Targeted long-read sequencing reveals function-restoring MYBA1 and MYBA2 mutations in white-to-red grape color somatic variants

Abstract

To diversify production, vinegrowers select somatic variants in grape color that arise spontaneously during the propagation of cultivars. This is the case of the red-berried bud sports that recently arose in the white-berried cultivars Albariño and Verdejo. To understand the molecular basis of grape color recovery, we combined real-time targeted Nanopore sequencing of a panel of anthocyanin metabolism and regulation genes, and a berry skin RNA-seq transcriptome comparison of the red variants and their corresponding ancestral white cultivars. In the red variant of Albariño, we identified overexpression of VviMYBA1 associated with the excision of the Gret1 retrotransposon that suppresses expression in the canonical white-grape allele. In the red variant of Verdejo, the frameshift InDel of the VviMYBA2 white-grape allele was restored, likely due to gene conversion between the two CR domains of the gene. Restoration of either VviMYBA1 or VviMYBA2 function activated the phenylpropanoid biosynthesis pathway, including the upregulation of VviPAL, VviCHS, VviFH3, VviLDOX, and VviUFGT in both red variants. Targeted UPLC-QqQ-MS/MS metabolomics confirmed specific accumulation of anthocyanins–predominantly dihydroxylated forms–in both red variants, with higher concentration in red Albariño than in red Verdejo. Among colorless flavonoids, the accumulation of trihydroxylated flavonols also increased in both red variants, whereas proanthocyanidins increased only in red Albariño and dihydroxylated flavonols only in red Verdejo. Our results reveal a recurrent recovery of VviMYBA1 expression and a rare restoration of the VviMYBA2 reading frame in independent white-to-red grape variants, suggesting potentially distinct roles of the two master regulators of anthocyanin biosynthesis in grapevine fruits.

Acknowledgements

This study was developed under the projects BIO-2017-86375-R and PID2020-120183RB-I00 funded by MICIU/AEI/10.13039/501100011033, and project PID2023-152513OB-I00 funded by MICIU/AEI/10.13039/501100011033 and by FEDER, EU. This study received funds from the Max Planck Society.