Amanda Padovan

Background: The cotton bollworm Helicoverpa armigera is a major global pest controlled by genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins, including Vip3Aa. While Vip3Aa is widely deployed, the genetic basis of resistance remains poorly understood. Previous work identified disruption of a thyroglobulin-like gene (HaVipR1) as one mechanism of resistance, suggesting additional loci may be involved. Results: Using linkage analysis, transcriptomics, long-read sequencing, and CRISPR-Cas9 gene editing, we identify a second thyroglobulin-like gene, HaVipR2, as a novel mediator of Vip3Aa resistance. Resistance in a field-derived H. armigera line was shown to be monogenic, recessive, and autosomal, mapping to chromosome 29. Long-read sequencing revealed a ∼16 kb transposable element insertion disrupting HaVipR2, which was undetectable using standard short-read approaches. CRISPR-Cas9 knockout of HaVipR2 conferred >900-fold resistance, confirming its causal role. Comparative analyses show that HaVipR1 and HaVipR2 share conserved domain architecture, indicating that thyroglobulin-domain proteins represent a recurrent target of resistance evolution. Conclusions: Our findings establish thyroglobulin-domain proteins as a new class of Bt resistance genes in Lepidoptera and demonstrate that transposable element insertions can drive adaptive resistance while evading detection by conventional methods. These results highlight the importance of long-read sequencing and accurate genome annotation for resistance monitoring and provide new insights into the molecular basis and evolution of Vip3Aa resistance.

Austropuccinia psidii is the causal agent of myrtle rust in more than 480 species within the family Myrtaceae. Lineages of A. psidii are structured by their hosts in the native range, and some have success in infecting newly encountered hosts. For example, the pandemic biotype has spread beyond South America, and proliferation of other lineages is an additional risk to biodiversity and industries. Efforts to manage A. psidii incursions, including lineage differentiation, rely on variable microsatellite markers. Testing these markers is time-consuming and complex and requires reference material that is not always readily available. We designed a novel diagnostic approach targeting eight selectively chosen loci including the fungal mating-type HD (homeodomain) transcription factor locus. The HD locus (bW1/2-HD1 and bE1/2-HD2) is highly polymorphic, facilitating clear biological predictions about its inheritance from founding populations. To be considered as potentially derived from the same lineage, all four HD alleles must be identical. If all four HD alleles are identical, six additional markers can further differentiate lineage identity. Our lineage diagnostics relies on PCR amplification of eight loci in different genotypes of A. psidii followed by amplicon sequencing using Oxford Nanopore Technologies and comparative analysis. The lineage-specific assay was validated on four isolates with existing genomes, on uncharacterized isolates, and directly from infected leaf material. We reconstructed alleles from amplicons and confirmed their sequence identity relative to their reference. Genealogies of alleles confirmed the variations at the loci among lineages/isolates. Our study establishes a robust diagnostic tool for differentiating known lineages of A. psidii based on biological predictions and available nucleotide sequences. This tool is suited to detecting the origin of new pathogen incursions.

The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study identifies the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1 ), as a crucial determinant of Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR-Cas9 knockout in susceptible lines confirmed the gene’s role in conferring resistance. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research demonstrates that HaVipR1 -mediated resistance operates independently of known resistance genes, including midgut-specific chitin synthase and the transcription factor SfMyb. The identification of HaVipR1 offers further insights into the Vip3Aa mechanism of action. This discovery is vital for devising strategies to counteract resistance and sustain the efficacy of Bt crops. Future research should focus on elucidating the biochemical pathways involving HaVipR1 and investigating its interactions with other resistance mechanisms. Our findings underscore the utility of analysing field-derived resistant lines in providing biologically relevant insights and stress the necessity for comprehensive management strategies to maintain agricultural productivity. Significance Statement: This is the first identification of a specific gene in Helicoverpa armigera which mediates resistance to the Bacillus thuringiensis (Bt) protein Vip3Aa. We identify that this gene is disrupted in two different ways in separate field-derived resistant lines, one being a large transposable element insertion in the first intron of the HaVipR1 gene, confirmed using long-read sequencing. Disruption of this gene using CRISPR-Cas9 knockout in a susceptible H. armigera line confers Vip3A resistance. The identification of a specific gene is important for molecular monitoring and management of H. armigera as well as other global pests of concern like Spodoptera frugiperda . These findings also offer insights for researchers aiming to understand how Vip3Aa functions, as the action pathway remains unclear.

Native to the Americas, the invasive Spodoptera frugiperda (fall armyworm; FAW) was reported in West Africa in 2016, followed by its chronological detection across the Old World and the hypothesis of an eastward Asia expansion. We explored population genomic signatures of American and Old World FAW and identified 12 maternal mitochondrial DNA genome lineages across the invasive range. 870 high-quality nuclear single nucleotide polymorphic DNA markers identified five distinct New World population clusters, broadly reflecting FAW native geographical ranges and the absence of host-plant preferences. We identified unique admixed Old World populations, and admixed and non-admixed Asian FAW individuals, all of which suggested multiple introductions underpinning the pest’s global spread. Directional gene flow from the East into eastern Africa was also detected, in contrast to the west-to-east spread hypothesis. Our study demonstrated the potential of population genomic approaches via international partnership to address global emerging pest threats and biosecurity challenges.

Native to the Americas, the invasive Spodoptera frugiperda (fall armyworm; FAW) was reported in West Africa in 2016, followed by chronological order of detections across the Old World and the hypothesis of an eastward Asia expansion. We explored population genomic signatures of American and Old World FAW and identified 12 maternal mitochondrial DNA genome lineages across the invasive range, while from 870 high-quality nuclear single nucleotide polymorphic DNA markers five distinct New World population clusters were identified, broadly reflected their native geographical ranges and absence of host-plant preferences. We identified unique admixed Old World populations, and admixed and non-admixed Asian FAW individuals, all of which suggested multiple introductions underpinned the pest’s global spread. Directional gene flow from the East into eastern Africa was also detected, in contrast to the west-to-east spread hypothesis. Our study demonstrated population genomic approaches via international partnership to address global emerging pest threats and biosecurity challenges.

Barriers to dispersal can disrupt gene flow between populations, resulting in genetically distinct populations. Although many marine animals have potential for long-distance dispersal via a planktonic stage, gene flow among populations separated by large geographic distances is not always evident. Polychaetes are ecologically important and have been used as biological surrogates for marine biodiversity. Some polychaete species are used as bait for recreational fisheries, with this demand supporting commercial fisheries for polychaetes to service the retail bait market. However, despite their ecological and economic importance, very little is known about the life history or population dynamics of polychaetes, and few studies have used genetic or genomic approaches to understand polychaete population connectivity. Here, we investigate the population structure of one commonly collected beachworm species used for bait on the eastern coast of Australia, namely, Australonuphis teres, by using genome-wide single-nucleotide polymorphism data. We sampled A. teres from hierarchical nested spatial scales along 900 km of the coast in New South Wales. We identified six genetic groups, but there was no clear geographic pattern of distribution. Our results suggest that there is considerable gene flow among the sampled populations. These high-resolution genomic data support the findings of previous studies, and we infer that oceanographic processes promote genetic exchange among polychaete populations in south-eastern Australia.

Somatic mutations can have important effects on the life history, ecology, and evolution of plants, but the rate at which they accumulate is poorly understood and difficult to measure directly. Here, we develop a method to measure somatic mutations in individual plants and use it to estimate the somatic mutation rate in a large, long-lived, phenotypically mosaicEucalyptus melliodoratree. Despite being 100 times larger thanArabidopsis,this tree has a per-generation mutation rate only ten times greater, which suggests that this species may have evolved mechanisms to reduce the mutation rate per unit of growth. This adds to a growing body of evidence that illuminates the correlated evolutionary shifts in mutation rate and life history in plants.

Somatic mutations can have important effects on the life history, ecology, and evolution of plants, but the rate at which they accumulate is poorly understood, and has been very difficult to measure directly. Here, we demonstrate a novel method to measure somatic mutations in individual plants and use this approach to estimate the somatic mutation rate in a large, long-lived, phenotypically mosaic Eucalyptus melliodora tree. Despite being 100 times larger than Arabidopsis , this tree has a per-generation mutation rate only ten times greater, which suggests that this species may have evolved mechanisms to reduce the mutation rate per unit of growth. This adds to a growing body of evidence that illuminates the correlated evolutionary shifts in mutation rate and life history in plants.

Summary: Terpenoid‐based essential oils are economically important commodities, yet beyond their biosynthetic pathways, little is known about the genetic architecture of terpene oil yield from plants. Transport, storage, evaporative loss, transcriptional regulation and precursor competition may be important contributors to this complex trait. Here, we associate 2.39 million single nucleotide polymorphisms derived from shallow whole‐genome sequencing of 468 Eucalyptus polybractea individuals with 12 traits related to the overall terpene yield, eight direct measures of terpene concentration and four biomass‐related traits. Our results show that in addition to terpene biosynthesis, development of secretory cavities, where terpenes are both synthesized and stored, and transport of terpenes were important components of terpene yield. For sesquiterpene concentrations, the availability of precursors in the cytosol was important. Candidate terpene synthase genes for the production of 1,8‐cineole and α‐pinene, and β‐pinene (which comprised > 80% of the total terpenes) were functionally characterized as a 1,8‐cineole synthase and a β/α‐pinene synthase. Our results provide novel insights into the genomic architecture of terpene yield and we provide candidate genes for breeding or engineering of crops for biofuels or the production of industrially valuable terpenes.

Terpene rich leaves are a characteristic of Myrtaceae. There is significant qualitative variation in the terpene profile of plants within a single species, which is observable as “chemotypes”. Understanding the molecular basis of chemotypic variation will help explain how such variation is maintained in natural populations as well as allowing focussed breeding for those terpenes sought by industry. The leaves of the medicinal tea tree, Melaleuca alternifolia, are used to produce terpinen-4-ol rich tea tree oil, but there are six naturally occurring chemotypes; three cardinal chemotypes (dominated by terpinen-4-ol, terpinolene and 1,8-cineole, respectively) and three intermediates. It has been predicted that three distinct terpene synthases could be responsible for the maintenance of chemotypic variation in this species. We isolated and characterised the most abundant terpene synthases (TPSs) from the three cardinal chemotypes of M. alternifolia. Functional characterisation of these enzymes shows that they produce the dominant compounds in the foliar terpene profile of all six chemotypes. Using RNA-Seq, we investigated the expression of these and 24 additional putative terpene synthases in young leaves of all six chemotypes of M. alternifolia. Despite contributing to the variation patterns observed, variation in gene expression of the three TPS genes is not enough to explain all variation for the maintenance of chemotypes. Other candidate terpene synthases as well as other levels of regulation must also be involved. The results of this study provide novel insights into the complexity of terpene biosynthesis in natural populations of a non-model organism.