation outcomes. This thesis aims to address this gap and compares floral and bee diversity, pollination services and pollination networks in revegetated landscapes ranging in habitat quality. In addition, novel molecular tools were explored to improve the ease of undertaking pollen identification and quantification, and applied these methods to describe pollination networks. Field experiments were used to compare floral and bee diversity in revegetation sites varying in quality, together with remnant habitat and cleared land in South Australia. Pollination services within the same sites were measured using two native phytometer species, one pollinated by native bees only, and the other by both native bees and introduced honey bees. Bee diversity and richness were found to be higher within sites that were higher in floral diversity. In addition, while pollination services provided by honey bees were uniform across treatments, pollination by native bees was higher in higher diversity revegetation compared with lower diversity revegetation. Pollination networks were then generated using the bee collected pollen from field surveys. Pollen identification is an important objective for many scientific fields, including pollination ecology and agricultural sciences, where the quantification of mixture proportions is sought after but remains challenging. Novel molecular hybridisation capture approaches can potentially improve upon current methods for identifying and quantifying taxa, and were applied to artificial pollen mixtures. This method uses complementary RNA baits to capture DNA barcodes of interest, and produces random length DNA fragments, which allow for the removal of PCR duplicates, reducing bias in downstream quantification. This metabarcoding approach was applied using two reference libraries for angiosperms (matK and RefSeq chloroplast) constructed from publicly available sequences. Taxon ID provided by the single barcode did not always have resolution to species or genus level. The RefSeq chloroplast database yielded better qualitative results at these taxonomic levels, but the database was limited in taxon coverage. This method was then applied to the native bee pollen. Pollination networks from these data revealed that high diversity revegetation sites had similar complexity and robustness to remnant revegetation, although the latter sites had much larger networks. Networks in low diversity revegetation were simple and potentially un-robust. The main results of this thesis indicate that higher quality revegetation characterised by the establishment of a more diverse set of plant species has the potential to restore native bees and associated pollination services and networks. However, there is still a gap between pollination levels and networks observed in high diversity revegetation compared to remnant vegetation, as well as a substantial difference in bee composition, suggesting that preserving remnant vegetation should be the highest priority conservation action in any landscape.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Improving the diversity of resistance mechanisms available in wheat to combat Fusarium ear blight disease

Fusarium ear blight (FEB) is a disease of wheat and small grain cereals, caused by the fungi Fusarium culmorum and Fusarium graminearum. The disease causes premature bleaching of spikelets and shrivelling of the grain can result in a direct yield loss. Mycotoxins such as deoxynivalenol produced by the fungus can reduce grain quality. Breeding for resistant wheat cultivars is considered one of the best control options. Previously identified resistance has been reported in the Chinese genotype Sumai 3. The principal aim of this project was to identify novel sources of resistance to FEB. Twenty four wheat genotypes were evaluated for resistance to FEB in this project. Molecular markers linked to previously identified QTL from Sumai 3 conferring resistance were used to confirm their absence in the genotypes under investigation, and revealed that none of the genotypes under investigation contained all of the QTL for resistance. Field trials conducted over two years screening for resistance demonstrated that, although not statistically similar to Sumai 3, levels of disease were below 10% in some of the Chinese genotypes. Follow up experiments using reporter strains of Fusarium graminearum explored the accumulation of fungal biomass and the expression of the gene Tri5, which is essential for DON biosynthesis. Fungal biomass levels were not significantly different between genotypes; however expression of the Tri5 gene was significantly lower in the genotype Alsen. A previously developed wheat leaf seedling bioassay was also explored. Scanning electron microscopy revealed the presence of fungal hyphae in advance of the visible lesion during the infection course of F. culmorum. Inoculation with a Tri5 mutant strain of F. graminearum demonstrated that a lack of mycotoxin production altered the lesion type. This project has successfully identified potential novel resistance mechanisms and the future prospects for the control of this disease are discussed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

DNA metabarcoding for the identification of species within vegetarian food samples

>Magister Scientiae - MScAims DNA metabarcoding has recently emerged as a valuable supplementary tool to ensure food authenticity within the global food market. However, it is widely known that highly processed food samples are one of DNA metabarcoding’s greatest shortfalls due to high DNA degradation, presence of PCR inhibitors and the incomplete removal of several undesirable compounds (such as polysaccharides) that makes the amplification of desired DNA challenging. This project has two main aims, the first of which was to determine and develop a cost and time effective DNA metabarcoding system that could successfully describe to species level the ingredient composition of highly processed vegetarian food products. The DNA metabarcoding system was thoroughly evaluated and tested by combining well-researched primers with varying concentrations into a multiplex reaction. The combination of plant and animal primers selected that yielded the best results were used to determine the species composition in the samples. The second aim is to determine the possible presence of meat contaminants within the highly processed vegetarian food samples. Numerous studies have shown that food adulteration is a wide-spread phenomenon throughout the world due to the economic gains it can provide. Animal primers were introduced into the multiplex reaction to aid in the identification of any meat products that could have been inserted into the vegetarian products to lower the overall cost to company. Methodology Thirty-two highly processed vegetarian food samples were collected in the Cape Town area from local and franchised supermarkets. DNA was extracted using the Chloroform/Isoamyl alcohol method best suited for plant-based samples followed by amplification of the following mini-barcoding regions: the mitochondrial 16S ribosomal rRNA, cytochrome B, tRNALeu – trnL – UAA intron and the ribosomal internal transcribed spacer region – ITS2 for plant and fungi identification. The PCR products were purified using the Qiaquick kit and library preparation and building was conducted using the TruSeq DNA PCR-free Library kit. Final purification was completed using AMPure XP kit and the pooled libraries were sequenced on an Illumina Miseq using 300bp paired-end run. Statistical and bioinformatic analysis on the NGS raw sequence reads was performed in R version 3.6.3. Results The results of the data analysis showed that the cytochrome B primer couldn’t detect any animal DNA in the vegetarian samples, however animal-derived sequences were detected in the positives present, validating the efficacy of the multiplex reaction. Mitochondrial 16S ribosomal rRNA was only able to detect plant-based DNA due to the structural homology between chloroplast and mitochondrial DNA. The fungal ribosomal internal transcribed spacer region – ITS2 detected sequences deriving from “Viridiplantae”. This result could have been due to the fungal and plant ribosomal internal transcribed spacer region – ITS2 sharing a reverse primer during amplification. The trnL region was able to detect the presence of undeclared coriander, mustard and wheat in 8 (29%), 6 (21%) and 5 (18%) samples respectively. Additionally, trnL was able to detect the presence of tobacco in 11 (35%) samples. This could have been due to cross-contamination between samples being co-extracted and amplified at the same time for separate studies. The PITS2 region was able to detect the presence of undeclared barley, mustard and wheat in 8 (25%), 4 (14%) and 4 (14%) samples respectively. Our results show the possibility of DNA metabarcoding for the authentication of a wide range of species present in highly processed vegetarian samples using a single assay. However, further optimization of the technique for the identification of both plant and animal species within vegetarian samples needs to be performed before the wide-spread implementation of this technology would be both feasible and viable. Eliminating primer biases, decreasing the risk of homology between different primers in the same assay as well as preventing the amplification of sequencing of undesirable DNA need to be further explored and ultimately mitigated before DNA metabarcoding can be widely seen as an effective and cost-effective method for authentication and food control

A taxonomic revision of 'Hypenia' (Mart. ex Benth.) Harley (Labiatae)

A monograph is presented of Hypenia in the plant family Labiatae. Hypenia was formerly a genus but in this study it is treated as 23 species in six sections of the genus Eriope. 22 of the 23 species treated in this study occupy relatively restricted ranges in the savannas of the central Brazilian plateau and neighbouring parts of the Serra do Espinaco in eastern Brazil. The remaining species occurs in dry scrubland in north-eastern Brazil with a disjunction to savanna in northern Venezuela. The previous classification and taxonomic history of Hypenia are outlined followed by a discussion of morphology and cytology in the group. Observations were made on pollination biology of selected species. Cladistic investigations were conducted using morphological characters and the ITS region of the nuclear genome. The morphological and molecular analyses were then combined in a single analysis. The cladistic analyses indicated that Hypenia was a paraphyletic genus and that generic boundaries between Hypenia and Eriope needed to be reconsidered. The classification of Hypenia was revised in the context of the phylogeny generated by the combined analysis and with consideration given to the theory of classification with particular reference to recent literature on the subject. The largest number of species in Hypenia belonged to the 'macrantha complex', a group of red-flowered species endemic to central Brazil. Considerable revision of the species in this group was required. The 'macrantha complex' was recognised as Eriope section Hypenia with two subsections. One of the subsections was characterised by resupination of the corolla, a previously overlooked character. Five new species of Hypenia are presented in this account. Distribution maps of all the species described were followed by a discussion of biogeographic patterns in the group