Horticulture Research

The efficient pyramiding of favorable alleles underlying complex traits remains a major challenge in crop breeding as most quantitative trait loci (QTLs) have not been resolved to causal genes, limiting their direct application in marker-assisted breeding. Although haplotypes provide more informative genetic units than individual markers, existing haplotype-based studies have largely focused on genetic interpretation and elite haplotype discovery, whereas computational frameworks for translating haplotypes into breeding decisions remain limited. Here, we developed HAPBDB, a haplotype-guided breeding framework that directly translates regional haplotypes into parental selection, cross design, and elite QTL pyramiding, and applied it to a lettuce genomic breeding panel. HAPBDB accurately reconstructed functional haplotypes at known loci and resolved elite haplotypes for five major QTLs controlling flowering time and yield. Integrating haplotype information across loci enabled systematic identification of accessions carrying complementary elite haplotypes and rational design of crosses that maximized favorable haplotype accumulation while minimizing segregating loci. Experimental validation using QTL-specific molecular markers demonstrated concordance between predicted and observed multi-locus genotypes across all designed F hybrids. Our results demonstrated that regional haplotypes can serve as practical breeding units even when the underlying causal genes remain unknown, thereby enabling the direct utilization of genetically mapped QTLs for precision breeding. By bridging the gap between genomic discovery and practical breeding, HAPBDB provides a practical framework for converting genomic information into breeding decisions and accelerating precision improvement of complex traits.

DNA-free genome editing is a promising strategy for the genetic improvement of horticultural crops and fruit trees because it enables targeted mutagenesis without stable genetic transformation. In planta particle bombardment (iPB) delivers CRISPR-Cas9 ribonucleoproteins (RNPs) directly into shoot apical meristems (SAMs), enabling heritable genome editing without the use of tissue culture-based transformation systems. However, the practical application of iPB-mediated editing in fruit trees is limited by the frequent occurrence of chimerism, which cannot be readily eliminated through sexual segregation while maintaining the genetic background of elite cultivars. To overcome this limitation, we combined iPB-mediated RNP delivery with regeneration from edited leaf tissues (iPB-REG). Using this approach, we targeted the self-incompatibility gene S9-RNase in the elite apple cultivar Fuji and efficiently recovered non-chimeric edited plants. These results establish iPB-REG as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.

Plants produce a variety of O-prenylated aromatics that exhibit biological activities beneficial to human health, and the presence of the O-prenyl moiety is often crucial to their functions. However, most aromatic O-prenylation genes remain unknown in plants. In this study, we report the molecular identification of an aromatic O-prenyltransferase (PT) involved in the biosynthesis of auraptene (7-geranyloxycoumarin), a citrus metabolite known for its preservative effect on human cognitive function. Based on in silico screening focusing on the membrane-bound PT family, CpPT4 was isolated as a candidate from grapefruit (Citrus x paradisi), an auraptene-rich species. Enzymatic characterization demonstrated that recombinant CpPT4 specifically catalyzes umbelliferone 7-O-geranyltransferase activity to form auraptene, which differs from the enzymatic functions of known O-PTs. This enzyme also catalyzed aromatic N-prenylation to produce a new-to-nature auraptene analog. Regarding organ- and organellar-specific localization, it is strongly suggested that CpPT4 functions in the outer pericarp plastids, where auraptene is expected be formed. Furthermore, we found that CpPT4 orthologs are widely distributed in citrus genomes. Intriguingly, mandarins and their descendant species possess dysfunctional orthologs, which is consistent with the low accumulation of auraptene and its downstream metabolites in these species. This study provides an example of the contribution of the UbiA superfamily to O-prenylated aromatic biosynthesis. Moreover, CpPT4 can be useful as a tool in the synthetic biology-based production of auraptene and its analogs, as well as a molecular marker in the breeding of auraptene-rich citrus varieties.

Capsaicinoids are synthesized in the placenta of Capsicum fruit, where vanillylamine aminotransferase (VAMT) catalyzes the formation of vanillylamine, the precursor of the pathway. The modulation of pungency has relied on genetic breeding and transgenic approaches, and this pathway has not been addressed by spray-induced gene silencing. The aim of this study was to evaluate whether a single non-invasive spray of double-stranded RNA (dsRNA) targeting VAMT allows the gene to be silenced and capsaicinoid accumulation to be modified in Capsicum chinense fruit. The molecule was designed in silico and applied at 10 days post-anthesis. Pedicel injection reduced the VAMT transcript in a dose-dependent manner, with three levels of inhibition distinguishable from one another. Spraying with surfactant reduced it by 86.2 %, a magnitude statistically indistinguishable from the 90.6 % obtained by injection, and also reduced the Pun1 transcript, a co-regulation previously described only as a difference between cultivars. Analysis by gas chromatography coupled to mass spectrometry showed reductions of 84.4 % in capsaicin and 68.5 % in dihydrocapsaicin, the loss of nonivamide and one further vanillylamine-derived compound, and the detection of capsiate and a second capsinoid, absent in control fruits. The siRNA was detected in non-treated tissues, and a single topical application is therefore sufficient to silence an endogenous biosynthetic gene and shift the metabolic profile of the fruit without genetic modification.

Caffeoylquinic acids (CQAs) are the major phenolic compounds in eggplant (Solanum melongena) fruit, with 5-O-caffeoylquinic acid (5-CQA; chlorogenic acid) as the predominant isomer, while crop wild relatives may offer novel variation for diversifying these profiles. Previous work detected a distinct phenolic acid profile in S. elaeagnifolium, a tertiary genepool wild relative of eggplant, and in advanced backcross materials carrying introgressions from this species. This profile was characterized by a secondary chromatographic peak eluting after 5-CQA, but the identity of this compound and the associated chromosome 1 region remained unresolved. Here, we combined HPLC-MS-based metabolite identification, fine mapping, and comparative microsynteny analysis to characterize this phenotype. The secondary peak was identified as 4-O-caffeoylquinic acid (4-CQA). Fine mapping across three consecutive selfing generations, involving 612 screened individuals and 21 informative recombinants, reduced the associated interval from 2.753 Mb to 0.211 Mb, delimiting a region containing 30 annotated genes. The 4-CQA peak was detected in both homozygous and heterozygous introgression-derived materials, consistent with dominant control of the trait. Structural comparison of the fine-mapped interval revealed broad conservation between S. melongena and S. elaeagnifolium, but also identified a localized structurally divergent block potentially underlying the secondary 4-CQA peak. Within this block, the S. elaeagnifolium interval harbors acetylajmalan esterase-like/GDSL-type genes and a transcript-supported putative novel donor-specific gene. Overall, this study highlights S. elaeagnifolium introgressions as a promising source of phenolic diversification and providing markers to facilitate the development of eggplant lines with a more diverse CQA profile and potentially enhanced functional quality.

Terpenoids are major contributors to grapevine berry and wine aroma, with mono- and sesquiterpenoids exhibiting substantial variation among cultivars. However, the genetic basis of cultivar-specific terpenoid profiles remains only partially understood due to gene duplications that expanded the terpenoid synthase (TPS) gene family across different cultivars. Here, we compared haplotype-resolved genome assemblies for seven cultivars to analyze copy number variation and expression profiles of terpenoid biosynthesis genes and terpenoid accumulation throughout flower and berry development. These assemblies resolved duplicated TPS loci at the haplotype level, revealing extensive copy number variation across cultivars and between haplotypes within a cultivar, with up to 44 mono-TPS genes on a single haplotype. We identified eight specific monoterpenoids as key drivers of cultivar-specific profiles in ripe berries. Total TPS transcript abundance correlated strongly with total terpenoid accumulation over time. In contrast, TPS copy number was largely decoupled from both expression and terpenoid accumulation, indicating that expression regulation, rather than gene dosage, underlies aroma differences among cultivars. Transcript-metabolite network analysis revealed both expected correlations between specific TPS genes and putative products as well as novel associations. We functionally validated selected candidate TPS in Nicotiana benthamiana, characterizing -farnesene, {beta}-ocimene, and multi-product -terpineol synthases. The in vivo characterization demonstrated that minor sequence variations can alter catalytic activity and product profile. Together, these findings show that the extensive duplication of TPS genes in grapevine does not itself determine terpenoid output; instead, cultivar-specific aroma arises from how these genes are expressed and from sequence differences that shape enzyme activity.

Understanding adaptation to environmental variation is fundamental to climate-resilient agriculture. Here, we analyzed a georeferenced collection of 167 Dioscorea accessions spanning a wide range of bioclimatic and biophysical variation, from dry sahelian environment to rainforest environment. Genome-wide association analyses revealed that the genetic architecture of environmental adaptation is largely driven by large-effect loci, although precipitation-related traits were associated with many small-effect loci. This mixed architecture could lead to multiple breeding strategies, including marker-assisted backcrossing and the targeted use of wild and semi-wild relatives. We identified a core collection of accessions that captures much of the genetic and environmental diversity, and highlighted accessions as potential parents for abiotic stress tolerance. Projections under future climate scenarios using genomic offset identified regions and accessions at elevated risk of maladaptation, as well as others with broad adaptive potentiality. Maximizing the use of available material may facilitate the development of climate-resilient cultivars.

Adventitious root formation (ARF) is a critical trait for the cost-effective propagation of grapevines in commercial nurseries. Poor rooting ability can limit the use and adoption of new rootstocks derived from underutilized Vitis species, constraining breeding efforts largely to the traditional trio: Vitis riparia, V. rupestris, and V. berlandieri. Despite its agronomic relevance, the genetic basis of ARF remains poorly characterized across the broader Vitis genus. In this study, we evaluated 308 accessions representing 18 Vitis species over three growing seasons, quantifying rooting performance at two developmental stages, callus-stage and post-transplant, alongside root biomass, cutting weight, and a derived transplant-response index. We observed extensive phenotypic variation both within and across species, and species rankings depended on the trait considered. V. riparia, V. rupestris and V. californica ranked among the top five species for all four rooting traits, whereas V. cinerea and V. candicans ranked among the lowest for root weight and post-transplant rooting. V. arizonica and V. acerifolia rooted well at the callus stage but were intermediate after transplanting, and V. berlandieri was among the weakest at the callus stage yet intermediate for post-transplant rooting. Repeatability was moderate to high for root weight (0.74) and callus-stage rooting (0.66), and lower for post-transplant rooting (0.47), reflecting both genetic control and season-to-season variation. Between-species differences accounted for 68% of the genetic variance in callus-stage rooting but only 10% in cutting weight. Rooting was associated with the climate of each accession's wild site of origin: after removing differences among species, accessions originating from sites with lower dry-season precipitation rooted better and produced more root biomass. Genome-wide association analysis using 3.4 million SNPs identified 54 significant SNPs resolving into 18 independent loci across four traits, with root weight contributing 12 of them. Candidate genes in linkage with these loci include a mitogen-activated protein kinase, a SCARECROW-LIKE GRAS transcription factor, PASTICCINO1, expansin A1, an AP2/ERF-RAV1 transcription factor, a tandem array of caffeoyl-CoA O-methyltransferases, and several sugar, peptide and nitrate transporters, implicating auxin-linked cell proliferation, cell wall and lignin remodeling, and solute transport. Genomic and phenomic prediction models yielded moderate accuracies across traits and seasons; up to r = 0.67 for post-transplant rooting within a season and r = 0.65 for previously unevaluated accessions. Moreover, the integration of spectral and genotypic data further improved predictive performance. Prediction accuracy was essentially flat between 5,000 and 50,000 markers. This study establishes a foundational framework for the genetic improvement of grapevine rootstocks, promoting broader use of resilient, high-performing, and clonally-propagable germplasm in viticulture.

A better understanding factors regulating agricultural production is a research priority, given the pace of environmental change and global human population growth. Fruit orchards may be particularly vulnerable to climate change owing to the strong temperature-dependency of reproduction in temperate trees. In this study, we explored climate influences on the reproductive dynamics of apple (Malus domestica), one of the most widely-grown, economically-important fruit crops worldwide. Observations of individual-tree fruit-set, a key indicator of final yield, were acquired for three apple cultivars in an eight-year census of ~44,900 trees in >5,500 orchards spanning wide climate gradients across the Republic of Korea. With maximum-likelihood models, we quantified temporal and climate-driven patterns in fruit-set, investigated evidence for spatially-synchronized production, and conducted simulations of orchard vulnerability to climate change. We found annual fruit-set oscillated around modal levels and was synchronized between orchards within 25 km. Higher temperatures had contrasting influences, reducing fruit-set during the cold-season (consistent with climate-induced phenological shifts), while increasing fruit-set during the season of bud initiation (prior spring). Simulations predict that higher future temperatures during bud initiation increase average fruit-set, despite the constraints of cold-season warming, but that such enhancements level-off by late-century and are accompanied by heightened volatility. Decelerating fruit-set and greater instability in production have implications for future societal needs, as robust supply systems depend as much on consistent production as on high average output. Our analyses also imply that future climate patterns promoting synchrony in fruit-set among orchards may accentuate the negative consequences of fluctuating production.

Background: Rvi6, introgressed from Malus floribunda 821, is the most widely deployed apple scab resistance gene, but Venturia inaequalis races overcoming it are widespread. Transcriptomic differences between Rvi6-effective and Rvi6-breaking interactions in one host genotype remain unresolved. Aims: To compare these interactions in cv. Ametyst and nominate genes for subsequent validation. Methods: Own-rooted Ametyst plants were mock-inoculated or inoculated with an Rvi6-avirulent isolate (Rin; incompatible) or an Rvi6-virulent isolate (Rola; compatible) and harvested at 4 days post-inoculation (21 mRNA libraries). Expression was quantified using GDDH13 reference-based and de novo Trinity strategies, followed by DESeq2, WGCNA and GSEA. Results: Rin vs Rola yielded 1,339 candidate genes (more than two-fold change, FDR < 0.01) in the reference-based analysis. Curation of Rin-induced defense candidates retained 151 genes, 92 of which concentrated in a single co-expression module. Thirty-one also passed the Rin-vs-Rola thresholded test and were prioritized for genetic validation. Conclusions: The incompatible interaction was associated with a coherent defense program and the compatible interaction with a distinct transcriptional state. With one isolate per class, isolate identity and Rvi6 virulence cannot be separated.

Apple scar skin disease (ASSD), caused by Apple scar skin viroid (ASSVd), is characterized by peel scarring, cracking, dappling, and fruit deformation, resulting in reduced fruit quality and marketability. Despite its economic importance, the physicochemical and metabolic alterations underlying disease progression remain poorly understood. To address this knowledge gap, apple fruits representing four stages of ASSD (healthy, lightly infected, moderately infected, and highly infected) were comprehensively characterized. ASSVd infection was confirmed by RT-PCR, amplicon sequencing, and phylogenetic analysis. Fruit morphology and quality attributes, including firmness, total soluble solids (TSS), pH, titratable acidity (TA), and total phenolic content (TPC), were evaluated. ASSVd infection significantly reduced fruit weight and firmness and altered TSS and TA, indicating progressive deterioration of fruit quality. To investigate the underlying metabolic changes, peel and pulp tissues were analysed separately using gas chromatography-mass spectrometry (GC-MS), while major soluble sugars were quantified by 1H nuclear magnetic resonance (1H NMR) spectroscopy. Integrated metabolomic analyses revealed distinct tissue-specific metabolic reprogramming during disease progression. Major soluble sugars declined significantly during early infection, followed by tissue-dependent recovery at later stages, whereas organic acids, amino acids, phenolics, lipids, polyols, and pentacyclic triterpenoids exhibited dynamic stage-dependent changes. Notably, lupeol accumulated progressively, whereas ursolic acid and oleanolic acid declined, indicating disease-associated alterations in host triterpenoid metabolism. Multivariate analyses demonstrated clear metabolic separation among disease stages. Lupeol, ursolic acid, and chlorogenic acid were identified as candidate discriminatory metabolites in the peel, whereas myo-inositol, chlorogenic acid, and aspartic acid were identified in the pulp. Collectively, these findings demonstrate that ASSD induces coordinated, tissue-specific physicochemical and metabolic reprogramming that compromises postharvest fruit quality and reshapes defence-associated metabolism. This study provides the first integrated metabolomic characterization of ASSD progression and identifies potential metabolic biomarkers for disease diagnosis and severity assessment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/741181v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@92e453org.highwire.dtl.DTLVardef@1cc2436org.highwire.dtl.DTLVardef@15d7e27org.highwire.dtl.DTLVardef@1056568_HPS_FORMAT_FIGEXP M_FIG C_FIG

We performed a screening to evaluate the responses to water deficit before the flowering stage in a panel of 31 genotypes composed of one commercial hybrid, one wild species, multiple ex-PVP (vintage) lines, several commercial heirlooms from the Northeast USA, Mexican landraces from hot and humid environments and genome edited lines with altered flowering and inflorescence traits. We characterized 18 different morphological, physiological and biochemical traits to assess the response to 40% water deficit. We observed diverse morphological, physiological and biochemical responses across the 31 genotypes screened, suggesting the presence of useful variation for breeding for drought tolerance in cultivated tomato. Interestingly, some heirlooms exhibited superior performance under drought, suggesting they can be used as donors to discover novel genes involved in drought responses. Unexpectedly, we found mutants in SlCLE9, a paralog of the stem cell regulator SlCLV3 known to co-regulate meristem proliferation in tomato, exhibited enhanced drought tolerance. Notably, the slcle9 mutant also conferred drought tolerance when used as rootstock with grafted scions from our reference cultivar M82, suggesting SlCLE9 influence root architecture and drought response. Consistent with this, we found SlCLE9 is transcriptionally upregulated in roots and leaves during drought stress, and exogenous application of SlCLE9 modifies root growth and response to drought. Altogether, our screening uncovered new potential trait donors for breeding against drought stress and describes a novel role for SlCLE9 in drought tolerance.

Table beet (Beta vulgaris subsp. vulgaris var. vulgaris), also known as red beet, garden beet, or beetroot, is an important crop with both nutritional and agricultural value, yet high-quality genomic resources for this taxon are limited. We present the chromosome-scale genome assembly RefTab-1 based on PacBio and Hi-C data of the inbred table beet genotype W357B. The assembly has a size of 668.6 Mbp and an N50 of 72 Mbp, with 96% of the assembly anchored to nine pseudo-chromosomes. We annotated 64% repetitive DNA, 24,863 protein-coding genes, and 10,405 non-coding RNA loci including a 5S rRNA cluster on chromosome 4. Genome-wide methylation profiling using EM-seq revealed high levels of cytosine methylation in repeats and context-specific methylation in gene bodies and promoters. We propose three terpene synthase candidate genes based on functional annotation and based on their location near a genetic marker on chromosome 8 previously associated with geosmin concentration, responsible for the earthy aroma of table beet. Comparative analysis with sugar beet indicated overall structural conservation and a 9 Mbp inversion on chromosome 9. These high-quality resources for table beet will enhance genetic, epigenetic, and comparative studies in Beta vulgaris, advancing beet biology and breeding programs.

Genetic transformation of citrus is essential for functional genomics and trait improvement, yet remains limited by prolonged regeneration cycles, genotype-dependent responses, and inefficient screening of transformed tissues. Here, we established an Agrobacterium-mediated indirect organogenesis-based transformation system for Carrizo citrange and evaluated two visible reporter systems, the anthocyanin regulator ROSEA1 and the betalain biosynthetic cassette RUBY, for monitoring transformed tissues throughout regeneration. An optimized regeneration protocol enabled visible callus ini-tiation within 7 days following a 5-day pre-culture treatment and recovery of PCR-confirmed transgenic plantlets within 5 months. Both visible reporters were compatible with callus development and shoot regeneration throughout the indirect organogenesis workflow. During callus proliferation, visible pigmentation closely corresponded with GFP fluorescence, enabling rapid, non-destructive identification of transformed tissues. However, reporter performance diverged during shoot regeneration: RUBY maintained stable pigmentation throughout regeneration, whereas ROSEA1-associated pigmentation progressively declined despite continued GFP fluorescence. Consequently, RUBY exhibited a 4.7-fold higher pigmenta-tion-based detection rate than ROSEA1 at the shoot stage and showed closer agreement with GFP-based detection. Putative transgenic events were confirmed by PCR in independent lines of both constructs, together with GFP fluorescence in leaves and root tips. Together, these findings establish an efficient indirect organogenesis-based transformation platform for citrus and demonstrate that both ROSEA1 and RUBY are effective visual reporters during callus proliferation, whereas RUBY provides more reliable visual identification during shoot regeneration and plant recovery.

- Domestication can profoundly reshape plant defense strategies and host-parasite interactions, but its consequences for resistance and coevolution in perennial crops remain poorly understood. We investigated how domestication altered apple responses to its major pest, the rosy apple aphid (Dysaphis plantaginea). - We compared wild (Malus orientalis, two geographically differentiated M. sylvestris populations) and cultivated (cider and dessert M. domestica) apple genotypes against two genetically distinct aphid genotypes, combining controlled infestation experiments with phenotypic measurements, paired host-aphid RNA-seq, co-expression network analysis, and genome-wide selection scans. - Aphid fitness and host resistance varied strongly across host populations: domesticated apples supported higher aphid performance, whereas Romanian M. sylvestris showed the highest resistance, revealing marked host-aphid genotype-specific asymmetric compatibility. Cultivated apples have developed extensive inducible transcriptional defense responses, whereas wild populations showed weaker perturbation despite lower aphid fitness, consistent with more constitutive defense. Host-aphid co-expression analyses identified candidate coevolutionary modules linking host resistance genes under balancing selection with aphid genes under positive selection. - Together, these results demonstrate that apple domestication reshaped defense-associated regulatory networks and modified the adaptive landscape experienced by aphid populations, providing new insight into the evolutionary consequences of perennial crop domestication on host-parasite interactions.

Potato is the worlds fourth major staple crop, and tuber starch is a critical dietary energy source and a food processing raw material. In this study, hybrid progenies of the high-starch cultivar Huasheng No.7 and China main commercial varieties were used as materials, high-throughput sequencing was performed on tuber samples at tuber bulking, maturation and storage stages. Combined with bioinformatics analysis, parent-progeny resequencing, SNP screening, the regulatory mechanism of tuber starch metabolism was explored. The results showed significant stage-specific transcriptional reprogramming in potato tubers: few differentially expressed genes (DEGs) were identified at tuber bulking and maturation stages, but massive transcriptional changes occurred during storage. XET family genes regulated cell wall remodeling and carbon translocation across all stages, and WRKY transcription factors specifically controlled starch homeostasis in stored tubers. The DEGs at different stage were induced by the SNPs from their parent, and the allele from the high-starch parent, Huasheng 7 may contribute the high-starch allele to the offsprings. One key gene, Soltu.DM.02G019910, which encoded {beta}-glucosidase, have a negatively relationship with tuber starch concentration. The low-starch potato breeding tubers shown higher significantly {beta}-glucosidase activity than high-starch breeding population at maturation stage. This study clarifies the molecular regulatory network of potato tuber starch metabolism and screens core regulatory genes from the tuber bulking stage to tuber storage, thereby providing theoretical support and genetic resources for molecular breeding of high-starch and storage-resistant potato varieties.

Plants perceive external and internal signals via receptors to modulate their growth, development, and defenses. Leucine-rich-repeat receptor-like-kinase (RLKs) and receptor-like-proteins (RLPs) are two major cell-surface receptors in plants. RLPs are a unique gene family (well-studied and characterized) and play an important role in plant development and defense activities against pests and pathogens. Yet their comparative analyses are hampered by a lack of reproducible and incomplete annotation tools. Here we present the PlantLRR-PRR, a reproducible and standardised RLP/RLK annotation pipeline. It outperforms previous tools and helps to dissect the RLP variation among Solanum spp. We investigated RLP diversity in eight genomes from five wild tomato Solanum sect Lycopersicum. We found limited intra- but moderate inter-specific copy number variation displaying a possible long-term diversification (gain and loss) of RLPs driven by host, environment, and pathogen interactions. Interestingly, we observed a dual-evolutionary pattern characterized by conservation and diversification of developmental- and defense-related RLPs, respectively. Overlapping with this, we found transposable elements (TEs) highly enriched around defense-related RLPs, supporting a strong role of TEs in promoting loss, gain, and structural variations. Further, zooming into the known Cf5 and CuRe1 RLP gene cluster revealed signatures of typical birth and death patterns. Comparative analysis of RLPs in wild tomato species revealed RLP diversity that is not apparent from cultivated tomato alone, highlighting the value of wild germplasm for understanding RLP family evolution. Together, our results provide a comparative framework for understanding the evolutionary divergence and conservation in the RLP family and establish a foundation for linking receptor evolution with functional resistance, and can form a stepping stone for translational applications in crop improvement.

Eggplant (Solanum melongena L.) displays extensive fruit color diversity, in which chlorophyll-related pigmentation contributes to both external appearance and market value. Previous genetic studies identified SmAPRR2 and SmGLK2 as major candidate genes controlling uniform green pigmentation and green netting in fruit, respectively, but their individual and combined functional contributions had not been validated through targeted mutagenesis in a common genetic background. Here, we established a multiplex CRISPR/Cas12a system in eggplant accession MEL3, representing, to our knowledge, the first application of this nuclease for genome editing in eggplant. Transformation efficiency was 2.0%, but all 15 genotyped regenerants were edited, yielding four SmAPRR2 and six SmGLK2 alleles. Segregation and crossing enabled the recovery of six transgene-free lines carrying single or combined edited alleles. Disruption of SmGLK2 abolished the reticulated green netting pattern while preserving a uniformly green peel and the internal green ring. Conversely, disruption of SmAPRR2 reduced the background uniform peel pigmentation and eliminated the green ring while retaining green netting. Double mutants carrying disruptive alleles at both loci produced white fruits lacking internal green pigmentation, whereas putatively hypomorphic SmAPRR2 and SmGLK2 alleles generated intermediate phenotypes. Whole-genome resequencing identified only two predicted off-target sites under a canonical TTTV PAM search allowing up to four mismatches. Both were fully covered, and no edited-line-specific candidate variants were detected. These findings establish complementary and partially separable roles for SmAPRR2 and SmGLK2 in fruit peel and flesh chlorophyll pigmentation and demonstrate the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.

Plant pathogen genomes adapt across coding and non-coding regions in response to their hosts, environments, and disease management practices. Venturia inaequalis, the causal agent of apple scab, has evolved alongside its Malus host throughout apple domestication. We generated 17 high-quality genome assemblies, including the first chromosome-scale assembly of V. inaequalis, to define the species' complete gene content and investigate the role of structural variation in genome evolution. Together with a previously published scaffold-level reference, these assemblies were used to construct a gene-based pangenome containing 215,301 predicted protein-coding genes clustered into 13,240 orthogroups, 88.11% of which were core (73.86%) or soft-core (14.25%), 11.88% shell (present in 2-16 isolates), and 0.1% private, indicating a highly conserved gene repertoire. Virulence-associated effectors were significantly enriched in the soft-core and shell components, and accessory apoplastic effectors displayed elevated nucleotide diversity and nonsynonymous-to-synonymous substitution ratios (Ka/Ks) relative to core effectors, consistent with diversification. We further identified a putative accessory chromosome of variable size (23-132.6 kb) in European and U.S. isolates encoding genes with virulence function. A graph-based pangenome identified 38,799 non-redundant structural variants (SVs), largely driven by Gypsy LTR retrotransposons. We used the graph-based pangenome to genotype 136 globally distributed V. inaequalis isolates and identified variants differentiating populations associated with domesticated apple (Malus domestica) from those associated with its Central Asian wild progenitor, M. sieversii. Together, these resources establish a foundation for future studies of diversity, host adaptation, and genome evolution in V. inaequalis.

Canola breeding has been shaped by strong selection for oil quality, yet the origins of the known oil quality alleles and genomic consequences of their selection are not fully resolved. By integrating pedigree reconstruction with graph pan-genomics we trace inheritance of ancestral genomic regions across historical and contemporary germplasm. Surrounding the low erucic acid allele in BnA08.FAE1, we identify a 17.23 Mb haplotype that approached fixation in Australian canola in the early 2000s. Contradicting the prevailing model, this haplotype predates modern breeding and was likely widespread in ancestral B. napus in the early 1900s. Genomic analyses implicate centromeric recombination suppression and structural variation in its long-term persistence, which has led to megabase-scale diversity loss through hitchhiking of neighbouring alleles. The haplotype contains extensive structural variation and multiple alleles associated with polygenic disease resistance. Together, these findings reveal the long-term consequences of repeated selection on standing variation during crop improvement.