Queensland fruit fly (Q-fly) ( Bactrocera tryoni ) is the most damaging insect pest of horticulture in Australia. The sterile insect technique (SIT) provides an effective, species-specific, and environmentally benign method for regional management of Q-fly outbreaks. Genetically engineered strains have recently been developed (1) to enable male-only releases through sorting by sex differences in pupal colour (Grey-Scale Exon-3 [GS Exon-3], Genetic Sexing Strain [GSS]) and (2) to enable phenotypic marking through yellow body colour (Yellow-1, Yellow-2). The present study assesses field cage mating competitiveness and mating compatibility, and routine quality control parameters, of these engineered strains, comparing them with the unengineered laboratory strain that is currently used in SIT programs. GS Exon-3, GSS, and Yellow-1 strains performed as well as, or better than, the unengineered strain in mating competitiveness, mating compatibility, copula latency, copula duration, and indices of sexual isolation when paired with both wild and laboratory flies, as well as for emergence, flight ability, and egg hatch rate for all except GSS, which had reduced egg hatch rate. However, Yellow-2 had lower mating performance, percentage of emergence, flight ability, and hatch rate, indicating lower suitability for SIT programs. These results support continued investigation of GSS and Yellow-1 as promising candidates for deployment in SIT programs, offering potential improvements in cost-effectiveness, operational safety, and efficiency through male-only release or phenotypic marking.

Key message Zebularine-treated wheat uncovered a phenotype with characteristics of an epigenetically regulated trait, but major chromosomal aberrations, not DNA methylation changes, are the cause, making zebularine unsuitable for epigenetic breeding. Abstract Breeding to identify disease-resistant and climate-tolerant high-yielding wheats has led to yield increases over many years, but new hardy, higher yielding varieties are still needed to improve food security in the face of climate change. Traditional breeding to develop new cultivars of wheat is a lengthy process taking more than seven years from the initial cross to cultivar release. The speed of breeding can be enhanced by using modern technologies including high-throughput phenomics, genomic selection, and directed mutation via CRISPR. Here we test the concept of modifying gene regulation by transiently disrupting DNA methylation with the methyltransferase inhibitor, zebularine (Zeb), as a means to uncover novel phenotypes in an elite cultivar to facilitate breeding for epigenetically controlled traits. The development and architecture of the wheat inflorescence, including spikelet density, are an important component of yield, and both grain size and number have been extensively modified during domestication and breeding of wheat cultivars. We identified several Zeb-treated plants with a dominant mutation that increased spikelet density compared to the untreated controls. Our analysis showed that in addition to causing loss of DNA methylation, Zeb treatment resulted in major chromosomal abnormalities, including trisomy and the formation of a novel telocentric chromosome. We provide evidence that increased copy number of the domestication gene, Q , is the most likely cause of increased spikelet density in two Zeb-treated plants. Collateral damage to chromosomes in Zeb-treated plants suggests that this is not a viable approach to epigenetic breeding.

Roots provide the critical interface where plants acquire nutrients and water, but our limited understanding of the genetic controls modulating root system architecture (RSA) in crop species constrains opportunities to develop future cultivars with improved root systems. However, there is vast knowledge of root developmental genes in model plant species, which has the potential to accelerate progress in crops with more complex genomes, particularly given that genome editing protocols are now available for most species. PIN-FORMED2 (PIN2) encodes a root-specific polar auxin transporter, where its absence resulted in roots being unable to orient themselves using gravity, producing a significantly wider root system. To explore the role of PIN2 in a cereal crop, we used CRISPR/Cas9 (where CRISPR is clustered regularly interspaced short palindromic repeats) editing to knockout of PIN2 in barley (Hordeum vulgare). Like Arabidopsis, the roots of barley pin2 loss-of-function mutants displayed an agravitropic response at seedling growth stages, resulting in a significantly shallower and wider root system at later growth stages. Notably, despite the significant change in RSA, there was no change in shoot architecture or total shoot biomass, with an insensitivity to the effects of higher planting density. We discuss the future challenges and opportunities to harness the PIN2 pathway to optimize RSA in crops for a range of production scenarios without a shoot trade-off.

Bactrocera tryoni, the Queensland fruit fly, is among the most damaging insect pests to the Australian horticultural industry as larvae infest ripening fruits or vegetables prior to harvest. Genetic biocontrol using Sterile Insect Technique (SIT) programs have been used to successfully suppress populations, via mass release of factory-reared sterile males that mate with wild females. Bi-sex flies are currently used for releases, although the efficiency of these control programs could be improved through using genetic sexing strains that eliminate females early during development, as they are not required for SIT. Here we used CRISPR/Cas9 mutagenesis to modify two nucleotides in the B. tryoni gene shibire, which created a proline to serine amino acid substitution and produced a temperature sensitive phenotype. Shibire is an essential GTPase required in endocytosis and synaptic vesicle recycling, and classical mutagenic screens in the vinegar fly Drosophila melanogaster previously identified temperature sensitive alleles including shits4 that results in adult paralysis. In B. tryoni, the shits4 mutant strain exhibited similar adult paralytic phenotypes when exposed to high temperatures, as well as temperature dependent lethality at egg, larval and pupal stages when subjected to heat treatment above standard rearing temperatures. These temperature sensitive phenotypes could be adapted to develop a SIT genetic sexing strain for conditional elimination of females prior to sterile releases, to improve efficiency and reduce costs.

Proteins play indispensable roles in cellular function, acting as both structural components and catalysts for essential biological processes. Their proper folding into three-dimensional structures is critical for functionality. To ensure correct folding, proteins interact with chaperones and folding catalysts such as Protein Disulfide Isomerases (PDIs), which assist in the formation and rearrangement of disulfide bonds that stabilize proteins by linking cysteine residues. PDIs are part of the thioredoxin (TRX) superfamily and are characterized by a conserved CXXC motif that contributes to their redox potential. They exhibit isomerase and oxidoreductase activities, that enable them to rearrange and form new disulfide bonds. PDI family members in sorghum (SbPDI) present a broad and largely unexplored diversity in domain order, structure, and architecture between or even within species. To shed light on this diversity, we identified and characterized PDI family members in sorghum in silico to explore their domain architecture, three-dimensional structure and functionality.

Root system architecture (RSA) plays a crucial role in crop adaptation and yield stability, especially in the context of climate change and variable growing conditions. Despite this, the genetic basis of RSA remains poorly understood in barley (Hordeum vulgare L.), necessitating the need for more research to better characterize this architecture and explore the potential of diverse germplasm for trait improvement. In this study, we aimed to dissect the genetic basis of seminal root angle (SRA) by examining natural variation within a diverse global collection of 816 barley accessions, including both landraces and modern cultivars. Using a haplotype-based mapping approach, which reflects the recombination patterns considered in breeding programs, we identified chromosomal regions associated with SRA variation. Notably, two major genomic regions on chromosome 5H were identified as novel, while a previously reported region, RAQ1, was confirmed on chromosome 3H. Our analysis revealed significant genetic diversity for SRA within the global collection, with accessions from distinct geographic origins exhibiting unique haplotype combinations. This finding underscores the quantitative nature of the SRA trait and suggests the likelihood of inadvertent selection through polygenic traits related to canopy or yield in commercial breeding programs. To further explore the genetic potential of SRA, we employed a simulation approach to evaluate the feasibility of creating an "ultimate genotype" for narrow SRA. Our results highlight the challenges associated with significantly altering SRA through traditional breeding approaches due to its quantitative, polygenic nature. Consequently, we recommend the integration of predictive and precision breeding techniques, such as genomic selection and gene editing, to effectively capture genetic diversity and accelerate RSA improvement in barley.

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. This study integrates classical and modern genetics to show that the ebony gene is linked to the black pupae phenotype in tephritids, offering new insights into Ebony’s role in the evolutionary developmental biology of this diverse group of flies

Roots provide the critical interface where plants acquire nutrients and water, but our limited understanding of the genetic controls modulating root system architecture (RSA) in crop species constrains opportunities to develop future cultivars with improved root systems. However, there is vast knowledge of root developmental genes in model plant species, which has the potential to accelerate progress in crops with more complex genomes, particularly given that genome editing protocols are now available for most species. PIN-FORMED2 ( PIN2 ) encodes a root specific polar auxin transporter, where its absence resulted in roots being unable to orient themselves using gravity, producing a significantly wider root system. To explore the role of PIN2 in a cereal crop, we used CRISPR/Cas9 editing to knockout of PIN2 in barley ( Hordeum vulgare ). Like Arabidopsis, the roots of barley pin2 loss-of-function mutants displayed an agravitropic response at seedling growth stages, resulting in a significantly shallower and wider root system at later growth stages. Notably, despite the significant change in RSA, there was no change in shoot architecture or total shoot biomass. We discuss the future challenges and opportunities to harness the PIN2 pathway to optimise RSA in crops for a range of production scenarios without a shoot trade-off. ### Competing Interest Statement The authors have declared no competing interest.

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. ### Competing Interest Statement The authors have declared no competing interest.

Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light-induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut-off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript-specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis.

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of ‘DH-Pahang’ reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent ‘Ma850’ and a susceptible line ‘Ma848’, to show that the STR4 resistance co-segregated with marker ‘28820’ at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as ‘Pahang’, ‘SH-3362’, ‘SH-3217’, ‘Ma-ITC0250’, and ‘DH-Pahang/CIRAD 930’. Additional screening in the International Institute for Tropical Agriculture’s collection suggests that the dominant allele is common among the elite ‘Matooke’ NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.

Accessible chromatin regions are critical components of gene regulation but modeling them directly from sequence remains challenging, especially within plants, whose mechanisms of chromatin remodeling are less understood than in animals. We trained an existing deep learning architecture, DanQ, on leaf ATAC-seq data from 12 angiosperm species to predict the chromatin accessibility of sequence windows within and across species. We also trained DanQ on DNA methylation data from 10 angiosperms, because unmethylated regions have been shown to overlap significantly with accessible chromatin regions in some plants. The across-species models have comparable or even superior performance to a model trained within species, suggesting strong conservation of chromatin mechanisms across angiosperms. Testing a maize held out model on a multi-tissue scATAC panel revealed our models are best at predicting constitutively-accessible chromatin regions, with diminishing performance as cell-type specificity increases. Using a combination of interpretation methods, we ranked JASPAR motifs by their importance to each model and saw that the TCP and AP2/ ERF transcription factor families consistently ranked highly. We embedded the top three JASPAR motifs for each model at all possible positions on both strands in our sequence window and observed position- and strand-specific patterns in their importance to the model. With our cross-species “a2z” model it is now feasible to predict the chromatin accessibility and methylation landscape of any angiosperm genome.

DNA methylation is a chromatin modification that plays an essential role in regulating gene expression and genome stability and it is typically associated with gene silencing and heterochromatin. Owing to its heritability, alterations in the patterns of DNA methylation have the potential to provide for epigenetic inheritance of traits. Contemporary epigenomic technologies provide information beyond sequence variation and could supply alternative sources of trait variation for improvement in crops such as sorghum. Yet, compared with other species such as maize and rice, the sorghum DNA methylome is far less well understood. The distribution of CG, CHG, and CHH methylation in the genome is different compared with other species. CG and CHG methylation levels peak around centromeric segments in the sorghum genome and are far more depleted in the gene dense chromosome arms. The genes regulating DNA methylation in sorghum are also yet to be functionally characterised; better understanding of their identity and functional analysis of DNA methylation machinery mutants in diverse genotypes will be important to better characterise the sorghum methylome. Here, we catalogue homologous genes encoding methylation regulatory enzymes in sorghum based on genes in Arabidopsis, maize, and rice. Discovering variation in the methylome may uncover epialleles that provide extra information to explain trait variation and has the potential to be applied in epigenome-wide association studies or genomic prediction. DNA methylation can also improve genome annotations and discover regulatory elements underlying traits. Thus, improving our knowledge of the sorghum methylome can enhance our understanding of the molecular basis of traits and may be useful to improve sorghum performance.

Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes.

SUMMARYThe DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs) are crucial for RNA‐directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element (TE) content. CRISPR‐based genome editing approaches were used to create loss‐of‐function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. Double mutant (drm1ab) plants exhibit some morphological abnormalities but are fully viable. Whole‐genome methylation profiling provided evidence for the widespread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild‐type plants. Evidence was also found for the locus‐specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified genes with altered expression in the drm1ab mutants. However, the majority of genes with high levels of CHH methylation directly surrounding the transcription start site or in nearby promoter regions in wild‐type plants do not have altered expression in the drm1ab mutant, even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of TEs identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons are likely to require active RdDM for the maintenance of transcriptional repression.

Epigenomics encompasses a broad field of study, including the investigation of chromatin states, chromatin modifications and their impact on gene regulation; as well as the phenomena of epigenetic inheritance. The epigenome is a multi-modal layer of information superimposed on DNA sequences, instructing their usage in gene expression. As such, it is an emerging focus of efforts to improve crop performance. Broadly, this might be divided into avenues that leverage chromatin information to better annotate and decode plant genomes, and into complementary strategies that aim to identify and select for heritable epialleles that control crop traits independent of underlying genotype. In this review, we focus on the first approach, which we term ‘epigenome guided’ improvement. This encompasses the use of chromatin profiles to enhance our understanding of the composition and structure of complex crop genomes. We discuss the current progress and future prospects towards integrating this epigenomic information into crop improvement strategies; in particular for CRISPR/Cas9 gene editing and precision genome engineering. We also highlight some specific opportunities and challenges for grain and horticultural crops.

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light–induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of the photosystem II (PSII) D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

Sterile Insect Technique (SIT) is a biocontrol strategy that has been widely utilized to suppress or eradicate outbreak populations of insect pests such as tephritid fruit flies. As SIT is highly favored due to it being species-specific and environmentally friendly, there are constant efforts to improve the efficiency and efficacy of this method in particular at low pest densities; one of which is the use of genetically enhanced strains. Development of these desirable strains has been facilitated by the emergence of the CRISPR/Cas genome-editing technology that enables the rapid and precise genomic modification of non-model organisms. Here, we describe the manual microinjection of CRISPR/Cas9 reagents into tephritid pest Bactrocera tryoni (Queensland fruit fly) embryos to introduce ideal traits as well as the molecular methods used to detect successful mutagenesis.

CRISPR-Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR-Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.