Horticulture Research

Apple (Malus x domestica Borkh.) production faces many challenges stemming from abiotic and biotic stresses. Abiotic stressors, such as extreme temperatures, droughts, and spring frosts, can lead to diminished yields and tree loss, while biotic stresses like fire blight and pest infestations further reduce tree health and fruit quality. To lessen the threat of these challenges, plant breeders aim to introduce resistance and resilience genes into cultivated varieties. However, high-relatedness among cultivated varieties and breeding lines, coupled with the long juvenility and generation times in apples, hinder the breeding process. The introduction of resistance traits from wild relatives is also constrained by these factors, as well as the lack of genomic resources that could assist in accelerating the introgression process. Herein, we report the assembly and annotation of Malus angustifolia, the Southern Crabapple, one of Eastern North Americas native species. Using a combination of Pacific Biosciences High Fidelity reads, Next-generation short read sequencing, as well as chromatin conformation capture sequencing, we achieve an extremely contiguous haplotype-resolved assembly. We perform comparative haplotypic analyses to identify SNPs and large structural variants, shedding light on the genomic landscape of M. angustifolia. Finally, we explore the phylogenetic and syntenic relationships between Eurasian Malus progenitors and the recently sequenced North American species, contributing valuable insights to the broader understanding of apple evolution and potential breeding strategies.

Apple scab, caused by Venturia inaequalis, remains one of the most damaging diseases in apple orchards, driving intensive pesticide use worldwide. Reducing this dependence requires the deployment of durable resistance, ideally through the combination of major resistance genes (R genes) with quantitative trait loci (QTL) that confer partial and potentially complementary protection. Yet, few apple scab QTLs have been functionally validated, and their underlying mechanisms remain largely unresolved. Here, we refined and functionally described, with transcriptomic data, five resistance QTLs in a biparental population of 1,970 individuals derived from the cross TN 10-8 x Fiesta. Using 43 newly developed KASP markers, QTL locations were substantially precised through high-resolution genotyping and phenotyping with two V. inaequalis isolates exhibiting contrasting virulence. Four QTL (qT1, qF11, qF17, qT13) were validated, while qF3 was not confirmed. Transcriptomic data comparison revealed the expression of candidate genes within the narrowed intervals, including receptor-like proteins in qT1, and RNAi- and signaling-related genes in qF11 and qF17, suggesting a diversified and complementary defense network. These findings refine the genetic architecture of apple scab resistance and suppose the existence of shared molecular pathways between major R gene, such as the well-described Rvi6 gene, and quantitative resistance, with for instance the QTL qT1. The identified loci and markers provide robust tools for marker-assisted and genomic breeding aimed at developing apple cultivars with complementary and potentially durable resistance pathways.

European viticulture faces two critical challenges currently: climate change and its direct and indirect impacts in the one hand and the need to dramatically reduce pesticide use in the other hand. In response, breeding programs, such as the INRAE-ResDur program, have prioritized the development of grapevine varieties with disease resistance and improved adaptability to climate change. We took the opportunity of the important plant material produced during the INRAE-ResDur program over twenty years to dissect the genetic basis of key agronomic traits using a comprehensive approach. We conducted genome-wide association studies (GWAS) modeling both additive and non-additive genetic effects, alongside classical QTL mapping and a meta-GWAS that integrates data across multiple environments and pleiotropic effects. These analyses identified numerous loci associated with important traits related to yield, phenology, and stress tolerance. Notably, several loci showed significant genotype-by-environment (GxE) interactions, while others showed pleiotropic effects influencing multiple traits. Overall, 39 genomic regions were detected in QTL analysis in biparental populations, GWAS with additive and non-additive effects, and meta-GWAs analysis, accounting for the 13 agronomic traits. These results provide valuable molecular markers that can be applied in marker-assisted selection or knowledge-assisted genomic selection. This study highlights a complex polygenic architecture underlying agronomic traits in grapevine, involving multiple loci with both additive and interaction effects and underscores the need for integrative breeding strategies to achieve sustainable improvements in future viticulture.

Powdery mildew is one of the most serious diseases in apple production. SAR has a broad-spectrum immunity in plants against pathogen. Plants activate SAR against pathogen invasion and also prevent over-immunity. The relevant mechanism is still unknown in apple. In this study, we isolated and identified powdery mildew pathogen from the field and preserved them on the apple tissue culture seedlings. We performed DAP-seq of powdery mildew-inducible WRKY40. WRKY40 positively regulates NPR3like by directly binding to the W-box element of its promoter. NPR3like represses the expression of the PR1 gene in the presence of SA by competing with TGA2 for binding to NPR1. WRKY1 positively regulates WRKY40 by directly binding to the dual W-box element of its promoter, while WRKY1 positively regulates NPR3like by directly binding to the W-box element of its promoter. The expression trends of WRKY1, WRKY40, and NPR3like were basically the same as that of PR1 within 24 h after powdery mildew and SA treatments. Besides, WRKY1 increased SA content by positively regulating EPS1. After inoculation with powdery mildew, the up-regulation of PR1 in RNAi-silenced plants of WRKY1 was more slowly compared with the wild type, and the number of spores and mycelium increased significantly. In summary, we established a new model of NPR3like inhibition of NPR1 activity positively regulated by the WRKY1-WRKY40 module and found that the WRKY1-EPS1 module accelerated the up-regulation of PR1 by increasing the SA content. Finally, we elucidated WRKY1 confers resistance to powdery mildew by accelerating SAR and preventing over-immunity in apple.

BackgroundEnsete ventricosum, also known as the "tree against hunger" plays a key role in Ethiopian food security and farming systems, feeding more than 20 million people. Since domestication via clonal selection in the south-west Ethiopian highlands, todays diverse enset landraces contribute multiple benefits including food, fibre by-product, animal bedding and cattle fodder to farmers and local communities. Improved genomic resources for this highly drought-tolerant plant are essential to supplement the conventional clonal selection-based breeding programme and pave the way towards targeted breeding. ResultsWe sequenced the genome of enset landrace Mazia, which is partially resistant/tolerant to Xanthomonas wilt and predicted 38,940 protein-coding genes. The Mazia assembly (540.14 Mb) is more complete than the previously published genome assembly of landrace Bedadeti (451.28 Mb) and displayed 1.41% heterozygosity and 64.64% repetitive DNA content. Comparative analyses with the Bedadeti assembly and chromosome-level genome sequences of the two main banana progenitors (Musa acuminata, AA genome; Musa balbisiana, BB genome) unexpectedly revealed [~]25% of the Mazia genome is unique to enset. Gene Ontology (GO) and sequence similarity search analysis of enset-specific protein-coding genes identified distinct functional signatures that underpin the lifestyle, adaptation, and corm productive quality of enset, including functions related to DNA integration, carbohydrate metabolism, disease resistance and transcriptional regulation. In contrast, Musa-specific genes showed enrichment for defence response, protein phosphorylation and fruit development pathways. Focusing on the classical nucleotide binding site leucine rich repeat (NLR) disease resistance genes, we identified and characterised NLRs in enset and Musa species genomes, revealing a considerable expansion in the Musa acuminata genome. We also identified unique genes in enset and banana genomes whose functional and evolutionary roles are yet to be determined. ConclusionsHere, we report a de novo genome assembly for the enset (Ensete ventricosum) landrace Mazia and provide a high-quality annotation of both Mazia and the previously published assembly of the landrace Bedadeti. Collectively, these genomic resources provide a valuable foundation for comparative genomics within the Musaceae family and open new opportunities for the development of marker-assisted breeding strategies to accelerate the improvement of agronomically important traits in enset.

Tomato is one of the most produced and consumed vegetables globally due to its nutritional benefits, sensory characteristics, and cultural importance. However, tomato fruit has a short shelf-life, which can be extended by postharvest techniques, but often at the expense of fruit quality, leading to consumer dissatisfaction. To address this challenge, we modified the upstream regulatory regions of Ripening inhibitor (RIN), a master regulator of tomato fruit ripening, utilizing a CRISPR/Cas9 multiplex system. This approach enabled the creation of a population of tomato fruit with mutations of varying severity, which could have far-reaching effects on the RIN-induced gene regulatory network in tomato fruit, leading to downstream changes in fruit traits. We have generated 264 first-generation (T0) transgenic lines of RIN promoter mutants with diverse genetic lesions and RIN transcriptional levels. Our study revealed a non-linear relationship between promoter mutations and gene expression, highlighting the potential roles of certain types of mutations in regulating RIN transcription. Future work will focus on evaluating fruit traits from mutants with pronounced changes in RIN expression, as well as performing transcriptomic analysis to explore the mechanisms underlying fruit quality modifications due to genome editing.

Apple (Malus domestica) is a globally significant crop and a vital dietary component worldwide. During ripening, apples exhibit a longitudinal gradient, ripening first at the stalk cavity and extending toward the calyx pit. Over-ripening in the stalk cavity leads to early senescence, characterized by peel browning, which diminishes fruit quality. This study examines the natural senescence process in 6-year-old Ruixue apples by screening transcriptome data to uncover senescence-related genes and validate their molecular functions. Our analysis of antioxidant capacity and reactive oxygen species in different peel regions revealed that malondialdehyde, hydrogen peroxide, and superoxide anion levels increased with senescence, especially in the stalk cavity. Transcriptome clustering and enrichment analyses across developmental stages revealed MdWRKY70L, MdSAG101, and MdZAT12 as key regulators of peel senescence. Further in vitro and in vivo studies demonstrated that MdWRKY70L is phosphorylated at Ser199 by MdMPK6/02G, enhancing its stability and promoting peel senescence. These findings offer insights for developing strategies to delay fruit senescence and improve postharvest quality control.

Apple is one of the major cultivated fruit crops in temperate regions. To better support breeding programs and facilitate the development of improved cultivars, we generated a new haplotype-resolved version of the Golden Delicious genome, one of the major founders of many modern apple lineages. The assembly features the separation of the two haplotypes, with a total size of 647.3 Mb and 649.2 Mb, respectively. The phasing was accurately validated with 10,321 curated SNPs. Telomere-to-telomere continuity was verified by the analysis of telomeric sequence composition at the end of each chromosome. Gene prediction identified a total of 45,116 genes in haplotype 1 and 45,063 genes in haplotype 2. A pangenome analysis employing 6 haplotype resolved genomes identified both common and unique gene families. The availability of a phased genome enabled the assessment of genome-wide allelic specific expression. Our case study, focusing on Md-PG1 (a key regulator of fruit softening), revealed that the allelic form on haplotype 2 (GDH2-10g24673) was the dominant contributor to total gene expression. In addition, the phased genome also showed specific miRNA chromosomal distribution patterns, as well as a distinct methylation profile. Altogether, these genomic resources provide new insights into the allelic regulation of key agronomic traits and represent a valuable tool to accelerate apple breeding.

Citrus is one of the most important tree crops worldwide and citrus production is threatened by Huanglongbing (HLB), a devasting citrus disease caused by Candidatus Liberibacter. HLB is a pathogen-triggered immune disease, in which the pathogen initiates systemic and chronic immune responses including the excessive production of reactive oxidative species, which subsequently lead to cell death of phloem tissues and HLB disease symptoms. Here, we identified putative genetic determinants of HLB pathogenicity by integrating citrus genomic resources to characterize the pan-genome of accessions that differ in their response to HLB. Genome-wide association mapping and analysis of allele-specific expression between susceptible, tolerant, and resistant accessions further refined candidates underlying the response to HLB. To enable these analyses we first developed a phased diploid assembly of Citrus sinensis Newhall genome and produced resequencing data for 91 citrus accessions that differ in their response to HLB. These data were combined with previous resequencing data from 356 sequenced accessions for genome-wide association mapping of the HLB response. Genes with HLB pathogenicity were associated with the host immune response, ROS production, and antioxidants. Overall, this study has provided a significant recourse of citrus genomic data and we have identified candidate genes to be further explored to understand the genetic determinants of HLB pathogenicity and to generate HLB resistant/tolerant citrus varieties.

The grapevine is one of the most ancient and economically important horticultural crops in the world. The grapevine species Vitis vinifera is cultivated globally; however, due to its susceptibility to pathogens and environmental stresses, wild Vitis species and their hybrids are often used as rootstocks in vineyards. Here, we report the genome analysis of a Vitis strain named Maeve, which was identified in a vineyard in Japan, though its genetic origin remains unclear. We performed haplotype-resolved de novo assembly of the Maeve genome using PacBio HiFi sequencing and Hi-C assembly. Our genome analysis revealed that Maeve has originated from an interspecific hybridization between an unknown V. vinifera cultivar and the V. riparia Gloire cultivar, likely arising through breeding or natural pollination. This novel cultivar has a potential to expand wine grape production across a wider range of environmental conditions where conventional cultivars are unsuitable for viticulture.

CRISPR genome editing has shown tremendous potential in genetic improvement of citrus. So far, citrus genome editing has been conducted using juvenile tissues resulting in genome-edited citrus plants that require multiple years before they can produce flowers and fruit. Here we tested whether citrus genome editing via mature tissue transformation can overcome such a hurdle. CsLOB1 is a susceptibility gene for citrus canker caused by Xanthomonas citri subsp. citri (Xcc). The transcription activator-like effector PthA4 of Xcc activates CsLOB1 by binding to the effector-binding element in its promoter (EBEpthA4-CsLOBP). In Valencia sweet orange, two CsLOB1 promoter alleles are present: TI CsLOBP, and TII CsLOBP. We specifically utilized a CRISPR/Cas9 construct (GFP-p1380N-Cas9/sgRNA:CsLOBP2) targeting EBEpthA4 in TI CsLOBP but not TII CsLOBP to test genome editing efficacy and off-target mutations. GFP-p1380N-Cas9/sgRNA:CsLOBP2 function was first validated using Xcc-facilitated agroinfiltration in Valencia leaves. The construct was subsequently introduced into Valencia mature internodal stem segments via Agrobacterium-mediated transformation, generating three independent transgenic lines (#V2, #V3 and #V5). Targeted mutations in EBEpthA4-TI CsLOBP were detected in all three lines with mutation frequencies of 100%, 21.43% and 41.94% in #V2, #V3 and #V5, respectively, while no mutations were detected in TII CsLOBP. Infection with Xcc{Delta}pthA4:dCsLOB1.3, carrying a designer TALE that specifically activates TI CsLOBP, resulted in reduced canker symptoms in #V2. Importantly, all three EBEpthA4-TI CsLOBP edited lines flowered within 15 months. In sum, these results demonstrate that CRISPR/Cas9-mediated genome modification through mature citrus transformation can achieve high genome editing efficacy and overcome the juvenility.

The pear (Pyrus spp.) is a remarkable fruit, well known for its diverse flavors, textures, culinary versatility, and global horticultural importance. However, the genetic diversity responsible for its extensive phenotypic variations remains largely unexplored. Here, we de novo assembled and annotated the genomes of the maternal (PsbM) and paternal (PsbF) lines of the hybrid Yuluxiang pear and constructed the first pear pangenome of 1.15Gb by combining these two genomes with five previously published pear genomes. Using the constructed pangenome, we identified 21,224 gene PAVs and 1,158,812 SNPs in the non-reference genome that were absent in the PsbM reference genome. Compared with SNP markers, we found that PAV-based analysis provides additional insights into the pear population structure. In addition, we also revealed that some genes associated with pear fruit quality traits have differential occurrence frequencies and differential gene expression between Asian and European populations. Moreover, our analysis of the pear pangenome revealed a mutated SNP and an insertion in the promoter region of the gene PsbMGH3.1 potentially enhances sepal shedding in Xuehuali which is vital for pear quality. This research helps further capture the genetic diversity of pear populations and provides valuable genomic resources for accelerating pear breeding.

Heat stress threatens tomato productivity by reducing pollen viability, fruit set, and consequently, overall yield. While heat-tolerant traits are predominantly found in wild tomatoes, the introgression of heat tolerance into elite cultivars remains challenging due to linkage drag. To address this issue, the World Vegetable Center developed a Multi-parent Advanced Generation Inter-Cross (MAGIC) population, derived from crosses between four heat-tolerant and disease-resistant cultivars. Phenotypic evaluations revealed that fruit number accounted for approximately 57% of reproductive output, making it a critical selection index for heat tolerance. A total of 16,350 SNPs were developed for the MAGIC population, and genome-wide association studies (GWAS) identified 50 QTLs linked to the evaluated traits. Notably, three QTLs on chromosomes 1, 3 and 11 emerged as hubs influencing multiple reproductive traits, underscoring their critical role in heat tolerance. SL4.0CH11_47205149 associated with fruit number was converted into a Kompetitive Allele-Specific PCR (KASP) marker and validated for its significant association with yield. The identification of key QTLs and the prioritization of fruit number as a primary determinant of reproductive success offer valuable insights for targeted breeding strategies. HighlightNumber of fruits is crucial for selecting heat-tolerant tomatoes in open fields. A marker linked to high fruit production helps develop high-yielding tomatoes better suited to withstand climate change.

Climate change poses an increasing threat to the cultivation of deciduous fruit trees, placing greater demands on modern pear breeding. Using pear germplasm adapted to diverse environments, we assembled 11 chromosome-level genomes. In combination with 13 publicly accessible pear genomes, we analyzed presence-absence variations (PAVs) and constructed a graph-based pangenome for pear. By performing a PAV-eQTL analysis of the fruit of 123 pear accessions, we identified PAVs significantly associated with expression levels of genes that may be involved in regulating agronomic traits. Population analysis of 268 pear accessions revealed two stop-gained variants in DAM1 of independent origin, which may function in advancing the blooming date and reducing the chilling requirement. We detected complex PAVs at the NOR1 locus, including two copy-number variations and one deletion. These PAVs contributed to the rapid diversification of the NOR1 locus and the fruit development period through regulating ARF5 and other ripening-related genes. We revealed the selection history of the NOR1 locus and developed novel pear individuals that accumulated alleles for low chilling requirement, early blooming date, and short fruit development period. The results provide valuable resources for pear genomics research and offer a guideline for breeding modern pears with climate resilience.

Grape breeding programs are mostly focused on developing new varieties with high production volume, sugar contents, and phenolic compound diversity combined with resistance and tolerance to the main pathogens under culture and adverse environmental conditions. The Niagara variety (Vitis labrusca x Vitis vinifera) is one of the most widely produced and commercialized table grapes in Brazil. In this work, we selected three Niagara somatic variants with contrasting berry phenotypes and performed morphological and transcriptomic analyses of their berries. Histological sections of the berries were also performed to understand anatomical and chemical composition differences of the berry skin between the genotypes. An RNA-Seq pipeline was implemented, followed by global coexpression network modeling. Niagara Steck, an intensified russet mutant with the most extreme phenotype, showed the largest difference in expression and showed selection of coexpressed network modules involved in the development of its russet-like characteristics. Enrichment analysis of differently expressed genes and hub network modules revealed differences in transcription regulation, auxin signaling and cell wall and plasmatic membrane biogenesis. Cutin- and suberin-related genes were also differently expressed, supporting the anatomical differences observed with microscopy.

Breeding for new peach cultivars with enhanced traits is a prime target in breeding programs. In this study, we used a discovery panel of 90 peach accessions in order to dissect the genetic architecture of 16 fruit-related traits. ddRAD-seq genotyping and the intersection between three variant callers yielded 13,045 high-confidence SNPs. These markers were subjected to an exhaustive association analysis by testing up to seven GWAS models. Blink was selected as the most adjusted, simultaneously balancing false positive and negative associations. Totally, we identified 16 association signals for six traits showing high broad-sense heritability: harvest date, fruit weight, flesh firmness, contents of flavonoids, anthocyanins and sorbitol. By assessing the allelic effect of significant markers on phenotypic attributes, nine SNP alleles were denoted favorable. A promising marker (SNC_034014.1_7012470) was found to be simultaneously associated with harvest date and fruit firmness conferring a positive allelic effect on both traits. We anticipate that this marker could be used to improve firmness in late harvested cultivars. Candidate causal genes were shortlisted when fulfilling the following criteria: i) position within the linkage disequilibrium block, ii) functional annotation and iii) expression pattern. A bibliographic review of previously reported QTLs mapping nearby the associated markers allowed us to benchmark the accuracy of our approach. Despite the moderate germplasm size, ddRAD-seq allowed us to produce an accurate representation of peachs genome resulting in SNP markers suitable for empirical association studies. Together with candidate genes, they lay the foundation for further genetic dissection of peach key traits.

The Pacific crabapple (Malus fusca) is a wild relative of the commercial apple (Malus x domestica). With a range extending from Alaska to Northern California, M. fusca is extremely hardy and disease resistant. The species represents an untapped genetic resource for development of new apple cultivars with enhanced stress resistance. However, gene discovery and utilization of M. fusca has been hampered by the lack of genomic resources. Here, we present a high-quality, haplotype-resolved, chromosome-scale genome assembly and annotation for M. fusca. The genome was assembled using high-fidelity long-reads and scaffolded using genetic maps and high-throughput chromatin conformation capture sequencing, resulting in one of the most contiguous apple genomes to date. We annotated the genome using public transcriptomic data from the same species taken from diverse plant structures and developmental stages. Using this assembly, we explored haplotypic structural variation within the genome of M. fusca, identifying thousands of large variants. We further showed high sequence co-linearity with other domesticated and wild Malus species. Finally, we resolve a known quantitative trait locus associated with resistance to fire blight (Erwinia amylovora). Insights gained from the assembly of a reference-quality genome of this hardy wild apple relative will be invaluable as a tool to facilitate DNA-informed introgression breeding.

Crop pests have profoundly deleterious effects on crop yield and food security. However, conventional pest control depends heavily on the utilization of insecticides, which develops strong pesticide resistance and concerns of food safety. Crop and their wild relatives display diverse levels of pest resistance, indicating the feasibility for breeding of pest-resistant crop varieties. In this study, we integrate deep learning (DL)/machine learning (ML) algorithms, plant phenomics and whole genome sequencing (WGS) data to conduct genomic selection (GS) of pest-resistance in grapevine. We employ deep convolutional neural networks (DCNN) to accurately calculate the severity of damage by pests on grape leaves, which achieves a classification accuracy of 95.3% (Visual Geometry Group 16, VGG16, for binary trait) and a correlation coefficient of 0.94 in regression analysis (DCNN with Pest Damage Score, DCNN-PDS, for continuous trait). We apply DL models to predict and integrate phenotype (both binary and continuous) along with WGS data from 231 grape accessions, conducting Genome-Wide Association Studies (GWAS). This analysis detects a total of 69 QTLs, encompassing 139 candidate genes involved in pathways associated with pest resistance, including jasmonic acid (JA), salicylic acid (SA), ethylene, and other related pathways. Furthermore, through the combination with transcriptome data, we identify specific pest-resistant genes, such as ACA12 and CRK3, which play distinct roles in resisting herbivore attacks. Machine learning-based GS demonstrates a high accuracy (95.7%) and a strong correlation (0.90) in predicting the leaf area damaged by pests as binary and continuous traits in grapevine, respectively. In general, our study highlights the power of DL/ML in plant phenomics and GS, facilitating genomic breeding of pest-resistant grapevine.

Land plants produce a cuticle, an extracellular hydrophobic layer that covers aerial organs and is involved in many critical protective roles, most notably in preventing desiccation. The predominant component of the cuticle is the lipidic polyester, cutin, which is deposited in the epidermal primary cell wall. Most of cutin of tomato fruit, a model for cuticle research, is polymerized by the extracellular GDSL-hydrolase enzyme CUTIN SYNTHASE-LIKE 1 (CUS1). However, other enzymes involved in cutin assembly remain to be identified and characterized. In this current study, we investigated whether other GDSL-hydrolases that are highly expressed in fruit epidermis might also contribute to cutin polymerization and restructuring. Candidates include homologs of Arabidopsis thaliana CUTICLE DESTRUCTIVE FACTOR 1 (CDEF1), which has been reported to catalyze cutin hydrolysis, as well as other phylogenetically diverse and distantly related GDSL-hydrolases. We determined that members of the CUS and CDEF families can catalyze the transesterification of cutin precursors in vitro, and can modify tomato fruit cutin structure in semi-in vivo assays. Tomato mutant knockout lines of CUS and CDEF genes generated by CRISPR/Cas9 and cross mutations with cus1 (previously cd1) were found to exhibit different fruit and flower phenotypes related to cutin assembly, including an effect on cutin monomer esterification, composition and content, cutin nanoridge formation in flowers, fruit cuticle permeability and permeance. Characterization of the mutant phenotypes, in combination with the enzyme analysis and bioassays, revealed distinct differences in the contribution of CUS and CDEF enzymes to cutin biosynthesis and remodeling. Our analysis also revealed unexpected spatiotemporal variation in cutin polymerization and structure coordinated by distinct GDSL-hydrolase enzymes over the fruit surface, which further suggests great complexity in cutin deposition and cuticle functions during organ development. HighlightsO_LICutin polymerization in tomato is catalyzed by coordinating the spatiotemporal expression of CUTIN SYNTHASE enzymes in different organs, including during fruit development. C_LIO_LIExtracellular cutin polymerization is not a function limited to the canonical CUTIN SYNTHASE family members but can be also be catalyzed by other GDSL-hydrolase enzymes, as suggested by evidence in vitro. C_LIO_LITomato CDEF enzymes, a clade within the GDSL-hydrolase superfamily, are involved in remodeling cutin structure during fruit development. C_LIO_LIThe biosynthesis and remodeling of cutin over the tomato fruit surface is spatially heterogeneous. C_LI

Softening is a hallmark of ripening in fleshy fruits, and has both desirable and undesirable implications for texture and postharvest stability. Accordingly, the timing and extent of ripening and associated textural changes are key targets for improving fruit quality through breeding. Previously, we identified a large effect locus associated with harvest date and firmness in apple (Malus domestica) using genome-wide association studies (GWAS). Here, we present additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1, may cause variation in these traits. First, we confirmed our previous findings with new phenotype and genotype data from [~]800 apple accessions. In this population, we compared a genetic marker within NAC18.1 to markers targeting three other firmness-related genes currently used by breeders (ACS1, ACO1, and PG1), and found that the NAC18.1 marker was the strongest predictor of both firmness at harvest and firmness after three months of cold storage. By sequencing NAC18.1 across 18 accessions, we revealed two predominant haplotypes containing the single nucleotide polymorphism (SNP) previously identified using GWAS, as well as dozens of additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a protein with high similarity to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato (Solanum lycopersicum). To test whether these genes are functionally orthologous, we introduced both NAC18.1 transgene haplotypes into the tomato nor mutant and showed that both haplotypes complement the nor ripening deficiency. Taken together, these results indicate that polymorphisms in NAC18.1 may underlie substantial variation in apple firmness through modulation of a conserved ripening program.