# Pigment-Related Mutations Greatly Affect Berry Metabolome in San Marzano Tomatoes
## 1 Introduction
The phenotype, that emerged during a breeding program in 1965, is controlled by a single recessive gene [25] that has not yet been identified. Several wild tomato species yield anthocyanin-pigmented fruits, and this trait has been introressed into cultivated forms in the past [26; 27; 28]. The dominant allele _Aft_, originating from _S. chilense_ ([PERSON] [PERSON], was firstly described by [PERSON] et al. [26]; it initiates anthocyanin accumulation in the immature green fruit that continues during the development with increased pigmentation that spreads uniformly across the fruit, resulting in tomato berries with purple spots where the skin is exposed to sufficient light levels [29]. The _Aft_ locus was mapped in the long arm of Chr10 [30; 31; 32]. More recently, [PERSON] et al. [33] revealed the genetic basis of the _Aft_ trait, consisting of an R2R3 MYB transcription factor encoding gene, _SIAN2 like_, in which a mutation of the wild-type allele underlies the lack of anthocyanin pigmentation in cultivated tomato. The _atv_ variant was derived from _S. cheesmaniae_ ([PERSON]) [PERSON], where it regulates the phytochromeresponse by causing purple pigmentation in foliage and rarely in the fruit [34]. _Atv_ has been mapped in Chr7 of tomato [35]. Recent research demonstrated that the _atv_ phenotype is genetically associated with the mutation of the gene encoding the R3-MYB protein, named _SIMYB-ATV_[36; 37]. In the mutant, the lack of a functional repressor is responsible for the misregulated accumulation of anthocyanins wherever their production is induced. Both _Af_ and _atv_ do have characteristics typical of the high-photoperiod response mutants, and, when they are combined into the same genotype (_Af_atv_), an intensely purple fruit phenotype is generated. Pigmentation in _Af_atv_ is significantly stimulated by high light intensity and is limited to the epidermis and epicarp of the fruit, which may have both purple and red regions, depending on exposure of the fruit to the light [29; 38]. Although the described mutations have been characterized at both the molecular and biochemical level, a comparison of their effect in a common genetic background at a \"global\" metabolomic level has not been carried out. In this context, a breeding program carried out in our research group has produced 20 new tomato lines by backcrossing variant alleles affecting fruit pigmentation and ripening coming from different donors to SM, the cultivar used as recurrent parent. The collection and how it was obtained were widely described elsewhere [39]. Here, we focus on the metabolomic characterization of four of these lines, carrying _hp-1_, _hp-2_, _pd_, and the _Af_atv_ combination. To date, most of the metabolic characterization works have been focused on detecting and quantifying mainly carotenoids for _hp_ mutations and anthocyanins for _Af_atv_ lines [40; 41; 42; 43; 44; 45]. Our aim was to expand the analysis of the effects generated by this set of mutations to a wider spectrum of metabolites belonging to different biochemical classes and fractions to identify all the possible effects with the purpose to bring an added value to the SM variety and generate novel fruit variants.