2019 ASHS Annual Conference

Muscadine grapes (Vitis rotundifolia Michx.) are a specialty crop cultivated in the southern United States. Muscadines (2n=40) belong to the subgenus Muscadinia and are distantly related to ‘bunch’ grapes of the subgenus Euvitis (2n=38), including V. vinifera. Muscadines are often processed into juices and wines; however, they have poor color stability during storage. Fruit color in muscadines is determined by anthocyanin accumulation in berry skins. While the candidate genes for berry color in V. vinifera map to chromosome 2, the color locus in muscadine was mapped to a 0.8 Mbp region syntenic with chromosome 4 of V. vinifera in two mapping populations segregating for berry color. The objective of this research was to identify and characterize the candidate gene for color variation in muscadine berries. Of the 21 predicted genes spanning the 0.8 Mbp locus, glutathione S-transferase (GST4) was identified as a likely candidate gene involved in anthocyanin biosynthesis pathway. Other members of the GST family have essential roles in anthocyanin transport and accumulation including Bz2 in maize, An9 in petunia, Fl3 in carnation, and TT19 in Arabidopsis. PCR and KASP genotyping assay identified a single intragenic SNP (C/T) marker corresponding to a proline to leucine171 mutation within the muscadine GST4 (VrunGST4) sequence that differentiated black from bronze muscadines in 64 breeding selections, 32 cultivars, and 320 progeny from the two mapping populations. Anthocyanin profiling on a subset of the progeny with CC (homozygote black), CT (heterozygote black), or TT (homozygote bronze) genotypes indicated no correlation between color and anthocyanin content in black muscadine berries. Furthermore, VrunGST4 action was found to be completely dominant, and no difference was observed in the anthocyanin content or composition in muscadine berries with CC and CT genotypes. VrunGST4 had higher expression in the ripe berries compared to unripe berries of black muscadines, unlike in the bronze muscadine. These results suggest that berry pigmentation in muscadines may be regulated by a mechanism distinct from V. vinifera. The VrunGST4 sequence from both black and bronze muscadines was successfully cloned into pET19b vector for expression of the VrunGST4 protein. Further research is in progress to determine differences in the catalytic and/or ligandin activity between the black and bronze VrunGST4 proteins. Work is also in progress using RNA seq to identify other potential candidate genes involved in the diglucoside anthocyanin pathway of muscadine. These findings will have important implications in muscadine breeding programs and the processing industry.