Significant IL’s were identified as those with a median transcript level .1 SD from the mean of all genes across all IL’s in the module.Differentially expressed genes with enriched transcript levels in laser-microdissected SAM/P0 versus P1 samples or hand-dissected samples of the SAM + P0-P4 or P5 sampled over developmental time were obtained from Chitwood et al. . Genes for which a differential expression call could be made were merged with detected eQTL using the merge function in R . For bootstrapping, cis- and transregulated transcripts were analyzed separately. Merged transcript abundance patterns were randomly permuted using the sample function against bin identity. For each test, 10,000 permutations were sampled to count the times that a particular transcript expression pattern was assigned to a bin more than the actual count. Resulting frequencies, representing a probability value, were multiple test adjusted using the BenjaminiHochberg method using p.adjust. Those bins with multiple test adjusted P values, 0.05 were analyzed further using visualizations created with ggplot2 .The rise of agriculture c. 7000 BC ensured a stable food supply, allowing human civilizations to develop and populations to grow . The challenge of feeding a growing population is exacerbated by climate unpredictability, with drought and temperature increases, leading to decreased crop yield . The narrow genetic base of most crops, combined with selection for performance under optimal conditions, has reduced the genetic variability in environmental stress responses, best indoor plant pots and the modern cultivars of tomato are no exception .
The wild relatives of tomato have the genetic ability to adapt to extreme habitats, and many heirloom cultivars also retain this ability as a result of directed breeding with wild species, and less selection for commercially valuable traits Heirloom tomatoes are defined as varieties, which have been passed down through multiple generations of a family . Improvement in tomato has focused on flowering, fruit traits, and disease resistance probably as a result of a perceived negative correlation between fruit size and sugar content . Thus, potential impacts of other factors on yield and fruit quality are relatively ignored . In a previous study by Chitwood et al. , a meta-analysis on a set of introgression lines linked leaf complexity and leaflet shape in tomato to fruit sugar content measured on the same lines by other researchers . This correlation showed that plants with complex and rounder leaflets also had increased fruit sugar content . Because leaves are the primary site of photosynthesis, it is possible that leaf shape changes may impact photosynthetic capacity and therefore result in different sugar content and yield in fruits. In addition to photosynthesis, sugar transport, and distribution to sinks are other potential sites of regulation in leaf function as source tissue. While sugar transport in plants is well described, distribution among different sink tissues is not fully understood . We analyzed tomato cultivars with varied yield and fruit quality, photosynthetic capacity, leaflet shape, and other vegetative traits and found that leaflet shape was strongly correlated to overall fruit quality assessed as a composite measure of BRIX and yield , with rounder leaflets positively correlated with higher BY values.
Photosynthesis, on the other hand, had a negative correlation with yield. Based on our analysis, leaf shape seems to play an important role in the distribution of photo assimilates. Additionally, we performed phylogenetic network analysis on 23 cultivars, including eight identified as having the rounder Potato Leaf Morph , known to be caused by a mutation in the C-locus , to determine their breeding histories and identify any potential selection for this trait.As photosynthesis is the primary source of sugar production in plants, a time course for photosynthesis, stomatal conductance , PARi and ɸPS2 was performed on all cultivars using a LI-6400XT . Additionally, we analyzed leaf sugar and vasculature for these lines in glasshouse conditions . Fig. 3 shows photosynthesis by gst and the trend is similar among all cultivars, with photosynthesis reaching a maximum rate after 0.6–0.8 gst, which is a standard response curve . Optimal photosynthetic performance regardless of light conditions has been observed in a forest tree species , and we saw the same at different levels of the canopy. Fig. 3 shows the PARi and ɸPS2. ɸPS2 had an overall downward trend across the season, as the amount of light used in photosystem II decreased with age. This corresponds well with the increase in vegetative biomass , and the increased PARi . Individual leaf contribution to overall photosynthesis, and therefore photons used in PSII, decreases as the leaf area of the plant increases. Because of this trend, we calculated the whole-plant photosynthesis, as photosynthesis/area, corrected for the green area visible in overhead images, and gst to capture the total rates and not just specifically measured leaves . The trend is linear for photosynthesis vs gst when the whole plant-exposed green area is incorporated, compared with Fig. 3 where the trend is more logarithmic. This corresponds to our previous observation that individual leaf contribution decreases as the total vegetative mass increases.
Leaf shape was shown to be strongly correlated with fruit BRIX and sugar accumulation in a metaanalysis 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 R 2 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, blueberry container size 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 VARIANCE EFFECT 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 breedingof 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, a nonPotato Leaf heirloom with very distinct leaf morphology. These cultivars come from a similar region of eastern Europe , and our WGS phylogeny supports a regionspecific 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 . 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 confidence 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 R 2 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.