Genetics - Theses
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ItemGenetic variation in field populations of the cotton bollworm, Helicoverpa armigera (H�bner) (Lepidoptera: Noctuidae)(University of Melbourne, 2008)Within the noctuid moth genus Helicoverpa, H. armigera from the Old World and H. zea of the New World are considered as the most devastating agricultural pests, while species such as H. assulta and H. punctigera are also significant agricultural pests but are limited geographically and/or in their host plant range. Due to their morphological similarity, these Helicoverpa pest species are only reliably distinguishable through adult genitalia morphologies. Despite inflicting significant annual agricultural and socioeconomic costs associated with crop damage and with the application of pesticides in outbreak control measures, fundamental questions such as the single species status of H. armigera throughout its distribution range, its evolutionary relationship with H. zea, and the population genetic structure of H. armigera have not been adequately addressed. Difficulties in obtaining adequate sample sizes to address single species status and inter-species evolutionary relationships have hindered the progress of basic evolutionary genetic research in Helicoverpa pest species. Furthermore, despite significant efforts invested by various research groups in China and Australia in marker development, poor efficacies of microsatellite DNA markers developed to-date represented a significant underlying factor in the lack of consistent advance in population genetic research on H. armigera. In this study, the biology, ecology, population genetics, and problems associated with microsatellite DNA markers of H. armigera are critically reviewed in Chapter I. Chapter II reports on the single species status of H. armigera and of its relationships with other Helicoverpa pest species including that with the New World H. zea based on the mtDNA barcoding gene Cytochrome Oxidase I (COI). Results from the mtDNA COI phylogeny supported the single species status of H. armigera across its Australian, Asian and African distribution range, while high levels of genetic similarity between H. armigera and H. zea suggested that H. zea evolved from a H. armigera founder event around 1.5 million years ago. Chapter II also reports on the patterns of restriction fragment length polymorphisms (RFLPs) of partial mtDNA COI and Cytochrome b (Cyt b) genes produced by endonucleases BstZ17l and Hphl in the four major Helicoverpa pest species. This provides a rapid species identification PCR-RFLP technique which is highly reliable and effective irrespective of insect life stages, and offers support to conventional taxonomic differentiation based on morphological characters. Chapter III reports molecular characterisation of selected published H. armigera microsatellite DNA loci in order to identify factors affecting marker efficacies. Allele characterisation by DNA sequencing and bioinformatic analyses revealed complicating factors caused by single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) at primer binding sites, large INDELS due to Insertion Sequences, and the presence of microsatellite DNA families that potentially associated with transposable elements (TEs). The development of alternative, exon-primed, intron-crossing (EPIC) PCR markers in H. armigera is also presented in Chapter III. The robustness of the EPIC PCR markers was demonstrated through mapping family studies and cross-species amplification, with their suitability as population genetic markers determined from field H. armigera populations. Preliminary analysis of limited Indian H. armigera populations using EPIC PCR markers provided evidence of heterogeneous population substructure that may reflect the diversity of Indian cropping systems and associated geographic heterogeneity. Results from this preliminary population genetic study identified factors fundamental to the success of future H. armigera population genetic studies relevant to the complex Indian agricultural system.
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ItemCharacterisation of lipase genes in Helicoverpa armigera( 2006)Helicoverpa armigera (cotton bollworm) is a major agricultural pest in Australia, Asia and Africa. Analysis of a midgut cDNA library revealed a surprising diversity of genes encoding lipases, enzymes that metabolise lipids. Prior to and during this project, 13 neural lipases and three acidic ones were discovered. These were classified and compared to other insect lipases using sequence alignments and phylogenetic analysis. Novel non-catalytic subfamilies were discovered, including one containing an H. armigera lipase. At least three other subfamilies were found to contain H. armigera lipases. Lipidopteran lipases were found to be almost entirely distinct from Dipteran ones.
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ItemThe role of cytochrome P450s in pyrethroid resistance in the AN02 strain of Helicoverpa armigera( 2003)Helicoverpa armigera is a lepidopteran moth species that is one of the largest agricultural pests in the world. In Australia, it is especially damaging to the cotton industry. Previous research on pyrethroid resistance in Helicoverpa armigera has suggested several resistance mechanisms present in the field. Research on a specific strain of H. armigera, AN02, suggested that resistance to pyrethroids in this particular strain is due to metabolism of the insecticide by cytochrome P450. This thesis describes work undertaken to gain further knowledge of the resistance mechanism present in this strain. Chapter 2 describes expression testing and elimination of the CYP6B cluster of P450s as candidates for resistance in the AN02, which were the most likely candidates available at the time. Chapter 3 describes construction, probing and sequence analysis of a midgut cDNA library of H.armigera to clone more P450 sequences and analyse the general transcriptome of one of the tissues where detoxification is likely to occur. Chapter 4 describes expression testing of new P450 sequences obtained from different sources to determine whether any of them have increased expression that is associated with the resistance genotype. Several new P450 genes were found to have increased expression and are candidates for further study.