††slugcomment: Draft version Seriola lalandi comprises three genetically distinct clades, with S. lalandi lalandi inhabiting the Southern Hemisphere. While a chromosome-level genome exists for the Northwestern Pacific S. lalandi aureovittata, no such resource is available for S. lalandi lalandi, limiting genetic research and aquaculture advancements. This study presents a high-quality chromosome-level genome assembly of S. lalandi lalandi using Illumina short-read, long-read (ONT) and Hi-C. A total of 289.12 Gb of raw genomic data was generated, and a final assembly of 644 Mb was produced with 99.64% of the assembled bases anchored into 24 pseudo-chromosomes. Genome annotation identified 24,600 protein-coding genes, with 96.1% completeness based on 3,640 BUSCO orthologs. Additionally, the complete mitogenome was assembled and annotated for S. lalandi lalandi. This genomic resource will help to inform comparative genomic studies, improve aquaculture breeding programs, and support appropriate fishery strategies, particularly in Aotearoa New Zealand, where S. lalandi lalandi is a culturally significant taonga species for M¯aori.

Flower production is a key determinant of yield in many fruit crops, including kiwifruit (Actinidia chinensis var. deliciosa). Floral development is influenced by both genetic and environmental factors, with biotic stressors such as pathogens also playing a role in reproductive success. In kiwifruit, the bacterial pathogen Pseudomonas syringae pv. actinidiae (Psa) has been linked to floral bud rot (FBR), a phenomenon that may affect flower opening and overall yield. However, the effects of Psa on flower development and fruit set have not yet been thoroughly investigated. We examined the development of Psa-induced FBR on yield potential within an A. chinensis var. deliciosa seedling population. Two novel methods were employed to determine the severity of FBR symptoms in a kiwifruit seedling population. The first method involved detailed daily measurements of FBR on individual flower buds, recorded over a flowering season. The final fate of each flower bud (successful or unsuccessful fruit set) and the shape of the resulting fruit (normal or deformed) were recorded. The second, more efficient method used less detailed scoring of the FBR symptoms. Results showed that the development of FBR symptoms was not correlated with the severity of the final FBR expression, with an average of 11 days from the symptom onset to the final FBR expression. Flower buds with over 50% of the sepals affected by FBR have a lower chance of successful pollination and, if pollination does occur, there is a higher chance that a deformed fruit will form. Both detailed and simplified methodologies effectively scored FBR symptoms within an A. chinensis var. deliciosa seedling population, with the simplified four-category scoring system being the more efficient.

Breeding elite apple cultivars with scab resistance is a key global goal, as reliance on fungicides is unsustainable. The causal fungus, Venturia inaequalis, evolves rapidly, threatening cultivars with single-gene resistance. Since the 1980s, breeding programmes have introduced novel resistance sources via backcrossing. Here, we generated a haplotype-phased genome assembly of Russian apple R12740-7A and an Oxford Nanopore assembly of the Rvi2-resistance accession TSR34T15, enabling detailed dissection of the Rvi2 resistance locus. Fine-mapping using a ‘Royal Gala’ × TSR34T15 segregating family delimited Rvi2 to a narrow genomic interval, within which we identified a 10 041 bp long terminal repeat retrotransposon (LTR-RT) insertion—an insert-based structural variant (SV) strongly linked with Rvi2. Notably, this LTR-RT harbours an FPPS gene, a member of the farnesyl pyrophosphate/geranylgeranyl pyrophosphate (FPP/GGPP) synthase family, located 2 kb from a key candidate defence gene. Although the FPPS gene exhibits stable expression, its integration within the retrotransposon suggests a cis-regulatory role, potentially priming adjacent defence genes for robust up-regulation upon pathogen attack. We validated the marker derived from this SV in diverse germplasms and successfully implemented it in marker-assisted selection across extensive seedling cohorts. This marker will streamline the development of scab-resistant apple varieties.

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is a persistent problem for pear (Pyrus spp.) growers across most production regions around the world. Growing resistant cultivars is one of the best options for managing fire blight. The resistant P. communis cultivars ‘Potomac’ and ‘Old Home’, and the hybrid selection NJA2R59T69 were used in a previous study to identify quantitative trait loci (QTLs) linked to the resistance. The major chromosome 2 QTLs identified in the ‘Potomac’ and ‘Old Home’ sources overlap with QTLs that were previously identified in ‘Harrow Sweet’ and ‘Moonglow’, while that of NJA2R59T69 (through P. ussuriensis ‘Pai Li’) mapped to a nearby location on chromosome 2. In the current study, genes associated with disease resistance in the two chromosome 2 QTL regions were cloned in 23 accessions representing resistant and susceptible cultivars, and progeny from the three sources. Alleles unique to resistant cultivars in these genic regions were targeted by SeqSNP and used to genotype a diversity set of 382 pear accessions with known fire blight disease responses from multiple sources. Association mapping was conducted across subsets of the 382 accessions based on the shared fire blight resistance source. Association mapping identified a marker (Chr2_3601869) that was predictive with 88.0

Flowering in perennial crops is a trait influenced by genetics, environmental cues and plant vigor. Here, we studied the genetic and environmental control of repeat flowering (RF) in blueberry (Vaccinium corymbosum). RF was measured in a full-sib population from a cross between repeat and non-repeat flowering cultivars (‘Hortblue Petite’ and ‘Nui’, respectively). Longitudinal phenotypes were used to model the area under the curve for the first and second flowering peaks. We found that RF was strongly influenced by bush size and vigor, which we then incorporated into the area under the flowering curve models. Quantitative trait loci linked to both first bloom and RF were detected at three hotspots on chromosomes 4 and 10, and genes of interest known to regulate flowering under both temperature and photoperiod control were discussed. The phenotyping protocol and statistical modelling method reported here are an effective strategy for the investigation of complex interactions between multiple genetic loci and environmental variables on developmental traits, such as flowering. Experimental designs with replicated multi-environment and multi-year measurements are now needed to corroborate our results and further elucidate the determinism of RF in blueberry.

Context. Wetland restoration is critical for the recovery of biodiversity, ecosystem functions and services. In highly modified landscapes, conservation must navigate complex trade-offs among these goals. Local-scale information on the genetic structure and trajectory of species can support the design of restoration strategies that maximise diversity. Objectives. We investigated population genetics of Syzygium maire (maire tawake), a critically threatened canopy tree of swamp forests in the Greater Wellington Region of Aotearoa New Zealand. Methods. We sampled leaves of adult trees and seedlings in 12 sites across the region applying low-coverage whole genome resequencing to each individual. We characterised genetic structure in the adult and seedling cohorts, examining patterns of kinship and diversity using hierarchical Analysis of Molecular Variance. Further, we compared the decay in spatial autocorrelation among trees in this region to that of a relatively large and intact reference population. Results . Our data suggest historical homogeneity and admixture across extensive pre-fragmentation swamp forests, but fine-scale genetic structure and decay of spatial autocorrelation within a few kilometres indicates recent fragmentation now constrains gene flow. Small yet significant reductions in genetic diversity and increased inbreeding in seedlings point to early genetic erosion in remnant habitat patches. Although genetic loss is not yet severe, continued isolation risks accelerating degradation in future generations. Conclusions. Restoration should prioritise reconnecting fragmented populations, maximizing genetic diversity by replanting with broadly sourced seed and reestablishing habitat for avian seed-dispersers. These findings highlight how genetic information can inform restoration planning and improve the long-term resilience of habitat specialist.

Agathis australis (D.Don) Lindl. (kauri) is an endemic conifer species of Aotearoa New Zealand and is among the largest of the forest trees. We used genotyping‐by‐sequencing to genotype 231 samples collected from across its natural range. Population structure analysis using 1337 high‐confidence SNPs recovered two regional provenances of kauri from Northland/Auckland and Bay of Plenty. Low levels of admixture were observed between individuals from all locations. Ancestral admixture was apparent between individuals of all sampled populations, suggesting high levels of gene flow and integration between individuals and populations. Pairwise F st values were low, with the highest value measured being 0.0564. A weak positive, but significant, relationship was identified between genetic and geographic distance, via isolation by distance analysis. This reflects a north–south pattern of variation in the genetic data. We suggest these SNP data and the current distribution reflect in situ refugia populations persisting throughout its range during the Last Glacial Maximum and subsequent local expansion from these in the later Holocene. There is no support for southward post‐glacial migration from a northernmost refugia. Investigating the genetic diversity and structure of kauri informs its conservation and can be used to assist with managing the threats presented by kauri dieback.

The restoration of swampland is vital for the recovery of both biodiversity and cultural values in Aotearoa New Zealand. Syzygium maire, an endemic wetland tree species, is a focus of many wetland restoration efforts. Formerly widespread, extant populations are small, fragmented, and under pressure from myrtle rust. Restoration initiatives may be unknowingly compounding these threats to the species by failing to represent the complete genetic diversity of populations. What genetic diversity remains in remnants and how it is distributed is not known. We therefore aimed to assess the national scale population structure, genetic diversity, and adaptive potential of S. maire to inform species conservation. We identified over 760,000 high-quality single nucleotide variants in 269 reproductive age trees from across the species' range, using low coverage whole genome resequencing. At a national scale, we found five distinct regional-scale genetic clusters, which in turn exhibit local structure and admixture. In the North Island: Northland, Bay of Plenty in the central east, Taranaki in the central west, and Greater Wellington/Manawatū in the south. A single cluster was identified in the South Island, Marlborough. Within-cluster substructure was particularly evident for Greater Wellington/Manawatū. Genetic diversity and fixation indices (F ST) were relatively uniform across all clusters, and there was some evidence of north to south increase in kinship and shorter time since radiation. These patterns are likely to reflect glaciation cycles that resulted in complex contractions into local microrefugia and subsequent re-radiations of the species over time. Genotype by environment analysis detected genetic variants potentially contributing to environmental adaptation, notably precipitation seasonality. Restoration and conservation goals would best be served by capturing diversity within regional clusters. Information on the geographic and environmentally structured distribution of this tree's genetic diversity supports conservation and restoration strategies through ensuring the complete extant diversity is captured, identifying regions at most risk of genetic degradation, and facilitating planning regarding the movement of adaptive diversity in a changing environment.

Blueberry is promoted as a super food with several health properties derived from chlorogenic acid and anthocyanin. Previous studies indicated that anthocyanin acylation and the content of chlorogenic acid could affect their level of absorption and biological activity. In this study, a genome-wide association study was performed to identify loci associated with anthocyanin and chlorogenic acid and characterize the candidate genes controlling anthocyanin acylation. Two stable loci controlling anthocyanin acylation and glucose specific glycosylation were confirmed on chromosomes 2 and 4, respectively, while no stable loci associated with chlorogenic acid were identified. Two acyl-CoA acyltransferases named VcBAHD-AT1 and VcBAHD-AT4 were identified as best candidate genes controlling anthocyanin acylation. Interestingly, the two genes clustered in acyl-CoA acyltransferases clade III, a clade that is not commonly associated with anthocyanin acylation. A virus-induced gene silencing approach optimized for silencing VcBAHD-AT1 and VcBAHD-AT4 in the whole blueberry fruits, confirmed the role of these two genes in anthocyanin acylation. Overall, this study establishes the foundation to develop a molecular marker to select for higher acylated anthocyanin and delivered a method for rapid functional characterization of genes associated with other fruit related traits in blueberry. Also, the study adds evidence that during the evolution of acyl-CoA acyltransferases multiple routes led to the emergence and/or fixation of the anthocyanin acyltransferase activity. These outcomes advance knowledge about the genes controlling anthocyanin acylation in blueberries and that extend to other plants. Selecting new blueberry cultivars with higher acylated anthocyanin levels could potentially increase absorption of this health-related bioactive.

Self-incompatibility (SI) in plants has evolved independently multiple times and S-RNase-based gametophytic self-incompatibility (GSI) is most common. The Rosaceae family possesses both self-recognition (Prunus) and nonself-recognition (Malus) GSI systems, and the latter is widespread in flowering plants. Gillenia trifoliata is a Rosaceae species related to Prunus and Malus, providing utility for understanding SI evolution. Gillenia is sister taxon to Malus, but unlike Malus, has not undergone polyploidisation. In addition, the common ancestor of Gillenia and Prunus is close to the origin of the subfamily. Using a highly contiguous Gillenia genome, orthologous regions to both Malus and Prunus S-loci were identified. Only the Prunus-like S-locus was highly polymorphic and had signatures of a functional S-locus including positive selection of the S-RNase. This suggests a self-recognition system controls SI in Gillenia, and the common ancestors of Gillenia and Prunus, and Gillenia and the apple tribe, likely had a self-recognition SI system. Comparative genomics between Gillenia and Malus suggest apple lost the self-recognition mechanism, and a nonself-recognition mechanism evolved independently from a rudimentary locus with at least one male S-determinant. Repetitive sequences in the Malus-like S-locus in Gillenia may facilitate illegitimate recombination, suggesting putative mechanisms of evolution of nonself-recognition S-loci.

Abstract Blueberry is promoted as a super food with several health properties derived from chlorogenic acid and anthocyanin. Previous studies indicated that anthocyanin acylation and the content of chlorogenic acid could affect their level of absorption and biological activity. In this study, a genome-wide association study was performed to identify loci associated with anthocyanin and chlorogenic acid and characterize the candidate genes controlling anthocyanin acylation. Two stable loci controlling anthocyanin acylation and glucose specific glycosylation were confirmed on chromosomes 2 and 4, respectively, while no stable loci associated with chlorogenic acid were identified. Two acyl-CoA acyltransferases named VcBAHD-AT1 and VcBAHD-AT4, were identified as best candidate genes controlling anthocyanin acylation. Interestingly, the two genes clustered in acyl-CoA acyltransferases clade III, a clade that is not commonly associated with anthocyanin acylation. A Virus-induced gene silencing approach optimized for silencing VcBAHD-AT1 and VcBAHD-AT4 in the whole blueberry fruits, confirmed the role of these two genes in anthocyanin acylation. Overall, this study establishes the foundation to develop a molecular marker to select for higher acylated anthocyanin and delivered a method for rapid functional characterization of genes associated with other fruit related traits in blueberry. Also, the study adds evidence that during the evolution of acyl-CoA acyltransferases multiple routes led to the emergence and/or fixation of the anthocyanin acyltransferase activity. These outcomes advance knowledge about the genes controlling anthocyanin acylation in blueberries and that extend to other plants. Selecting new blueberry cultivars with higher acylated anthocyanin levels could potentially increase absorption of this health related bioactive.

Petunia hybrida is the world’s most popular garden plant and is regarded as a supermodel for studying the biology associated with the Asterid clade, the largest of the two major groups of flowering plants. Unlike other Solanaceae, petunia has a base chromosome number of seven, not 12. This along with recombination suppression has previously hindered efforts to assemble its genome to chromosome level. Here we achieve a chromosome-level assembly for P. hybrida using a combination of short-read and long-read sequencing, optical mapping (Bionano) and Hi-C technologies. The resulting assembly spans 1253.6 Mb with a BUSCO score of 99.8%. A total of 35,089 genes were predicted and of those 29,655 were functionally annotated. Syntenic regions between petunia, tomato and pepper were identified, highlighting rearrangements that have occurred since their divergence indicating that the 12 chromosomes of Solanaceae did not originate from whole genome duplication of an ancestral species with seven chromosomes like petunia. This assembly will enhance trait mapping efficiency and serve as a valuable resource for functional genomic studies.

Blueberry has a diversity of anthocyanins that confer its characteristic blue-coloured skin. Whilst most cultivars produce only anthocyanin glycosides, some can add aliphatic or aromatic groups to the sugar moiety to create acylated anthocyanins. Due to their enhanced stability, acylated anthocyanins represent an attractive breeding target in blueberry. In this study, a haplotype-resolved assembly of a previously identified quantitative trait locus on chromosome 2 of 'Hortblue Petite' (Vaccinium corymbosum) was created to identify candidate anthocyanin acyltransferase genes. One full-length gene (VcAAT1a) was selected based on quantitative PCR expression profiling and transient expression in tobacco leaves and in strawberry and blueberry fruit flesh. In all three systems, VcAAT1a was able to produce a range of acylated anthocyanins in planta. Recombinant VcAAT1a protein demonstrated that, while VcAAT1a was able to act on both anthocyanin 3-O-glucosides and 3-O-galactosides, it could only utilize acetyl-CoA as an acyl donor. Protein modelling using AlphaFold suggested that this restricted range in acyl donors may be due to a spatially restricted sub-pocket in the acyl-binding site of VvAAT1. Finally, LUC/REN promoter activation assays revealed that the VcAAT1a promoter was transactivated by the VcMYBPA1 and VcMYBPA2 transcription factors, further expanding our knowledge of anthocyanin regulation in blueberry.

Blueberry (Vaccinium spp.) is one of the most economically important berry crops worldwide. Validation of genetic mapping studies is often hindered by asynchronous marker technology. The development of a standardized genotyping platform that targets a specific set of polymorphic loci can be a practical solution to unify the scientific and breeding community toward blueberry improvement. The objective of this study was to develop and evaluate a targeted genotyping platform for cultivated blueberries that is affordable, reproducible, and sufficiently high density to warrant large-scale adoption for genomic studies. The Flex-Seq platform was developed in a two-step procedure that resulted in 22,000 loci that yielded 194,365 single nucleotide polymorphisms when assessed in a diversity set of 192 samples including cultivated and other related wild Vaccinium species. Locus recovery averaged 89.4% in the cultivated polyploid blueberry (northern highbush [NHB], southern highbush [SHB], and rabbiteye [RE]) and on average 88.8% were polymorphic. While recovery of these loci was lower in the other Vaccinium species assayed, recovery remained high and ranged between 60.8% and 70.4% depending on the taxonomic distance to the cultivated blueberry targeted in this platform. NHB had the highest mean number of variants per locus at 9.7, followed by RE with 9.1, SHB with 8.5, and a range between 7.7 and 8.5 in other species. As expected, the total number of unique-in-state haplotypes exceeded the total number of variants in the domesticated blueberries. Phylogenetic analysis using a subset of the SNPs and haplotypes mostly conformed to known relationships. The platform also offers flexibility about the number of loci, depth of sequencing for accurate dosage calling, loci and haplotype reconstruction from increased fragment length. This genotyping platform will accelerate the development and improvement of blueberry cultivars through genomic-assisted breeding tools.

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has decimated kiwifruit orchards growing susceptible kiwifruit Actinidia chinensis varieties. Effector loss has occurred recently in Psa3 isolates from resistant kiwifruit germplasm, resulting in strains capable of partially overcoming resistance present in kiwiberry vines (Actinidia arguta, Actinidia polygama, and Actinidia melanandra). Diploid male A. melanandra recognises several effectors, sharing recognition of at least one avirulence effector (HopAW1a) with previously studied tetraploid kiwiberry vines. Sequencing and assembly of the A. melanandra genome enabled the characterisation of the transcriptomic response of this non-host to wild-type and genetic mutants of Psa3. A. melanandra appears to mount a classic effector-triggered immunity (ETI) response to wildtype Psa3 V-13, as expected. Surprisingly, the type III secretion (T3SS) system-lacking Psa3 V-13 ∆hrcC strain did not appear to trigger pattern-triggered immunity (PTI) despite lacking the ability to deliver immunity-suppressing effectors. Contrasting the A. melanandra responses to an effectorless Psa3 V-13 ∆33E strain and to Psa3 V-13 ∆hrcC suggested that PTI triggered by Psa3 V-13 was based on the recognition of the T3SS itself. The characterisation of both ETI and PTI branches of innate immunity responses within A. melanandra further enables breeding for durable resistance in future kiwifruit cultivars.

Aotearoa New Zealand’s swamp forests have experienced significant habitat loss in fewer than two hundred years. Many of the country’s tree species are endemic with sparse to no genetic information available to underpin conservation strategies. Syzygium maire, Aotearoa’s only endemic Syzygium species, is a culturally and ecologically important component of swamp forest habitats. Unfortunately, populations of S. maire have been greatly reduced, heavily fragmented and are susceptible to the emergent pathogen Austropuccinia psidii (myrtle rust), posing eminent danger of a further decline of the species. We sought to develop genomic resources to inform conservation management of S. maire. To this end, we used long read, high accuracy sequencing technology to produce a highly complete reference quality genome for S. maire. The genome sequence was named ‘Ngā Hua o te Ia Whenua’ by the local Māori tribe where the tree used for genome sequencing grows. We assess whether genome-level divergence with other Myrtaceae may have followed geographic isolation of the species. We detect conservation of large scale synteny between three Syzygium species and Eucalyptus grandis, providing support for the stability of Syzygium genomes across evolutionary time. We annotate genes implicated in fungal pathogen defence, identifying several hundred putative NLR genes, including putative homologs of previously identified Austropuccinia psidii resistance genes. Finally, we evaluate the genetic relationships of individuals of a small, isolated population of trees. We find evidence of high levels of kinship and inbreeding within small and isolated S. maire populations, informing local-scale conservation strategies for the species. Our findings enable practical conservation actions and provide resources for larger scale studies of S. maire and other Syzygium species in the future.

Rewarewa (Knightia excelsa, Proteaceae) is a tree species endemic to Aotearoa New Zealand, with a natural distribution spanning Te Ika-a-Māui (North Island) and the top of Te Waipounamu (South Island). We used the pseudo-chromosome genome assembly of rewarewa as a reference and whole genome pooled sequencing from 35 populations sampled across Aotearoa New Zealand, including trees growing on Māori-owned land, to identify 1,443,255 single nucleotide polymorphisms (SNPs). Four genetic clusters located in the northern North Island (NNI), eastern North Island (NIE), western and southern North Island (NIWS), and the South Island (SI) were identified. Gene flow was revealed between the SI and NIE genetic clusters, plus bottleneck and contraction events within the genetic clusters since the mid-late Pleistocene, with divergence between North and South Island clusters estimated to have occurred ~115,000–230,000 years ago. Genotype environment analysis (GEA) was used to identify loci and genes linked with altitude, soil pH, soil carbon, slope, soil size, annual mean temperature, mean diurnal range, isothermality, annual precipitation, and precipitation seasonality. The location of the SNPs associated with these environmental variables was compared with the position of 52,192 gene-coding sequences that were predicted in the rewarewa genome using RNA sequencing. This new understanding of the genetic variation present in rewarewa and insights into the genetic control of adaptive traits will inform efforts to incorporate the species in restoration plantings and for marketing rewarewa honey based on provenance.

AbstractPseudomonas syringaepv.actinidiaebiovar 3 (Psa3) has decimated kiwifruit orchards growing susceptible kiwifruitActinidia chinensisvarieties. Effector loss has occurred recently in Psa3 isolates from resistant kiwifruit germplasm, resulting in strains capable of partially overcoming resistance present in kiwiberry vines (A. arguta, A. polygama, andA. melanandra). Diploid maleA. melanandrarecognises several effectors, sharing recognition of at least one avirulence effector (HopAW1a) with previously studied tetraploid kiwiberry vines. Sequencing and assembly of theA. melanandragenome enabled the characterisation of the transcriptomic response of this non-host to wild-type and genetic mutants of Psa3.A. melanandraappears to mount a classic effector-triggered immunity (ETI) response to wildtype Psa3 V-13, as expected. Surprisingly, the type III secretion (T3S) system-lackingPsa3 V-13 ΔhrcCstrain did not appear to trigger pattern-triggered immunity (PTI) despite lacking the ability to deliver immunity-suppressing effectors. Contrasting theA. melanandraresponses to an effectorless Psa3 V-13 Δ33Estrain and to Psa3 V-13 ΔhrcCsuggested that PTI triggered by Psa3 V-13 was based on the recognition of the T3S itself. The characterisation of both ETI and PTI branches of innate immunity responses withinA. melanandrafurther enables breeding for durable resistance in future kiwifruit cultivars.