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Analysis of over 100 Cannabis samples quantified for terpene and cannabinoid content and genotyped for over 100,000 single nucleotide polymorphisms indicated that Sativa- and Indica-labelled samples were genetically indistinct on a genome-wide scale. Instead, we found that Cannabis labelling was associated with variation in a small number of terpenes whose concentrations are controlled by genetic variation at tandem arrays of terpene synthase genes.

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

An understanding of the relationship between the cultivated apple (Malus domestica) and its primary wild progenitor species (M. sieversii) not only provides an understanding of how apples have been improved in the past, but may be useful for apple improvement in the future. We measured 10 phenotypes in over 1000 unique apple accessions belonging to M. domestica and M. sieversii from Canada’s Apple Biodiversity Collection. Using principal components analysis (PCA), we determined that M. domestica and M. sieversii differ significantly in phenotypic space and are nearly completely distinguishable as two separate groups. We found that M. domestica had a shorter juvenile phase than M. sieversii and that cultivated trees produced flowers and ripe fruit later than their wild progenitors. Cultivated apples were also 3.6 times heavier, 43% less acidic, and had 68% less phenolic content than wild apples. Using historical records, we found that apple breeding over the past 200 years has resulted in a trend towards apples that have higher soluble solids, are less bitter, and soften less during storage. Our results quantify the significant changes in phenotype that have taken place since apple domestication, and provide evidence that apple breeding has led to continued phenotypic divergence of the cultivated apple from its wild progenitor species.