## 3 Discussion
### Individual Diglucoside Anthocyanin QTL Colocate  
Anthocyanin pigment content and composition are critical to the color of berries and resultant wines. The major anthocyanins (i.e., the aglycone component) found in grapes are malvidin, cyanidin, peonidin, petunidin, and delphinidin, which may exist in both mono- and diglucoside forms [48]. Anthocyanin diglucosides are at negligible concentrations in _V. vinifera_, but they are commonly found in wild _Vitis_ spp. and their hybrids [48]. Diglucosides are generally considered undesirable for wine grapes because they are less able to form stable polymeric pigment; therefore, their presence is used as a bio-marker to identify unallowable interspecific hybrids in certain wine regions [16; 49]. [PERSON] et al. 2015 [50] reported several minor QTL on multiple chromosomes which are not observed in this study. Individual and total diglucoside QTL were detected with a peak position near the _V. vinifera 5-O-GLUCOSYLTRANSFERASE_ (Vitvi09g00582) candidate gene at 6.52 Mb. This gene, described by [PERSON] et al 2009 [51], lacks 51 amino acids due to a premature stop codon and is non-functional in _V. vinifera_, resulting in the absence of anthocyanin 3,5-diglucosides in most _V. vinifera_. The 3,5- diglucoside anthocyanins are found in many of the other _Vitis_ species including _V. riparian_. Protein alignment of Vitvi09g000582 with the corresponding _V. riparian_, _V. amurensis_ and _V. rotundifolia_ sequences (Figure S6) indicates these native American species contain 51 amino acids in the region necessary for diglucoside development. Characterization of the _V. amurensis 5-O-GLUCOSYLTRANSFERASE_ (Va5 GT) _in vitro_ shows that it can synthesize diglucosidic anthocyanins [52]. There were no genotypes with black berries without diglucosides; however, several black fruited VRS-F\({}_{2}\) individuals were identified with very low concentration of diglucoside anthocyanins, providing opportunity for genotype selections for future crosses. QTL Mapping Identified Malic Acid Dehydrogenase Candidate Gene on Chromosome 1 and Suggests Potential Temperature Influence  
Most wild _Vitis_ spp. are known to have higher TA and malic acid berry concentrations than _V. vinifera_, resulting in excessively sour wines and lower wine quality [53]. In this study, there was a significantly high correlation (ranging from 0.71 to 0.96, _p_-value < 0.0001) between the TA and malic acid concentration in all years, indicating that variation in the TA in this population can largely be explained by the variation in malic acid. Malic acid is mainly synthesized through Krebs cycle/sugar metabolism and degrades during ripening through the TCA cycle and respiration [54]. The activity of several enzymes critical to malate respiration, including malate dehydrogenase, are known to increase with higher temperature. In this study of 30-day post-veraison berries, a QTL on chromosome 1 in 2018 and 2016 (LOD > 3) contributed significantly to the malic acid models, although no QTL for chromosome 1 was detected in 2013. It is noted that 2013 had lower preveraison temperatures than did 2016 and 2018; however, mean malic acid concentrations were similar across all three years, likely because these differences in temperature occurred before respiration commenced. The chromosome 1 QTL in VRS-F\({}_{2}\) contained three candidate genes; two _MALATE DEHYRDROGENASE CYTOPLASMIC 1_ genes (Vitvi01g02239, Vitvi01g02240) are located near the QTL peak position on chromosome 1 and a third gene (_MALATE DEHYROGENASE PRECURSOR_, Vitvi01g01744) is within the Bayesian confidence interval. The malic acid QTL on chromosome 6 was stable in the VRS-F\({}_{2}\) population across the three years. The QTL confidence interval contains _ALMT_ candidate genes (Vitvi06g00922, Vitvi06g00928), first identified with single year data in the GBS map [43]. The _ALMT_ gene plays a role in malate and tartrate accumulation [43; 55].