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.

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.

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.

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.

Apple (Malus x domestica) is one of the most important fruit crops in terms of worldwide production. Due to its self-incompatibility system and the long juvenile period, breeding of new apple cultivars combining traits desired by growers (e.g. yield, pest and disease resistance) and consumers (e.g. fruit size, color, and flavor) is a long and complex process. Genomics-assisted breeding strategies can facilitate the selection of germplasm leading to new cultivars. While the most complete apple genome assemblies available to date are from anther-derived homozygous lines, de novo assembly of apple genomes encompassing the natural heterozygosity remains challenging. Using long- and short-read sequencing technologies in combination with optical mapping, we de novo assembled a diploid and heterozygous genome of the apple cultivar Gala Galaxy. This approach resulted in 154 hybrid scaffolds (N50 = 34.3 Mb) spanning 999.9 Mb and in 414.7 Mb of unscaffolded sequences. Anchoring 31 scaffolds with a genetic map was sufficient to represent an entire haploid genome of 17 pseudomolecules (719.4 Mb). The remaining sequences were assembled in a second set of 17 pseudomolecules, which spanned 601 Mb, leaving 80.6 Mb of unplaced sequences. A total of 41,264 genes were annotated using 74,900 transcripts derived from RNA sequencing of pooled leaf tissue samples. This study provides a high-quality diploid reference genome sequence encompassing the natural heterozygosity of the widely popular cultivar Gala Galaxy. The DNA sequence resources and the assembly described here will serve as a solid foundation for fundamental and applied apple breeding research.

Phylogenomic evidence from an increasing number of studies has demonstrated that different data sets and analytical approaches often reconstruct strongly supported but conflicting relationships. In this study, hundreds of single-copy nuclear (SCN) genes (785) and complete plastomes (75) were used to infer the phylogenetic relationships and estimate the historical biogeography of the apple genus Malus sensu lato, an economically important lineage disjunctly distributed in the Northern Hemisphere involved in known and suspected hybridization and allopolyploidy events. The nuclear phylogeny recovered the monophyly of Malus s.l. (including Docynia); however, it was supported to be biphyletic in the plastid phylogeny. An ancient chloroplast capture event best explains the cytonuclear discordance that occurred in the Eocene in western North America. Our conflict analysis demonstrated that ILS, hybridization, and allopolyploidy could explain the widespread nuclear gene tree discordance. We detected one deep hybridization event (Malus doumeri) involving the ancestor of pome-bearing species and Docynia delavayi, and one recent hybridization event (Malus coronaria) between M. sieversii and a combined clade of M. ioensis and M. angustifolia. Furthermore, our historical biogeographic analysis combining living and fossil species supported a widespread East Asian-western North American origin of Malus s.l., followed by a series of extinction events in the Eocene in northern East Aisa and western North America. This study provides a valuable evolutionary framework for the breeding and crop improvement of apples and their close relatives.

Carmenere is a widely cultivated and internationally recognized grapevine cultivar in Chile, yet genetic variation among its clones remains poorly characterized. Early studies based on SSR and AFLP markers detected limited polymorphism, but these approaches interrogate only a small fraction of the genome, leaving the extent of clonal diversity unresolved. Here, we generated an improved chromosome-scale diploid genome assembly of Carmenere FPS clone 02 and characterized clonal genomic diversity by sequencing 36 biological replicates representing 12 clones maintained in Chile, including heritage selections rescued from old producer vineyards by Vina Santa Carolina as part of its Bloque Herencia conservation program, and commercial nursery-derived clones. Focusing on low-frequency variants and using replicate-aware consensus calling, we identified more than 9,000 private single nucleotide variants (SNVs) and small indels per clone, providing high-resolution markers for clonal identification. Although most variants were located in repetitive or intergenic regions, a subset affected coding sequences, with genes involved in plant-pathogen interactions, transport, and secondary metabolism most frequently impacted. While variant-affected genes associated with wine anthocyanin content, TA, pH, and alcohol percentage were identified, broader phenotypic characterization will be required to assess their biological significance. Overall, this study provides a genome-wide characterization of extant clonal diversity in Carmenere, with implications for clonal selection and genetic resource conservation.