Conservation of synteny and gene order

This project directly compares the genomes of two important agricultural pests – the European corn borer (Ostrinia nubilalis) and the cotton bollworm (H. armigera). The project was initiated within CESAR by David Heckel, in collaboration with Dr. Ingolf Schuphan (RWTH Technical University, Aachen, Germany). The current research is being carried out by Siu Fai Lee (University of Melbourne) in collaboration with David Heckel. 2000 new genes from Ostrinia were identified through an EST sequencing project. 90% of these have been fully sequenced. An AFLP (Amplified Fragment Length Polymorphism) map was created for corn borer, encompassing all 31 chromosomes. The chromosomal locations of 45 single copy orthologous genes (anchor loci) have been determined in both O. nubilalis and H. armigera to allow linkage map comparison. These data revealed near absolute synteny conservation and significant gene order conservation between Ostrinia and Helicoverpa. This conservation extends broadly within the Lepidoptera. Comparisons to the silk moth (Bombyx mori) and the butterfly (Heliconius) are in progress.

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H. armigera and H. zea – one species or two?

The use of mitochondrial DNA Cytochrome Oxidase I (mtDNA COI) gene in species identification has gained popularity in recent years, with its effectiveness in identifying cryptic and new species demonstrated in birds and Lepidoptera. The noctuid moths of the genus Helicoverpa include two of the most devastating agricultural pest species: H. armigera of the old world, and H. zea found exclusively in the north and south American continents. Both of these species are polyphagous targeting >150 crop species. Phylogenies of H. armigera and H. zea have to-date been constructed based on coding nuclear DNA sequences and morphological characters, but a mtDNA phylogeny of H. zea and H. armigera has been lacking. Differentiating H. zea and H. armigera based on morphological characters relies almost exclusively on characters of the male genitalia, although accurate identification has remained problematic due to over-lapping ranges in character measurements. H. zea and H. armigera are presently recognised as two separate species despite successful bi-directional, ‘inter-specific’, mating experiments that gave rise to viable offspring, and the trapping of H. zea males when using H. armigera sex pheromones in the North American continent. Using a 511 base pair sequence of a partial mtDNA COI gene, Gajanan Behere and Tek Tay analysed the phylogenetic relationships amongst 228 H. armigera individuals sampled from China, Australia, Africa, India and Pakistan, plus 14 H. zea from North America, H. punctigera from Australia and H. assulta from India, using Heliothis virescens as an outgroup. Our mtDNA COI phylogeny of Helicoverpa species indicates that H. punctigera is ancestral to H. assulta which is in turn ancestral to H. armigera and H. zea. Furthermore, the long branch-length of H. zea from the H. armigera clade suggests a recent bottleneck event in H. zea’s separation from H. armigera. H. zea and H. armigera show an intermediate level of nucleotide diversity, lying between expected values for intra-specific and inter-specific sequence comparisons, possibly suggesting rapid nucleotide divergence in H. zea due to selection pressures imposed on movement by agricultural practices. Combined H. armigera and H. zea have the highest economic cost to agriculture imposed by insects. While it may never be possible to unambiguously resolve the question – one species or two – our research indicates a remarkable similarity in the genomes. Research from one species can be used to inform and direct research in the other. More comparative research on these species is clearly warranted.

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Global variation in H. armigera

Gajanan Behere has been using mitochondrial DNA sequences from the Cytochrome Oxidase I and Cytochrome b genes to investigate global diversity in moths identified as H. armigera. H. zea, H. punctigera, H. assulta and Heliothis virsescens have been used as outgroups. A total of 228 samples were analysed - Australia (56), Africa (59) ( Burkina Faso (35) and Uganda (24)) and Asia (111) (China (34), India (67) and Pakistan (10)). The sequencing of 511 base pairs of Cytochrome Oxidase I revealed 31 haplotypes and 434 base pairs of Cytochrome b 26 haplotypes. The pattern of molecular variation observed is consistent with the hypothesis that moths called H. armigera in Africa, Asia and Australia are members of the same species. The population structure and movement of the species in Asia and Australia is under intense investigation within CESAR. The analysis has required the development of new molecular markers.

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Molecular markers in H. armigera

Microsatellite markers have been widely used to investigate population structure and movement in many species, including H. armigera. Tek Tay and David Heckel have shown that in H. armigera, for many of the published markers, the target DNA sequences are often repeated multiple times within the genome. Hence these microsatellites are not single locus markers and conclusions based on their use will need to be tested with other marker systems. Our current strategies for isolating single copy DNA markers within H. armigera include (1) random isolation of microsatellite DNA markers and testing for their single copy status by the Southern Blotting method, and (2) the identification of single copy genes for design of exon-primed intron crossing (EPIC) markers. EPIC markers are markers that flank the coding regions of single copy genes for detection of polymorphisms across non-coding regions. Over 70 microsatellite DNA markers originated from published and unpublished silk moth (Bombyx mori) and cotton bollworm DNA sequences, H. armigera bacterial artificial chromosome (BAC) and partial genomic DNA libraries, as well as over 60 single copy genes (EPIC markers) have been identified. These microsatellite DNA and EPIC markers are currently being tested for their feasibility to be used in H. armigera population and genomic organisation studies.

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Spatial and temporal variation in H. armigera in Australia

Nancy Endersby has completed a microsatellite characterisation of Australian population samples of this pest species and compared her data to that from recent publications, out of the University of Queensland, that suggest that populations are differentiated and relatively isolated. Nancy’s use of only robust genetic markers and her critical analysis makes it clear that genetic uniformity/homogeneity across Australia is the situation for this species, and that pest control-measure decisions that assume otherwise are destined to fail.

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Progress in gene manipulation in the cotton bollworm Helicoverpa armigera

Adam Williams is developing systems to analyse candidate genes involved in insecticide resistance by over-expression, ectopic expression and gene knockdown, methods routinely used in Drosophila. Using piggyBac vectors he has been able to transiently drive strong EGFP and dsRed in eggs and young larvae via several promoters and shown that the GAL4-UAS system is functional. A naturally occurring bollworm mutant, yellow eyes has been isolated which could provide a useful genetic background for germline transformation. The armigera homologue of the Drosophila white eye pigment transporter was cloned, however cDNA and Southern analysis revealed that white was not the yellow eyes lesion. A genetic interaction between the yellow eyes mutant and a dark eyes mutant is reminiscent of the Drosophila scarlet/brown interaction, producing a severely de-pigmented double mutant. The armigera homologue of scarlet is being cloned to determine if it is the lesion in the yellow eyes mutant. Adam Williams has successfully knocked down egg pigmentation by targeting the white gene with double stranded RNA interference and antisense morpholino oligonucleotides. Transient EGFP expression has also been knocked down using dsRNAi to the EGFP transcript.

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Genomics of Sheep Blowfly

The blowfly genome project has been funded by Australian Wool Innovation. A BAC genomic library consisting of 55,000 clones with an average insert of 100 kb has been produced and gridded. The clones are currently being fingerprinted to allow the assembly of contigs and the definition of a minimum tiling path.

cDNA libraries have been produced from embryos and first instar larvae. The initial sequencing of 4,756 clones from the embryonic library identified 1853 genes. 78% of these ESTs have sequence similarity to proteins in the GenBank database. The hybridization of cDNA clones to BAC filters will be used to create a high resolution genetic map of L. cuprina. cDNA arrays will be used to examine the expression patterns of every L. cuprina gene identified in this study.

Bioinformatic approaches are being used to identify potential insecticide targets. Our goal is to identify gene products that are (a) expressed early in development (b) required for viability, and (c) unique to L. cuprina. In order to test for viability effects we need to be able to manipulate gene expression in vivo. Our collaborator, Max Scott (Massey University, NZ), is working to improve the efficiency of germline transformation so that the function of individual genes can be investigated using double stranded RNA interference and overexpression in this species.

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Genomics of Head Lice

Stephen Barker and colleagues made progress towards winning funding to sequence the genomes of two important pests: the body louse and the cattle tick. First, the nuclear genome of the body louse Pediculus humanus was added to the high-priority list of the NIH in 2005; it will probably be sequenced in 2006. The nuclear genome of P. humanus is exquisitely small (about 1 megabase) and thus should be relatively easy to interpret. The prospect of comparing the genomes of P. humanus (body lice), P. capitis (head lice), D. melanogastor (vinegar fly) and other insect pests at CESAR in 2006-7 is very exciting. Second, publication of a white paper that proposed the sequencing of the cattle tick, Boophilus microplus was another step towards the sequencing of this genome (Guerrero et al 2006 Journal of Medical Entomology 43, 9-16).

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