Core Ideas A gene bank of heritage apple cultivars with historical data proves suitable for GWAS. GWAS showed associations between acetate esters and AAT1 and nearby paralogues. Sugar content and sugar composition associates with a vacuolar invertase gene. Harvest date correlates with a certain haplotype of the NAC18.1 transcription factor. Understanding the genetic architecture of fruit quality traits is crucial to target breeding of apple (Malus domestica L.) cultivars. We linked genotype and phenotype information by combining genotyping‐by‐sequencing (GBS) generated single nucleotide polymorphism (SNP) markers with fruit flavor volatile data, sugar and acid content, and historical trait data from a gene bank collection. Using gas chromatography–mass spectrometry (GC‐MS) analysis of apple juice samples, we identified 49 fruit volatile organic compounds (VOCs). We found a very variable content of VOCs, especially for the esters, among 149 apple cultivars. We identified convincing associations for the acetate esters especially butyl acetate and hexyl acetate on chromosome 2 in a region of several alcohol acyl‐transferases including AAT1. For sucrose content and for fructose and sucrose in percentage of total sugars, we revealed significant SNP associations. Here, we suggest a vacuolar invertase close to significant SNPs for this association as candidate gene. Harvest date was in strong SNP association with a NAC transcription factor gene and sequencing identified two haplotypes associated with harvest date. The study shows that SNP marker characterization of a gene bank collection can be successfully combined with new and historical trait data for association studies. Suggested candidate genes may contribute to an improved understanding of the genetic basis for important traits and simultaneously provide tools for targeted breeding using marker‐assisted selection (MAS).

Background Modern biological approaches generate volumes of multi-dimensional data, offering unprecedented opportunities to address biological questions previously beyond reach owing to small or subtle effects. A fundamental question in plant biology is the extent to which below-ground activity in the root system influences above-ground phenotypes expressed in the shoot system. Grafting, an ancient horticultural practice that fuses the root system of one individual (the rootstock) with the shoot system of a second, genetically distinct individual (the scion), is a powerful experimental system to understand below-ground effects on above-ground phenotypes. Previous studies on grafted grapevines have detected rootstock influence on scion phenotypes including physiology and berry chemistry. However, the extent of the rootstock's influence on leaves, the photosynthetic engines of the vine, and how those effects change over the course of a growing season, are still largely unknown. Results Here, we investigate associations between rootstock genotype and shoot system phenotypes using 5 multi-dimensional leaf phenotyping modalities measured in a common grafted scion: ionomics, metabolomics, transcriptomics, morphometrics, and physiology. Rootstock influence is ubiquitous but subtle across modalities, with the strongest signature of rootstock observed in the leaf ionome. Moreover, we find that the extent of rootstock influence on scion phenotypes and patterns of phenomic covariation are highly dynamic across the season. Conclusions These findings substantially expand previously identified patterns to demonstrate that rootstock influence on scion phenotypes is complex and dynamic and underscore that broad understanding necessitates volumes of multi-dimensional data previously unmet.

9 10 Apple (Malus spp.) is a widely grown and valuable fruit crop. Leaf shape and size are important 11 for flowering in apple and may also be early indicators for other agriculturally valuable traits. 12We examined 9,000 leaves from 869 unique apple accessions using linear measurements and 13 comprehensive morphometric techniques. We identified allometric variation in the length-to-14 width aspect ratio between accessions and species of apple. The allometric variation was due to 15 variation in the width of the leaf blade, not length. Aspect ratio was highly correlated with the 16 primary axis of morphometric variation (PC1) quantified using elliptical Fourier descriptors 17 (EFDs) and persistent homology (PH). While the primary source of variation was aspect ratio, 18 subsequent PCs corresponded to complex shape variation not captured by linear measurements. 19After linking the morphometric information with over 122,000 genome-wide SNPs, we found 20 high narrow-sense heritability values even at later PCs, indicating that comprehensive 21 morphometrics can capture complex, heritable phenotypes. Thus, techniques such as EFDs and 22 PH are capturing heritable biological variation that would be missed using linear measurements 23 2 alone, and which could potentially be used to select for a hidden phenotype only detectable using 24 comprehensive morphometrics. 25 26 Passiflora (8). Previous work used EFDs to assess apple fruit shape (9), but this technique has 47 not yet been applied to apple leaves. A newly developed morphometric technique, persistent 48 homology (PH), provides another method for estimating leaf shape. PH, like EFDs, is 49 normalized to differences in size, but it also could be orientation invariant. PH treats the pixels of 50 a contour as a 2D point cloud before applying a neighbor density estimator to each pixel. A 51 series of annulus kernels of increasing radii are used to isolate and smooth the contour densities. 52The number of connected components is recorded as a function of density for each annulus, 53 resulting in a curve (a reduced version of persistent barcode) that quantifies shape as topology. 54The topology-based PH approach can also be applied to serrations and root architecture, allowing 55

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 pre-harvest ripening and associated textural changes following harvest 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 3 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 that is orthogolous to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato (Solanum lycopersicum). 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.

22Highlight: 23 NAC18.1 is a member of a family of conserved transcriptional regulators of ripening that 24 underlies variation in fruit firmness and harvest date in diverse apple accessions. 25 26 Abstract: 27Softening is a hallmark of ripening in fleshy fruits, and has both desirable and 28 undesirable implications for texture and postharvest stability. Accordingly, the timing and extent 29 of ripening and associated textural changes are key targets for improving fruit quality through 30 breeding. Previously, we identified a large effect locus associated with harvest date and firmness 31 in apple (Malus domestica) using genome-wide association studies (GWAS). Here, we present 32 additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1, cause 33 variation in these traits. First, we confirmed our previous findings with new phenotype and 34 genotype data from ~800 apple accessions. In this population, we compared NAC18.1 to three 35 other ripening-related markers currently used by breeders (ACS1, ACO1, and PG1), and found 36 that the effect of the NAC18.1 genotype on both traits greatly exceeded that observed for the 37 other markers. By sequencing NAC18.1 across 18 accessions, we revealed two predominant 38 haplotypes containing the SNP previously identified using GWAS, as well as dozens of 39 additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a 40 protein with high similarity to the NON-RIPENING (NOR) transcription factor, an early 41 regulator of ripening in tomato (Solanum lycopersicum). To test whether these genes are 42 functionally orthologous, we introduced NAC18.1 transgenes into the tomato nor mutant and 43 showed that both haplotypes complement the nor ripening deficiency. Taken together, these 443 results indicate that polymorphisms in NAC18.1 underlie substantial variation in apple firmness 45 and harvest time through modulation of a conserved ripening program. 46 47