The introgression region was split into 18 loci and a representative primer pair was made for each locus and these were sequenced using GT-Seq . Whether the region was from M82 or S. pennellii was determined by the presence of SNPs and can be found in Table S8. All F3 and F4 plants were grown in the greenhouse and compared to M82. The bHLH032 promoter region in S. pennellii is 200-bp larger than that of M82. This region was sequenced to determine if the lines contained the M82 or S. pennellii version of the gene and all data binned by genotypes determined by sequencing. Leaf shape for the select lines showed that those line containing the S. pennellii version of the bHLH032 had rounder leaves while those with the M82 version were closer to the M82 control although still significantly different . Likewise, the vascular density showed that those plants which had the S. pennellii bHLH032 had lower vascular density than those with the M82 version, which had a density the same as the M82 control plants . For both leaf shape and vascular density the heterozygous plants were more like the S. pennellii homozygous bHLH032 plants than the M82 containing plants . The BY for the S. pennellii and M82 bHLH032 containing plants was higher than the control M82 plants, suggesting that other parts of the introgression were still present and likely played some role in the increase in BY . The plants heterozygous for bHLH032 had a BY similar to the M82 control plants but did have a wider range of values. The trait measurements and the genotype of the bHLH032 gene present suggested that there was a likely role for bHLH032 in the regulation of BY through modulation of leaf shape and vasculature. To confirm this possible correlation, blueberry in pot we made bHLH032 CRISPR mutants in the M82 background.
Two RNA guides were made within the bHLH032 gene for CRISPR mutation, one in the first exon on the 5’ end of the bHLH domain and the second at the 5’ end of the second exon . We obtained ten CRISPR lines, however only the second guide in exon 2 was successful in creating mutations. Of these ten lines we chose lines 05, 06, and 10 to carry forward for further characterization. bHLH032-05 has a one base pair deletion near the guide site that creates a stop codon and truncates the gene early Similarly bHLH032-10 has a two base pair deletion which also creates an early stop codon, but it is located 20 amino acids further downstream than that found in bHLH032-05, and it alters the amino acid sequence between the guide site and the truncation stop codon . bHLH032– 06 was unique from the other mutations as it had a three base pair deletion which created a two amino acid deletion and one amino acid insertion . The T1 plants from these lines were grown up in the greenhouse and their leaf shape and vasculature characterized. All three lines had significantly rounder leaflets than that found in M82 and bHLH032-06 and bHLH032-10 had significantly reduced vasculature . bHLH032-05 did not have significantly lower vasculature but it was lower than M82 following the same trend as the other mutants . All three lines were carried forward after confirming a phenotype similar to BIL 260 , however bHLH032-05 also had fruiting/seed set deffect that caused it to have difficulty making fruit or seed. Because of this phenotype we decided not to continue with bHLH05 progeny and work only with bHLH032-06 and bHLH032-10, neither of which had seed set or fruiting issues. For bHLH032-06 and bHLH032-10 the T3 and T4 plants were grown in the greenhouse for characterization of BY along with the leaflet shape and vascular density. These plants are in the process of the being sequenced to determine if they have the same mutation as the T0 parents, however a few plants have been genotyped and they will be discussed here. bHLH032-10 lines that have been genotyped so far contain the two base pair deletion resulting in a stop codon /10.
Leaf shape analysis shows that none of these lines are significantly different from M82, however homozygous plants had significantly lower vascular density than that found in M82 or the heterozygous plants . Figure 5d shows example leaflets and vascular images all three genotypes, and while the homozyogous leaflet trends towards rounder than M82 it is still very similar. The vascular density of homozygous mutant plants was much lower, replicating the phenotype seen in BIL 260, despite not having round leaflets . The BY for the homozygous mutant plants was significantly higher than either M82 or heterozygous plants, indicating that the previously identified correlation between decreased vascular density and the total output of the plants holds in these lines . Thus, the pattern of inverse relationship between vascular density and BY seen in the original BIL 260 plants was replicated in the bHLH032-10 CRISPR plants .The primary focus of crop improvement, specifically yield, has been on improving photosynthesis as photosynthetic rates tend to correlate strongly with total biomass . However, more recent studies have shown that photo assimilates, when available in large quantities as would be found with high photosynthesis, are primarily converted to vegetative biomass in tomato as well as rice . Other characteristics such leaf shape were explored here as well as in previous studies, that revealed a potential correlation between BY and the shape of leaflets found in tomato . However, while BIL 260 and sub IL 4-3-4 showed a marked increase in BY and yield respectively, other lines such as BIL 267 and 139 also had rounder leaflets than that found in M82 but did not show an increase in BY . While models such as the PLS-Path Model seen in Rowland et al. indicate a direct relationship between leaf shape and BY, there are likely other genetic or physiological factors that confound this correlation. Photosynthetic rates in BIL 260 were slightly elevated over M82 and sub IL 4-3-4 which suggests that an increase in photosynthesis, as has been previously suggested, could result in increased BY as seen here . However, the large variability in photosynthesis rates creates some doubt as to the efficacy of this claim, as the BIL 260 photosynthetic rate is 27 µmols m-2 s -1 , compared to that for M82 at 25 µmols m-2 s -1 . The opposite, however, appears to be true with lower photosynthesis, and therefore lower photo assimilates, resulting in lower yields and BRIX values . All these data together suggests that while photosynthesis is necessary to increase BY there may well be a hard cap on how much it can improve crop output. If photosynthesis itself does not improve BY beyond a certain point, then research should investigate other areas such as sugar transport and storage. The sugar and starch concentrations in the leaves of BIL 260 and sub IL 4-3-4 show an increased mobilization of starch during night hours but no coinciding increase in sugar, unlike M82 where the same starch mobilization led to an increase in leaf sugar during the same time period . This points to mobilization and export of sugar as a likely reason for the increased BY seen in BIL 260 and sub IL 4-3-4. Leaf sugar in BIL 260 is lower than that for both other lines during the 1 to 5 am time period, plastic planters wholesale and correlates nicely to the fact that BIL 260 has a higher BY than either other line . How leaf shape relates to sugar export could be tied to the vascular density found in the leaves, as leaf shape and vasculature are closely linked during development .
The vascular density of BIL 260 is significantly lower than that of M82 or sub IL 4-3-4, indicating that a lower amount of vasculature per mm2 results in increased sugar export, a trend seen in previous studies . How decreased vascular density increases export and what is regulating these changes remain open questions. To address these questions, we performed WGS and identified three transcription factors introgressed from S. pennellii unique to BIL 260 . Of these three only bHLH032 showed up as DEG in RNA-seq data for SAM, mature leaves, and young leaves . Analysis of gene expression using dimensionality reduction methods, showed the Cluster 19 from young leaves was enriched in sugar related genes and contained the bHLH032 transcription factor. This suggests bHLH032 may be a regulator of sugar related metabolism, similar to the mobilization and export processes seen previously . bHLH032 is peripherally connected to the network in M82, which means it acts as an input or regulator to the network, working through a gene similar to the Arabidopsis genes BDX/DGR2 which regulate leaf and vascular development . bHLH032 is not present in the BIL 260 network, likely due to low expression levels precluding it from being an input into the network, and causing alterations to vascular development, leaf development through the tie to vascular development, and subsequent sugar export and processing .To identify if bHLH032 was an important part of this pathway we generated back crosses of BIL 260 with M82 to break up the introgression region, and indeed those lines in the F3 population that contained the S. pennellii version of bHLH032 had rounder leaves, lower vasculature density, and higher BY . These data strongly suggest that bHLH032 is a master regulator of these morphological characteristics which lead to increased BY. In fact, the CRISPR knockout mutants of bHLH032 in the M82 background show a similar trend, with lower vascular density and increased BY . Interestingly, the leaf shapes of these CRISPR lines were not different from M82, suggesting that the mutation in bHLH032 separated the normal link between leaf shape and vasculature, and pointing to vascular density as the driving factor behind increased BY . The decrease in bHLH032 causes changes both leaf shape or vasculature which correlate with BY directly . This also breaks the long known trend of BRIX and yield being inversely correlated to allow for increase in both. Thus bHLH032 could be an important regulator of BY, opening an avenue for tomato improvement. It and other transcription factors functioning in the regulation of leaf shape and vascular density provide a target for future research to continue tomato improvement.Gas exchange measurements were made in the field on attached leaves after the plants had recovered from transplanting. For the 2015 field season measurements were made weekly from week 10 through week 13 , week 14 through 17 , and 18 – 21 , on approximately 5 plants per week. For the 2014 and 2016 field seasons terminal harvest was performed on 60, and 20 plants, respectively. For all field seasons measurements were made on leaves from the upper and lower portion of the plants to eliminate positional bias within the plant and measured for three leaves per plant. The A , gst , transpiration, and ɸPS2 of a 6 cm2 area of the leaflet was measured using the LI-6400 XT infra-red gas exchange system , and a fluorescence head . The chamber was positioned on terminal leaflets such that the mid-vein was not within the measured area. Light within the chamber was provided by the fluorescence head at 1500 µmol m-2 s -1 PAR, and the chamber air flow volume was 400 µmols s-1 with the chamber atmosphere mixed by a fan. CO2 concentration within the chamber was set at 400 µmols mol-1 . Humidity, leaf, and chamber temperature were allowed to adjust to ambient conditions, however the chamber block temperature was not allowed to exceed 36°C. Measured leaflets were allowed to equilibrate for 2 to 3 minutes before measurements were taken, allowing sufficient time for photosynthetic rates to stabilize with only marginal variation.For the 2015 field season three plants per cultivar were destructively harvested each week. The final yield and fresh vegetative weight of each plant harvested, was measured using a hanging scale in the field. Five leaves were collected at random from the bottom and top of the plant to capture all canopy levels, and approximately nine fruit were collected for BRIX measurements.