DOI: 10.5061/dryad.p2ngf1vpp
title: Tomato fruit quality traits and metabolite content are affected by reciprocal crosses and heterosis

description: Heterosis occurs when the F1 individuals exhibit increased performance for
 a number of traits compared to their parental lines. On the other hand,
 reciprocal hybrids are obtained by changing the cross direction (or the
 sexual role) of parental genotypes in a cross. Both biological phenomena
 could affect the external and internal attributes of fleshy fruits. This
 work aimed to detect reciprocal effects and heterosis in different tomato
 (Solanum lycopersicum) for fruit quality traits and metabolite content.
 Twelve agronomic fruit traits and 28 metabolites identified and estimated
 by 1H NMR were evaluated in five cultivars grown in two environments. Due
 to the genotype component was the most important over the phenotype, the
 traits were evaluated following a full diallel mating design among those
 cultivars, in greenhouse. Data demonstrated that hybrids performed a
 higher phenotypic diversity than parental lines. Although both type of
 traits displayed reciprocal effect and heterosis, the metabolites had a
 bigger impact on generating this variation, mainly the amino acids. This
 coincided with the fact that agronomic traits were more influenced by GCA
 and metabolites by SCA. Furthermore, a relationship between genetic
 distance between parental lines and reciprocal effect or heterosis was not
 found. Hybrids with heterosis and high content of metabolites related to
 tomato flavour and nutritional components were obtained. Results of this
 work highlight the impact of selecting the role of a cultivar as male or
 female in a cross to enhance the variability of fruit attributes through
 hybrids as well as the possibility to exploit heterosis. 
description: 1. Description of methods used for collection/generation of
 data: 1.1 Characterization of parental genotypes by DNA
 molecular markers<br>Genomic DNA of frozen leaves (40 mg) from five
 parental genotypes was extracted with a commercial kit (Wizard® Genomic
 DNA Purification Kit, Promega®, Madison, WI, USA).  Two plants per
 genotype were used for the DNA extraction according to the manufacturer
 protocol, with some minor modifications. Samples were incubated 15 min at
 65°C with 600 µl of Nuclei Lysis Solution. Then, 200 µl of protein
 precipitation solution was added and the samples were centrifuged for 3
 min at 13000 rpm. The supernatant obtained was transferred to a clean 1.5
 ml tube with 600 µl of room temperature isopropanol. The solution was
 gently mixed by inversion and centrifuged for 1 min at 13000 rpm. The
 supernatant was discarded and the pellet was washed with ethanol 70% v/v.
 The solution was centrifuged for 1 min at 13000 rpm and the pellet was
 dried at room temperature. Finally, the pellet was re-suspended in TE
 buffer (10 mM Tris pH 7.4 and 1 mM EDTA pH 8.0). Quantity and quality of
 DNA extracted was evaluated with agarose gel 1% dyed with SYBR® Safe
 (Thermo Fisher Scientific®, Waltham, MA, USA) by comparison with a Lambda
 DNA.<br>Molecular markers were used to estimate polymorphism and
 genetic distance among parental genotypes. A total of 16 SSR (Single
 Sequence Repeats) and 64 InDel (Insertion/ Deletion) markers covering the
 entire tomato genome were used (Cambiaso et al., 2019a,b). Also, four
 functional markers: FW2.2 (Blanca et al., 2015), FW3.2 (Chakrabarti et
 al., 2013), LC (Muños et al., 2011) and FAS (Rodríguez et al., 2011) were
 used. The PCR amplification protocol consisted of 60 s
 at 94°C, 7 cycles (30 s at 94°C, 30 s at 58 °C – 52 °C and 2 min at 72°C),
 followed by 30 cycles including 30 s at 94°C, 30 s at 52°C and 2 min at
 72°C. The last step was 5 min at 72°C. Amplified fragments were visualized
 in agarose gel 3% dyed with SYBR® Safe (Thermo Fisher Scientific®,
 Waltham, MA, USA). 1.2 Agronomic fruit traits
 Six agronomic fruit traits were evaluated at breaker stage
 (fruits with 10 to 30% of final ripe colour, according to Giovannoni
 (2004)) in at least eight fruits per plant of each genotype: weight (FW,
 in g), height (H, in cm), diameter (D, in cm), shape index (FS), firmness
 (F, measured on the equatorial plane with a tester type Shore A) and shelf
 life (SL, measured as the number of days elapsed from harvesting to
 visualization of the first symptoms of deterioration in the fruits stored
 at 25±3°C, according to Green et al. (2016); Cambiaso et al.
 (2019a). Six additional agronomic fruit traits were
 evaluated at red ripe stage (fruits with 90% of mature colour): locule
 number (LN), soluble solids content (SS, in ºBrix, determined with a hand
 refractometer in the homogenized juice from the pericarp tissue), pH,
 titratable acidity (TA, g of citric acid/100 g of homogenized juice) and
 the fruit colour. The fruit colour was estimated by the absorbance index
 (a*/b*, where a* is the absorbance at 540 nm and b* at 675 nm) and
 reflectance percentage (L*) with a Minolta Chroma Meter reflectance
 colorimeter (model CR-400, Minolta Co., Osaka, Japan), as the average of
 three measures per fruit. 1.3 Metabolic
 traits<br>First or second fruit from the 2nd and 3rd inflorescence
 was collected from three independent plants at ripe stage per genotype.
 The harvest was done from 10 AM to noon. Fruit tissue was conditioned by
 removing the exocarp and locular tissue; the seeds were discarded while
 the mesocarp was cut into small pieces, quick-frozen in liquid nitrogen
 and then stored at -80 °C. A 1H NMR was done according to the protocol
 described by López et al. (2015). One gram of frozen mesocarp was powdered
 and 300 µl of 1M phosphate buffer (pH 7.4) prepared in D2O was added. The
 samples were centrifuged at 12000 rpm for 30 min at 4°C and the
 supernatant was transferred to a new tube. The pH of solutions was
 adjusted to 7.4 using NaOH 1N. As internal standard 1mM of TSP
 (3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt) prepared in D2O
 was added. An NMR Bruker Avance II spectrometer (Bruker, Germany) was used
 for the spectral analysis at 600.13 MHz. Proton spectra were acquired at
 298 K by adding 512 transients of 32 K data points with a relaxation delay
 of 5 s. Intense signals corresponding to H2O protons were suppressed by
 pulse gradient techniques. The 90° flip angle pulse was always *10 ls. TSP
 was used for both, chemical shift calibration and quantitation, that is,
 proton spectra were referenced to the TSP signal (d = 0 ppm) and their
 intensities were scaled to that of TSP. Spectral calibration was made
 using the software TopSpin v.3.5 pl 7, Bruker BioSpin GmbH. The
 identification and quantification of specific metabolites were done using
 the software “Mixtures v2.0”, developed ad hoc as an alternative to
 commercial programs (Abriata, 2012). Metabolites concentration was
 expressed in µmol/gFW. 2. Environmental/experimental
 conditions: Eight plants of each parental genotype were transplanted to
 open field (2) and greenhouse (1). Tomato plants were pruned and conducted
 to a single stem. Eight plants of each hybrid were transplanted into a
 greenhouse together with the parental genotypes. Tomato plants were pruned
 to a single stem and tied vertically to a top wire. In both environments,
 the plants were distributed according to a completely randomized design
 with 1.4 m between rows and 40 cm between plants.<br>Plants were
 drip irrigated and supplemented with nitrogen, phosphorus and potassium +
 magnesium oxide + magnesium and trace elements fertilizer once a week. The
 crop was grown according to the standard cultural recommendations for the
 area at the Estación Experimental ‘‘José F. Villarino’’ (33° SL and 61°
 WL), Argentina in 2015-2016.