GWAS analysis of genetic regulatory loci and candidate genes for β-damascenone in wine grapes

【Objective】β-damascenone is a key norisoprenoid compound responsible for the floral and fruity aromas in grapes and wine, which significantly contributes to the sensory quality and consumer acceptance of wine. However, the complex genetic regulatory mechanisms underlying its biosynthesis and accumulation in grape berries remain largely unclear. This study aims to analyze its genetic loci and provide a theoretical basis for molecular marker- assisted breeding of aromatic traits in wine grapes. 【Methods】This study used the F1 population derived from a cross between“Cabernet Sauvignon 685” and“Syrah 100”as the experimental material. After verifying the authenticity using fresh leaves collected in April 2018, mature berries were collected from August to September during three consecutive years (2018—2020), ultimately yielding 151 qualified samples. Solid-phase microextraction combined with gas chromatography/mass spectrometry (SPME- GC/MS) was used to determine the contents of free and glycosylated β-damascenone in the samples as phenotypic data. Combined with high- quality SNP loci obtained from whole-genome resequencing, genome-wide association study (GWAS) was conducted using a mixed linear model (MLM) to screen for significant SNP loci associated with β-damascenone (P＜8.99×10-7 ). The genes within 100 kb upstream and downstream of the SNP sites were annotated for gene function using the 12X.2 PN40024 grapevine reference genome. Candidate genes were further screened using KEGG enrichment analysis and transcriptomic data from grape berry developmental stages. Finally, functional validation was performed by transient homologous overexpression of one of the candidate genes in grape leaves and berries.【Results】This study detected both (E)-β-damascenone and (Z)-β-damascenone in the F1 population, with the content of the (E)-β-damascenone being significantly higher than that of the (Z)-β-damascenone in grape berries. To better characterize the genetic control loci controlling the β-damascenone content, the contents of both isomers were summed across all samples for subsequent experiments, the results showed that β- damascenone content exhibited a skewed distribution toward low values in the F1 population overall, though there were a few individuals with extremely high content. The coefficient of variation over the three years was generally around 40%, and the broad- sense heritability was above 90% in all three years, and the genetic transmission ability was high, with an average transmission efficiency above 90% over the three years, suggesting strong genetic control of this trait. Through genome-wide association study (GWAS), five SNPs with extremely significant associations with β-damascenone content were identified, distributed across ChrUn (ChrUn:13846350, ChrUn:23214460), Chr1 (Chr1:12111316), Chr10 (Chr10:17566481), and Chr11 (Chr11:1006227). ChrUn:13846350 has two genotypes, AT and AA, corresponding to different phenotypic values, with AA significantly higher than AT; ChrUn:23214460 has two genotypes, AG and AA, with AA significantly higher than AG; Chr1:12111316 has two genotypes, AC and AA, with AA significantly higher than AC; Chr10:17566481 has two genotypes, AG and AA, with the AA genotype showing significantly higher phenotypic content than the AG genotype; Chr11:1006227 has three genotypes, GG, AG, and AA, with the AA genotype showing significantly higher phenotypic concentration than both AG and GG. All five SNP loci are non-synonymous mutations, with mutations at the SNP loci ChrUn:13846350 and Chr10:17566481 causing the original amino acid to be replaced by a stop codon, potentially leading to truncated protein products. Additionally, it was found that the β-damascenone content in the homozygous genotype (AA) of the F1 population was significantly higher than those in the heterozygous genotype and alternative homozygous genotypes. Based on gene localization within a ± 100 kb range of significant SNP loci in the PN40024 grapevine reference genome (12X.2), a total of 86 genes were identified near to these significant SNP loci. KEGG enrichment analysis was first used for screening, and only one gene related to monoterpene biosynthesis was identified (mannitol dehydrogenase, VIT_200s1389g00010), and no known genes associated with the isoprenoid metabolic pathway were identified. Therefore, transcriptomic data from grape berry development were used to screen for candidate genes positively correlated with norisoprenoid accumulation during grape fruit development. Combined with gene functional annotations from the UniProt database, five candidate genes were selected: Mannitol dehydrogenase (VIT_200s1389g00010): ChrUn:23214460; Heavy metal- associated isoprenylated plant protein (VIT_210s0116g01340): Chr10:17566481; Cytochrome P450 72A225 (VIT_ 211s0016g01290): Chr11:1006227; Transcription factor MYB1 (VIT_211s0016g01310): Chr11: 1006227; and Transcription factor MYB1 (VIT_211s0016g01320): Chr11:1006227. Among these, except for the Heavy metal-associated isoprenylated plant protein, which was annotated as related to iso-prene, Mannitol dehydrogenase was annotated as involved in monoterpene biosynthesis via KEGG enrichment analysis; additionally, both the CYP and MYB family have been reported in the literature to be associated with monoterpene production in plants. Finally, one candidate gene, VvMYB1 (VIT_ 211s0016g01310), was selected for homologous overexpression experiments in grape leaves and fruits. The results showed a significant increase in norisoprenoids in grape leaves and fruits, with β-damascenone levels increasing by 5-fold and 1.2-fold in leaves and fruits, respectively, compared with the controls. Additionally, the expression of VvCCD1, VvCCD4a, and VvCCD4b in grape fruits was significantly upregulated as detected by qPCR, confirming that VvMYB1 promotes the synthesis of norisoprenoids by activating CCD gene expression.【Conclusion】Five significant SNP loci associated with β-damascenone content were identified in wine grape fruits. After screening, five candidate genes were obtained, and one of them, VvMYB1, was found to be involved in the accumulation of norisoprenoids in grape fruits, providing a new target for molecular selection of grape aroma traits.