The initial 40 tomato accessions
In an initial experiment 40 tomato genotypes, including cultivars and wild accessions (Table S1), were tested for their biomass under different lighting conditions (Ouzounis et al. 2016; Ji et al. 2020). These 40 accessions were grown in climate rooms (Ji et al. 2020) with artificial lighting. The plants were pre-grown in potting soil under fluorescent light for seven to nine days after sowing and then transferred to sterile river sand and grown in one of the following LED conditions: white light with additional UVB (ca. 0.16 W m-2) and FR (ca 26 μmol m-2 s-1) or R/B LEDs at a 16h photoperiod (light intensities ca. 130 µmol m-2 s-1). The white light was created with white/red LEDs (GreenPower LED-TL-DR/W-MB-VISN, Philips, The Netherlands), and the additional UVB and FR was provided by Philips GreenPower LED production modules far-red (GreenPower LED -PM-FR-150), cool fluorescent tubes (38W, Philips) and Philips Broadband TL 20W/12 RS Ultraviolet-B lamps. The R/B LEDs had 88% red light and 12% blue light (GreenPower LED-PM-DR/B-MB-150, Philips). The temperature and humidity of the climate room were approximately 22°C and 70%, respectively. Destructive measures for total dry biomass (roots, shoots and leaves) were done three weeks after transplanting.
Development of four RIL populations
From the 40 accessions, genotypes were selected that showed contrasting responses to the different light environments. The following four combinations of contrasting genotypes were selected for development of RIL populations: Ailsa Craig (LA2838A) and Kentucky Beefsteak (TR00021) hereafter abbreviated with “A ~ K”; LA1578 (EA00674; S. pimpinellifolium) and Rutgers (EA00465; S. lycopersicum) “L ~ R”; Moneymaker (CGN14330; S. lycopersicum) and Momotaro (Tough Boy; TR0003; S. lycopersicum) “Mm ~ Mo”; and Kentucky Beefsteak (TR00021; S. lycopersicum) and NunhemsFM001 (S. lycopersicum) “K ~ N”. For each of these cross-combinations a reciprocal cross was performed, swapping the two parents, and therefore each RIL population consisted of two subgroups differing in maternally inherited plastids and mitochondria. All individuals of the populations were grown via single seed decent at least to the F5 generation (the Mm ~ Mo RIL population was an F6). The F1 hybrids were made at Wageningen University and Research (WUR), whereas the RIL populations were developed in greenhouses in the Netherlands by Nunhems Netherlands B.V. (BASF Vegetable Seeds), Rijk Zwaan Nederland B.V., and Bejo Zaden B.V. For each subpopulation ca. 75 individual RILs were grown, leading to ca. 150 RILs per QTL analysis.
Growth conditions for the four RIL populations analysed in young stage
For the screening of the RIL populations, the plants were grown in glasshouses at Unifarm, Wageningen University and Research. In each experiment the same set of RILs was grown in parallel in two different compartments, one with HPS supplemental lighting and one with LED supplemental lighting.
Plants for both supplemental light treatments were germinated under HPS-supplemental lighting. Of each genotype ca. 12 seeds were sown. The seeds were placed on stone-wool plugs (Gro Plug, Grodan Rockwool B.V., Roermond, The Netherlands), covered with Agra-Vermiculite (PULL Rhenen, Rhenen, The Netherlands) and a transparent foil for additional humidity for approximately the first five days. The plugs were soaked with nutrient solution: NH4 1.2 mM, K 7.2 mM, Ca 4 mM, Mg 1.82 mM, NO3 12.4 mM, SO4 3.32 mM, P 1 mM, Fe 35 μM (as mixture of Fe‐DTPA/ Fe‐EDDHSA), Mn 8 μM, Zn 5 μM, B 20 μM, Cu 0.5 μM and Mo 0.5 μM. The macronutrients were provided as a mixture of fertilizers from Yara Benelux BV (Rotterdam–Vlaardingen, The Netherlands) and the micronutrients were from the Agrispoor product line of Horticoop BV (Bleiswijk, The Netherlands). The pH was between 5.5 and 5.8 (adjusted with KOH) and the EC was approximately 2.0 dS m-1.
Seedlings with fully expanded cotyledons were selected for transfer after one week. The plants were transferred to 10 cm x 10 cm x 6 cm stone-wool blocks (Grodan Delta, Grodan Rockwool B.V., Roermond, The Netherlands). Upon transfer the plants were divided among the two compartments, one with supplemental HPS-lighting (Master green power, cgt 400W, Philips) and one with supplemental DR/LB LED-lighting (Green Power LED top lighting module DR/B LB, 190W, Philips).
The light spectra of the lamps were measured (Figure S1) with a Field Spectroradiometer (SS-110, Apogee Instruments, Logan, UT, USA) and PSS values calculated according to the method of Sager et al. (1988). For the analysis, blue light over the wavelength range 400-500nm, green over 500-600nm, red over 600-700nm and FR over 700-800nm were considered (individual intensity measures per wavelength were used for the average if above 0.05 µmol s-1 m-2). Percentages of those wavelengths in the spectrum were compared to the overall intensity in the PAR range from 400 to 800 nm and rounded to 5% intervals. To shield the experiment from light of adjacent compartments, the walls of the compartments were covered with white horticultural foil. The supplemental light intensity was about 200 µmol s-1 m-2 (in a wavelength range from 400 to 700 nm) during 16 h, starting 16 h before sunset each day. The compartments were heated and set to an air temperature of 22°C during the supplemental lighting period and of 19°C outside the supplemental lighting time. Climate data inside the greenhouse were measured by a climate box (PT500 Temp./ RH sensor, Hoogendoorn Automatisering) every 20 s and averaged over 5 min intervals. The incoming solar light and other environmental factors were recorded and averaged over time (Table S2). The solar light was measured on top of the greenhouse. Eight light measurements (light meter LI-250A from LI-COR) inside the empty greenhouse were taken at the approximate plant height on one day. With this data a coefficient between incoming light and the light measured on top of the glasshouse was calculated to estimate the incoming solar light for all experiments. In case the solar radiation (in the third and fourth experiment) outside the greenhouse was above 200 W m-2, an energy screen (Harmony 4215 O FR) was closed above the lamps in the greenhouse (leading to 42% less light at 100% screen-closure, the light-coefficient was adapted accordingly). For the mature plants the intensity of the solar light received by the plants was estimated using the measured solar radiation outside the greenhouse and a transmissivity factor of 0.62 (based on previous measurements). In addition, the measured solar radiation was reduced to 47%, as this is the approximate amount of photosynthetically active photon flux density in the solar radiation; and then assuming that 1 W m-2 solar light equals 4.57 µmol m-2 s-1 the conversion into µmol m-2 s-1 was done (McCree 1972; Britton and Dodd 1976).
The RILs of the populations L ~ R, A ~ K and Mm ~ Mo were grown together in three, repeated experiments between October 2018 and March 2019 with one replicate per RIL and experiment, giving three replicates in time. The population A ~ N was grown in one experiment in March/ April 2019 with three replicates per RIL simultaneously. The seven parents of those RIL populations were grown in each experiment with four replicates. The first experiment was harvested 5 weeks after sowing and the consecutive three experiments were harvested in week 4 after sowing. The individual plants of each experiment were divided into four batches and sown on four consecutive days each on one ebb-and-flood table. Destructive measurements were performed during four consecutive days as well, so that the growth-period was the same for all plants. All plants were distributed in a randomized design over the four tables. For the first three experiments the tables were divided into four areas (front, back and two middle parts of the rectangular tables) for the randomization pattern with similar numbers of plants from each RIL-population and reciprocal cross per area. From the parents one replicate was grown per table. The parents used for one population were situated close to each other on each table.
Plants were watered by flushing the stone-wool blocks for 5 minutes with nutrient solution (see above) on the ebb- and flood-tables. The watering schedule over the course of the experiment was irregular, it aimed at keeping the blocks always moist but not wet.
Phenotyping during the growth of the young plants
The operating quantum yield of electron transport of photosystem II (ΦPSII; “Phi PSII”) was estimated, using the closed FluorCam 800MF (Photon Systems Instruments, Brno, Czech Republic) and a self-written program to determine Ft under actinic light (ca. 200 µmol s-1 m-2) and Fm (after max. light pulse) with the software FluorCam7 (version 1.2.4.4 Photon Systems Instruments, Brno, Czech Republic). The measurement was done four or five weeks after sowing on the second or third leaflet of a fully expanded leaf that was as much as possible perpendicular to the light. Measurements were done between 09.00 and 14.00 o’clock, alternating between the HPS supplemental light and LED supplemental light greenhouses approximately every 30 minutes (measuring batches of the same 22 genotypes in both HPS and LED supplemental lighting). Flavonol, anthocyanin and chlorophyll indices were obtained for all plants three weeks after sowing with the Force-A Dualex Scientific+™ sensor (Dynamax Inc., Houston, USA).
Destructive measurements of biomass related traits of the young plants
At the end of the growth period, four or five weeks after sowing (depending on the experiment), the plant stems were cut at the height of the stone-wool blocks and leaves were separated from the stem. In the axil of the true leaves, side shoots developed in most genotypes. The numbers of true leaves (TL) and side shoots were counted separately from the second experiment onwards. The leaf/ shoot-structures were counted in case they were longer than ca. 5mm and the total respective fresh weights per plant determined. Final leaf area (of true leaves and side-shoots) was determined with an area meter (Li-cor, LI-3100C Area Meter, Li-cor Inc., Lincoln, Nebraska, USA). True leaves, side shoots and stems were put into separate paper bags and placed into a ventilated oven at 70°C for 24 hours followed by 105°C for 24 hours, after which the dry weight was measured (in the first experiment the side shoot and true leaf dry weight was determined together, in all the following experiments separately). The specific leaf area (SLA) was determined as the ratio between leaf area and leaf dry weight.
Growth conditions of the fruiting RIL population
For the phenotyping of fruits of the RIL population “K ~ N” (‘Kentucky Beefsteak’ ~ NunhemsFM001) the plants were grown in glasshouses of Unifarm (Wageningen University and Research). The plants were sown in September 2020. The RIL population was chosen for the similar sizes and weights of their fruits of its parental lines, as well as the resistance to tomato mosaic virus (TMV) of the NunhemsFM001 parent. The plants were pre-grown for four and a half weeks on ebb and flood tables in a greenhouse. If within a 16h light period the outside global radiation was below 250W m-2, the natural light was supplemented with approximately 100 µmol m-2 s-1 HPS light. Subsequently, the plants were divided among two compartments of 12 m by 12 m each at a plant density of approximately 2.2 plants per m2. For avoidance of border effects, at each side of the greenhouse a border row of plants was grown. In both compartments about 220 µmol m-2 s-1 supplemental lighting was applied of either HPS in one compartment (Master green power, cgt 400W, Philips) or 95% red and 5% blue LEDs (Green Power LED top lighting module DR/B LB, 190W, Philips) in the other compartment. The light was measured ca 2.5 m over ground which was the height of the canopy top. The plants were kept at a height of approximately 2.5 m in order to keep 1 m of distance to the lamps above, and obtain equal light distribution. The duration of supplemental light was gradually increased, starting at five hours one month after sowing and reaching a maximum of 15 hours of supplemental HPS/LED light per day from four months after sowing onwards.
Stone-wool (Rockwool Grodan B.V., Roermond, The Netherlands) was used as planting medium, and the plants were drip-irrigated using the same nutrient solution as for the young plants. The EC of the nutrient solution in the stone wool slab was on average 3.8 ds m-1 under HPS versus 3.2 ds m-1 under LED. The compartments were heated if the temperature dropped below ca. 22°C (in a later stage of the experiment 24°C) during the supplemental lighting period or 19°C outside the supplemental lighting time. In compartment with LED supplemental lighting, heating pipes below the plants and above the lamps were used, to compensate for the heat irradiance from the HPS lamps. The HPS compartment was only heated by pipes below the plants. For pollination we used bumblebees (Koppert, biological systems). Harvesting and measurements started in week 17 after sowing and continued up until week 28. In each compartment the same set of 136 RILs was grown with one replicate each, as well as 11 replicates of each parent of the RIL population.
Growth abnormalities in the young RIL populations
The RIL populations were grown under two different supplemental lighting conditions in the greenhouse, under standard HPS lighting and R/B supplemental LED lighting. In some plants the formation of callus like structures starting at the veins on the abaxial side of leaves, called intumescence, was observed in both supplemental lighting conditions (Figure S2). Although all populations had individual plants with light intumescence symptoms (Williams et al. 2016), in some populations the occurrence was more severe. Intumescence was scored from 0 (no intumescence) to 3 (most of the leaves affected) and because this invasive physiological trait could affect other measured traits, genotypes that had a score of 2 and above were excluded from further analysis (0.1 to 19.2% of all individual plants, depending on the population).
Measuring fruit traits
The number of fruit trusses was counted on week 17 and week 25 after sowing. The fruit trusses were labelled in order of development, starting at truss number 1. The number of fruits were counted on week 17 and week 25 after sowing as well. Internode length was measured once during the trial on week 17 after sowing. This length was measured in centimetres from the third truss to the fifth truss. Fruit fresh weight was measured throughout the trial with intervals of three to four days between measurements, with the measurements starting in week 17 and ending in week 28 after sowing. The fresh fruits were measured from the breaker stage and onwards. This breaker stage is defined as the phase at which the fruit starts developing orange/red colour on its surface skin. During the last harvest during week 28 after sowing, all fruits were collected, including the non-ripe ones. By adding the weight of all unripe fruits per plant to the yield of ripe fruits of that plant, a total fruit yield (both ripe and unripe) was calculated per plant. By taking the number of fruits per plant into account, the average fruit weight per plant was obtained. Dry weight was measured for fruits from the 3rd and the 5th truss. This was done by cutting the fruits and subsequent drying the cut pieces in a ventilated oven for the first 12 hours at 70 °C and followed for at least 84 hours at 105 °C. Subsequently the weight of the dried fruit was determined.
Genotyping of the RIL populations
The four RIL populations were genotyped by Bejo Zaden B.V., Rijk Zwaan B.V, and Nunhems Nederlands B.V. The genotyping was done with 185, 219, 183 and 177 KASP markers for the populations A ~ K, L ~ R, Mm ~ Mo, and N ~ K, respectively, with approximately 2.9%, 12.5%, 5.2%, and 6.7 % of the marker calls scored as heterozygous.
Statistical Analysis
A Wilcoxon rank sum test was used to determine the significance of the light condition or reciprocal cross effect on the individual populations, using R (version 3.5.0; www.r-project.org). Descriptive statistics were done with the R package psych (version 1.8.4; Revelle 2018). All graphics were made with the R package ggplot2 (version 3.0.0; Wickham 2016). For the relative trait value in HPS and LED light, the trimmed mean under LED light, excluding the phenotypically upper 10% and lower 10% of individuals of each population (for A ~ K, L ~ R and Mm ~ Mo over three experiments), was expressed as percentage of the trimmed mean in HPS light (MeanLED*100/ MeanHPS). For the relative trait range per population, the trimmed average (again with a fraction of 0.1 of extremes trimmed at each end) trait value of the top 10% and bottom 10% plants within each population was calculated, and the difference between those values in percent as (((top-bottom)/top)*100) was calculated. Equally the relative difference between the sub groups of the RIL population that were derived from the reciprocal crosses were calculated, by comparing the trimmed means per subgroup as ((HighGroupMean- LowGroupMean)/ HighGroupMean)*100. A Shapiro-Wilk normality test per condition, population and experiment showed that nearly 2/3 of the trait values were not normally distributed. Broad sense heritability was estimated by using the mean sum of squares values (Mean Sq) from a two-factor ANOVA (type III SS) on the rank-transformed data, that uses the RIL-genotypes and the experiment as main factors or in case of the population “K ~ N” from a single ANOVA with genotype as factor. The ANOVA was done with the R package car (version 3.0-0; Fox and Weisberg 2019). For the estimation of the heritability, we used the equation H2 =σ2g/(σ2g+σ2e). The Mean Sq of the residuals was used as environmental variance (σ2e) and ((Mean SqRIL-genotype-Mean Sqresiduals)/replicate number) gave the genotypic variation (σ2g). To test for an interaction of the light treatments and the subgroups derived from the reciprocal crosses, a two-factor ANOVA on the rank transformed data was done as described above per population, using the subgroups and light-environment as interacting factors.
The QTL analysis was performed using the R package R/qtl (Broman et al. 2003; Broman and Sen 2009). The genetic map was based on the position information from the companies and re-calculated for each RIL population. A single QTL analysis was performed with the non-parametric mapping function for both the young and mature RIL populations, using individual values per experiment as input. However, in case of the young population N ~ K for which all replicates were grown within the same experiment an average value per genotype was used for the QTL analysis. In addition, a single QTL analysis with the non-parametric mapping function was performed for differences in response under LED and HPS, using as parameter (xLED – xHPS) / xHPS. For the populations A ~ K, L ~ R and Mm ~ Mo we used the BLUE function from the R package polyqtlR (Bourke et al. 2021) in the following way: BLUE(data=data, model = pheno~geno, random = ~1|experiment, genotype.ID = "geno"). In case of the population N~K the block in which the replicates were grown was used as random effect.
Also, a multiple QTL analysis was performed for the young populations with the multiple imputation method of Sen and Churchill (Sen and Churchill 2001) using rank transformed data as input, and in case of the first young three populations the experiment number was used as covariate. All permutation tests for LOD-thresholds/ penalties were done with 1000 permutations. In the single QTL analysis, the LOD threshold was determined specifically per population, trait and condition. For the multiple QTL analysis, the automated stepwise procedure was used (assuming a maximum of 7 possible QTL) with a penalized LOD score criterion balancing heavy and light interaction penalties. For computational reasons the penalties were calculated based on one trait with a median level heritability under HPS light conditions in the population N ~ K. LED-specific QTL were selected based on the multiple QTL analysis for the young population. For the mature population, these were selected based on the single QTL analysis. For the young population, QTL on the same chromosome were considered different if their approximate 99% Bayes credible intervals did not overlap. The significance of the QTL that were only identified under LED light was verified with an ANOVA for the effect of the allele distribution at the peak-marker on the phenotype under LED light. Also, the allele by light-condition interaction of those LED-specific QTL was assessed with an ANOVA (using the peak marker and the light condition as interacting factors explaining the trait). For both ANOVAs the rank transformed data from the individual plants was used and for the young populations A ~ K, L ~ R and Mm ~ Mo, the experiment number was used as a block factor. The phenotypic mean difference of the two QTL with significant allele by light interaction was tested with a Tukey HSD-test with the package Agricolae (De Mendiburu 2020).