In addition to running the PLSPM, 1000 bootstraps were performed to obtain statistical significance and confidence intervals of the path coefficients and the R2 values of each latent variable. The path coefficients are the standardized partial regression coefficients , and represent the direction and strength of causal relationships of direct effects. Indirect effects are the multiplied coefficients between the predictor variable and the response variable of all possible paths other than the direct effect . To determine the best path model, the latent variables were combined using our best understanding of biological relationships, and a general model using all data was generated. The paths between LVs were altered until a best-fit model was found. PLS-Predict was then used on the dataset to ensure that the model did not over or under fit the data, and for predictive performance of each manifest variable . This structural model, and not the fit values, was retained for use in predictive modeling of a separate dataset. PLSPREDICT, with the structural model developed as described earlier, was used on a separate dataset to determine the efficacy of the model. Two commercial cultivars, M82 and Lukullus, were used and only the leaf shape values were entered as exogenous variables. The predicted values for each output variable were compared with the actual measured values to determine how well the model predicted these variables.Yield, and fruit BRIX were measured over 14 wk of the growing season . For most cultivars the yield remained at or below 5 kg of fruit per plant at the time of harvest . The exceptions to this were Bloody Butcher, Glacier, Brandywine, Prudens Purple, and Stupice. All had a yield higher than 5 kg per plant, large round pot with Stupice having the highest yield, at c. 20 kg per plant by final harvest . Fruit BRIX remained nearly constant for all cultivars across the growing season , with the exception of ABC Potato Leaf, which had a large increase at time of harvest.
To better quantify fruit quality, BRIX and yield were multiplied to obtain a BY value for each measurement . Bloody Butcher, Glacier, and Stupice all had a BY value > 60, with Stupice reaching near 100 at terminal harvest . The average BY value for harvest weeks was 16.39, while Bloody Butcher, Brandywine, Glacier, and Prudens Purple had an average of c. 23 . Stupice showed the highest deviation with a mean BY value of 37.86 , setting it apart as the highest fruit quality cultivar tested in this study. Stupice maintained a stable BRIX content in its fruit despite the large increase in yield , which resulted in the large increase in overall fruit quality compared with other cultivars. Vegetative traits such as total biomass and leaf area were measured for the growing season as well . Fig. 2 shows the vegetative biomass and leaf area over the course of the growing season, which remain stably linked, indicating that overall leaf area increase contributed to increased biomass of the plant. This trend appears common in heirloom tomatoes but is different in commercial tomatoes, which have determinate growth .Leaf shape was shown to be strongly correlated with fruit BRIX and sugar accumulation in a meta-analysis of an introgression population . How leaf shape contributed to fruit BRIX was unclear, as shape and size of leaves do impact photosynthesis directly , but direct links between leaf shape and fruit quality appear lacking. Here, the heirloom population used displayed a wide array of leaf shapes, from very large and narrow to small and round. To understand if this range of leaf shapes had any impact on the overall fruit quality we measured leaflet shape and size for c. 3733 leaflets. Fig. 4 shows the resultant PCs of all primary leaflets measured and their relationship to traditional shape measures. PC1 contributes 78% of all variation found in the population and is tightly correlated with leaflet size , indicating that size was the largest source of variation among the heirloom leaflets . PC1 was also correlated with solidity , contributing to the slight shape changes seen in this PC . PC2 and PC4, while having no traditional shape measure correlation, indicate the left- and right handedness of the lateral primary leaflets, as these leaflets are mirror images of each other and therefore this measure describes the overall variation in leaf symmetry .
PC3 accounts for 3.8% of all variation, but has a strong correlation with aspect ratio, or the width divided by the length of the leaflet, with an R2 of 0.8 . PC3 therefore represents the roundness or narrowness of the leaflets, one aspect previously shown to be linked to fruit quality . The heirloom cultivars analyzed here were described as ‘potato leaf’, having broader, smoother leaves and typically lack the serration and lobes seen in other tomato varieties . However, despite this they had a wide range of leaf shape and size as illustrated in the leaf shape analysis . The classical potato leaf mutation is caused by a 5 kb transposable element inserted into the third exon of the C locus . To determine if this locus harbored mutations in the selected lines, a subset of the higher performing cultivars were selected for WGS analysis. Other mutations at the C locus have been described, and cause varied leaf shape . Fig. 5 shows the location of the mutations found in the C locus in these select lines. While the full Rider insertion could not be directly determined as the reference genome lacks this insertion, overhangs on reads in the third exon matched the Rider TE sequence . It is possible that different sizes and fragments of Rider are present in different cultivars, as the length and sequence of the overhangs varied . The identified Rider sequences were present in all but two of the sequenced lines, Prudens Purple and Glacier. No mutations were found in Glacier despite it having a rounder leaflets, although these were smaller in size with higher overall leaf complexity . Prudens Purple had a novel single base-pair substitution in the first exon outside the MYB/SANT conserved domain which results in the amino acid change P42R . We analyzed this mutation using the PROTEIN VARIANCEEFFECT ANALYZER , and found that it is predicted to be deleterious to the protein with a value of 8.454 , predicted to result in either a nonfunctional or partially functional protein . Based on leaf shape analysis, Prudens Purple shows a Potato Leaf like phenotype , although it differs slightly from the classical Potato Leaf shape seen in the reference allele and is reminiscent of the other mutations in C that have varying leaf shapes . These data demonstrate that different mutations in C, coupled with genetic background differences, may give rise to a range of leaf shapes seen among some of these cultivars.Pedigrees would probably inform the overall leaf shape in addition to the source of the C-locus mutations, but were not readily available. To elucidate relationships among these cultivars we used the WGS data from the select cultivars as well as WGS data obtained from the 150 Genomes Project to assemble a phylogeny and perform phylogenetic network analysis . The phylogeny includes several commercial cultivars, commercial heirloom cultivars, Solanum pimpinellifolium, and Solanum lycopersicum var. cerasiforme. ABC Potato Leaf does not appear to cluster with other Potato Leaf heirloom cultivars analyzed here . Stupice, Glacier, and Bloody Butcher are closely related in this phylogeny, corresponding to their often being listed as closely related in popular literature , and congruent with phenotypic similarities they exhibit in fruit size and leaf shape.
Bloody Butcher and Stupice both have the Rider insertion in the third exon at the C locus, while Glacier does not , suggesting the presence of other modifiers to leaf shape, which may have been selected for during the breeding of Glacier. A similar situation is seen in Prudens Purple , which is closely related to Jerry’s German Pink and Green Pearl. While Jerry’s German Pink and Green Pearl carry the Rider insertion at C, a novel single-base-pair substitution in the first exon leading to a deleterious effect on protein function is seen in Pruden’s Purple. Included in the clade is Silvery Fir Tree, big round plant pot anonPotato Leaf heirloom with very distinct leaf morphology. These cultivars come from a similar region of eastern Europe , and our WGS phylogeny supports a region specific breeding history. The relationships between the Potato Leaf and nonPotato Leaf heirlooms are not well resolved in our WGS-based phylogeny, probably as a result of close relationships between the cultivars and interbreeding. To identify any breeding history specifically related to the Potato Leaf Morph, we performed PHYLONETWORKS analysis using the WGS SNPs . We identified four hybridization events, relating to C mutants . It is noteworthy that Prudens Purple with a unique mutation at the Clocus is not part of this series of hybridization events . These hybridization events suggest a breeding effort for desirable traits associated with this morphology. In addition we also analyzed chromosomes 1, 6 and 12 and found unique hybridizations for all of these chromosomes . These data suggest that analyzing a much larger group of tomato cultivars for hybridization history could be very fruitful.When doing large-scale field studies, it is difficult to understand how all the collected data points relate to each other, and what the causative relationships are . We performed several key correlation tests between measured traits , but to test all traits we would need to perform 91 independent correlation tests. As such, to decipher how all the physiological and morphological traits measured related to each other, we performed PLS-PM using SMARTPLS3.0 , which gives weighted causative paths with bootstrapping for con- fidence and significance values. In PLS-PM, each LV is a composite value of its associated MVs and forms an outer model . The inner model consists of the connections between LVs, with R2 values indicating the degree to which each endogenous LV is described by the connections to it . Here, the only exogenous LV is leaf shape, as it has only its associated MVs and is descriptive of other LVs. Some LVs are described by other LVs within the model . When the value of a causative LV increases, the corresponding connected LVs change in accordance with their relationship with the causative variable. Similarly, in the outer model, changes in MVs reflect a change in their LV, and thus connect the outer model with MVs to the inner model of LVs. For instance, the MV PC3 has a negative correlation with the LV leaf shape , so that as the value of PC3 decreases, it reflects as a corresponding increase in LV leaf shape . This change is represented as an increase in the roundness of the leaf. This then corresponds to a positive change in yield , which is in turn a reflection of fruit biomass . The model indicates that photosynthesis has a strong positive influence on both fruit BRIX and vegetative biomass but has a negative impact on fruit yield. As photosynthetic rates increase , fruit BRIX increases, but at the sacrifice of yield, an inverse relationship which has long been known . Leaf shape has a negative relationship with vegetative biomass, which corresponds to the decreased leaf complexity with the Potato Leaf Morph . However, leaf shape has a strong positive influence on both fruit BRIX and yield , suggesting that leaf shape influences fruit quality as seen previously by Chitwood et al. . The effect of leaf shape on fruit quality does not work through leaf sugar, as this correlation was not significant. Our leaf sugar measurements were completed in the glasshouse, owing to the complexity of the chemical analyses required, and as such the model was tested without leaf sugar. While our work does not implicitly study mechanisms, the negative relationship between leaf sugar and fruit BRIX is of interest, and may provide some avenues for future research into the mechanisms underlying impact of leaf shapes on fruit quality in tomato. Fig. 6 displays the effect of each trait on the overall output of the plants . Leaf shape has no strong contribution to vegetative biomass.