Christopher M. Ward

The remarkable diversity of insect pigmentation offers a captivating avenue for studying evolution and genetics. In tephritids, understanding the molecular basis of mutant traits is also crucial for applied entomology, enabling the creation of genetic sexing strains through genome editing, thus facilitating sex-sorting before sterile insect releases. Here, we present evidence from classical and modern genetics showing that the black pupae (bp) phenotype in the GUA10 strain of Anastrepha ludens is caused by a large deletion at the ebony locus, removing the gene’s entire coding region. Targeted knockout of ebony induced analogous bp phenotypes across six major tephritid agricultural pests, demonstrating that disruption of Ebony alone is sufficient to produce the mutant trait in distantly related species. This functional characterization further allowed a deeper exploration of Ebony’s role in pigmentation and development across life stages in diverse species. Our findings offer key insights for molecular engineering of sexing strains based on the bp marker and for future evolutionary developmental biology studies in tephritids.

The remarkable diversity of insect pigmentation offers a captivating avenue for exploring evolution and genetics. In tephritid fruit flies, decoding the molecular pathways underlying pigmentation traits also plays a central role in applied entomology. Mutant phenotypes like the black pupae (bp) have long been used as a component of genetic sexing strains, allowing male-only release in tephritid sterile insect technique applications. However, the genetic basis of bp remains largely unknown. Here, we present independent evidence from classical and modern genetics showing that the bp phenotype in the GUA10 strain of the Mexican fruit fly, Anastrepha ludens , is caused by a large deletion at the ebony locus resulting in the removal of the entire protein-coding region of the gene. Targeted knockout of ebony induced analogous bp phenotypes across six tephritid species spanning over 50 million years of divergent evolution. This functionally validated our findings and allowed for a deeper investigation into the role of Ebony in pigmentation and development in these species. Our study offers fundamental knowledge for developing new sexing strains based on the bp marker and for future evolutionary developmental biology studies in tephritid fruit flies.

Cultural exchange of fermentation techniques has driven the spread of Saccharomyces cerevisiae across the globe, establishing natural populations in many countries. Despite this, Oceania is thought to lack native populations of S. cerevisiae, only being introduced after colonisation. Here we investigate the genomic landscape of 411 S. cerevisiae isolated from spontaneous grape fermentations in Australia across multiple locations, years, and grape cultivars. Spontaneous fermentations contained highly recombined mosaic strains that exhibited high levels of genome instability. Assigning genomic windows to putative ancestral origin revealed that few closely related starter lineages have come to dominate the genetic landscape, contributing most of the genetic variation. Fine-scale phylogenetic analysis of loci not observed in strains of commercial wine origin identified widespread admixture with European derived beer yeast along with three independent admixture events from potentially endemic Oceanic lineages that was associated with genome instability. Finally, we investigated Australian ecological niches for basal isolates, identifying phylogenetically distinct S. cerevisiae of non-European, non-domesticated origin associated with admixture loci. Our results illustrate the effect commercial use of microbes may have on local microorganism genetic diversity and demonstrates the presence of non-domesticated, potentially endemic lineages of S. cerevisiae in Australian niches that are actively admixing.

Fungal and bacterial symbiosis is an important adaptation that has occurred within many insect species, which usually results in the relaxation of selection across the symbiont genome. However, the evolutionary pressures and genomic consequences associated with this transition are not well understood. Pathogenic fungi of the genus Ophiocordyceps have undergone multiple, independent transitions from pathogen to associate, infecting soft-scale insects trans-generationally without killing them. To gain an understanding of the genomic adaptations underlying this transition, long-read sequencing was utilized to assemble the genomes of both Parthenolecanium corni and its Ophiocordyceps associate from a single insect. A highly contiguous haploid assembly was obtained for Part. corni , representing the first assembly from a single Coccoidea insect, in which 97% of its 227.8 Mb genome was contained within 24 contigs. Metagenomic-based binning produced a chromosome-level genome for Part. corni ’s Ophiocordyceps associate. The associate genome contained 524 gene loss events compared to free-living pathogenic Ophiocordyceps relatives, with predicted roles in hyphal growth, cell wall integrity, metabolism, gene regulation and toxin production. Contrasting patterns of selection were observed between the nuclear and mitochondrial genomes specific to the associate lineage. Intensified selection was most frequently observed across nuclear orthologs, while selection on mitochondrial genes was found to be relaxed. Furthermore, scans for diversifying selection identified associate specific selection within three adjacent enzymes catalyzing acetoacetate’s metabolism to acetyl-COA. This work provides insight into the adaptive landscape during the transition to an associate life history, along with a base for future research into the genomic mechanisms underpinning the evolution of Ophiocordyceps .

Wine fermentation is a highly complex and competitive environment, imposing harsh selective pressures on fungal community ecology and diversity. The composition of fungal communities inhabiting the surface of grapes will directly impact fermentation progression, wine quality, and contribute to the distinctiveness between wines from different geographical regions. Despite this, the extent of microbial community diversity between geographies, termed ‘microbial terroir,’ remains highly debated. We amassed a large survey of grape spontaneous ferments over six years, encompassing 3105 fungal microbiomes across 14 geographically separated grape-growing regions, and nine grape cultivars. Investigation into the biodiversity of these ferments identified that few high abundance genera form the core of the initial grape microbiome. In line with previous studies, various consistent taxa were linked to specific geographical locations and grape varieties. However, these taxa accounted for a small portion of the overall diversity in the dataset. Through unsupervised clustering, we identified three distinct community types in the grape fungal microbiome, each exhibiting variations in the abundance of key genera. Analysing ferments across temporal and spatial scales revealed significant differences in species richness and compositional heterogeneity between wineries and grape growing regions. However, microbial communities were transient between years in the same winery, regularly transitioning between the three broad community types. We then investigated microbial community composition throughout the fermentative process and observed that initial microbial community composition is predictive of the diversity during the early stages of fermentation, with Hanseniaspora uvarum detected as the main non- Saccharomyces species within this large cohort of samples. Our results help to formulate a clear understanding of the spatial and temporal characteristics of the grape juice fungal microbiome and suggest that these communities are mainly defined by the grape niche and in a minor way shaped by local environmental conditions.

Soft scale insects and mealybugs are phloem feeding Hemipterans that are considered majors pests in agricultural and horticultural settings throughout the world. Viticulturally, scale are a major issue due to their ability to secrete honeydew, which facilitates the development of sooty mould and for their propensity as transmission vectors for several viral diseases of grapevine. To facilitate the rapid identification and quantification of vineyard-associated insects a metagenomic-based bioinformatic pipeline was developed for generalised ecosystem monitoring that automated the assembly and classification of insect mitochondrial genomes from shotgun sequencing data using the Barcode of Life Database API. Parthenolecanium corni (European fruit scale), which was thought to be absent from Australian grapevines, was identified as the dominant coccid species infesting all vines sampled, along with secondary infestation by Pseudococcus viburni (obscure mealybug) and Pseudo. longispinus (long-tailed mealybug). In addition, parisitoidism by Coccophagus scutellaris (Aphelinidae) wasps was also detected. The discovery of Parth. corni as a significant member of scale infestations in Australia has significant implications for the development of effective control strategies for this important group of pests.

Cultural exchange of fermentation techniques has driven the spread of Saccharomyces cerevisiae across the globe, establishing wild populations in many countries. Despite this, most modern commercial fermentations are inoculated using monocultures, rather than relying on natural populations, potentially impacting wild population diversity. Here we investigate the genomic landscape of 411 wild S. cerevisiae isolated from spontaneous grape fermentations in Australia across multiple locations, years, and grape cultivars. Spontaneous fermentations contained highly recombined mosaic strains that commonly exhibited aneuploidy of chromosomes 1, 3, 6 and 9. Assigning wild genomic windows to putative ancestral origin revealed that few closely related commercial lineages have come to dominate the genetic landscape, contributing most of the genetic variation. Fine-scale phylogenetic analysis of loci not observed in strains of commercial wine origin identified widespread admixture with the Beer2 clade along with three independent admixture events from potentially endemic Oceanic lineages that last shared an ancestor with modern East Asian S. cerevisiae populations. Our results illustrate how commercial use of microbes can affect local microorganism genetic diversity and demonstrates the presence of non-domesticated, non-European derived lineages of S. cerevisiae in Australian ecological niches that are actively admixing.

Background and Aims. Shiraz (Syrah) is a dark-skinned cultivar of the wine grape Vitis vinifera that forms the basis of some of the worlds most iconic wines. Worldwide, Shiraz is the fourth most planted grapevine cultivar; however, it represents the most planted cultivar in Australia. Given the importance of Shiraz to worldwide wine production, this study aimed to produce a reference genome for this cultivar while investigating the unique genetic variants and ancestral origins of this iconic variety. Methods and Results. Long-read ONT data were selected to produce a highly contiguous genome assembly for Shiraz. Phylogenetic reconstruction using high-quality genome assemblies for wine grape cultivars provided further support of a kinship between Shiraz and Pinot Noir. Harnessing long-read data, transposable element insertions potentially affecting gene function were characterized in Shiraz and assessed relative to other cultivars. This revealed a heterogenous landscape of transposon insertion points across cultivars and uncovered a specific combination of allelic variants at the VviTPS24 terpene synthase locus. Conclusions. This establishment of a Shiraz genome provides a detailed view of the genetics that underpin this cultivar, including the discovery of a specific combination of VviTPS24 variants, which when combined with appropriate environmental triggers may allow Shiraz to produce high levels of rotundone, the aroma compound responsible for the distinctive peppery characteristics of this cultivar. Significance of the Study. The availability of a reference genome for Shiraz expands the pool of genomes available for wine grapes while providing a foundation resource for whole-genome studies involving this iconic cultivar, including intracultivar variant identification and transcriptomic studies using a matching reference genome.

Shiraz (Syrah) is a dark-skinned cultivar of the wine grape Vitis vinifera that forms the basis of some of the world’s most iconic wines. Worldwide, Shiraz is the fourth most planted grapevine cultivar, however it represents the most planted cultivar in Australia. Given the importance of Shiraz to worldwide wine production, a long-read based reference genome assembly for this cultivar was produced and used to investigate the unique genetic diversity and ancestral origins of this iconic variety. Phylogenetic reconstruction using high-quality genome assemblies for wine grape cultivars provided further support of a kinship between Shiraz and Pinot Noir. Harnessing long-read data, transposable element insertions potentially affecting gene function were characterized in Shiraz and assessed relative to other cultivars. This revealed a heterogenous landscape of transposon insertion points across cultivars and uncovered a specific combination of allelic variants at the TPS24 terpene synthase locus, which when combined with appropriate environmental triggers, may allow Shiraz to produce high levels of rotundone, the aroma compound responsible for the distinctive peppery characteristics of this cultivar.

Vicia sativa L. (common vetch, n = 6) is an annual, herbaceous, climbing legume, originating in the Fertile Crescent of the Middle East and now widespread in the Mediterranean basin, West, Central and Eastern Asia, North and South America. V. sativa is of economic importance as a forage legume in countries such as Australia, China, and the USA, and contributes valuable nitrogen to agricultural rotation cropping systems. To accelerate precision genome breeding and genomics-based selection of this legume, we present a chromosome-level reference genome sequence for V. sativa, constructed using a combination of long-read Oxford Nanopore sequencing, short-read Illumina sequencing, and high-throughput chromosome conformation data (CHiCAGO and Hi-C) analysis. The chromosome-level assembly of six pseudo-chromosomes has a total genome length of 1.65 Gbp, with a median contig length of 684 Kbp. BUSCO analysis of the assembly demonstrated very high completeness of 98% of the dicotyledonous orthologs. RNA-seq analysis and gene modelling enabled the annotation of 53,218 protein-coding genes. This V. sativa assembly will provide insights into vetch genome evolution and be a valuable resource for genomic breeding, genetic diversity and for understanding adaption to diverse arid environments.