Field evaluation of CRISPR-Cas9-driven brachytic and jointless pedicel tomatoes identifies an association between the high extra-large-sized fruit yield of the brachytic-mediated shortened tomato and the jointless2

Fresh-market tomato is one of the most important vegetables in the United States (US), and novel, cost-effective labor solutions are an important issue in current fresh-market tomato production. The shortened stem driven by the brachytic (br) and the jointless pedicel driven by the jointless2 (j2) are two important traits that would contribute to knowledge-based breeding; based on these traits, an appropriate plant architecture can be designed for ground cultivation systems that will ultimately enable mechanical harvesting. We aimed to evaluate the effect of CRISPR-Cas9-driven shortened plant architecture and jointless pedicel on the yield and horticultural performance of fresh-market tomatoes using a ground cultivation system in an open field. We conducted field trials during three successive seasons in 2020 and 2021 using the fresh-market tomato variety Fla. 8059 (a non-brachytic jointed pedicel tomato; BR/BR:J2/J2; wild-type) and its three different CRISPR-Cas9-driven mutants, a brachytic jointed pedicel Fla. 8059 (br/br:J2/J2), a non-brachytic jointless pedicel Fla. 8059 (BR/BR:j2/j2), and a brachytic jointless pedicel Fla. 8059 (br/br:j2/j2). Field evaluations confirmed that the total yield of the mutants was not significantly different from the yield of the wild-type. However, there was a significant association between the high extra-large-sized fruit yield of the br mutant and the j2 mutation. Field evaluations also revealed a clear positive impact of the br mutation on the high proportion of fruits laying on the raised plastic bed. Our results contribute to the optimization of fresh-market tomato architecture for developing shortened jointless pedicel varieties and the adoption of low input management practices.


Introduction
The CRISPR-Cas9 gene-editing system is an effective tool used to generate gene mutation(s) (Wang et al. 2016). The system is able to precisely change the DNA sequence of tomato genes while minimizing unwanted changes to other DNA sequences (Lee 2018). These characteristics provide advantages over traditional approaches [e.g., isogenic lines (ILs)] when researchers evaluate a trait of interest. Genetically, an IL can carry several to many different linked traits that may or may not affect agronomic performance when compared to its recurrent parent. Furthermore, in the absence of a completely known pedigree for breeding materials, it can be difficult to predict whether unlinked gene(s) might impact a trait of interest.
Almost all field-grown fresh-market tomatoes in the US are grown using labor-intensive practices such as staking and Communicated by Sung-Chur Sim.
1 3 tying and are harvested by hand. Dependable labor is scarce, and this is unlikely to change (United States Department of Agriculture Economic Research Service 2016; Florida Tomato Committee 2022). Consequently, the industry is in need of new production systems that utilize farm machinery to achieve and sustain higher levels of productivity and market value. Specifically, the development of a shorter plant architecture with stem-free fruit harvest via a jointless pedicel for the ground cultivation system that does not require the support of stakes and ties and is ultimately suitable for once-over mechanical harvesting, has significant advantages (Kemble et al. 1994a(Kemble et al. , 1994bFrasca et al. 2014;Florida Tomato Committee 2022).
The BRACHYTIC (BR; ) and JOINT-LESS2 (J2; Soyk et al. 2017) loci control plant height (i.e., the br allele shortens plant height) and formation of pedicel abscission joint (the j2 creates a jointless pedicel) in tomato respectively, and they have been actively adopted in freshmarket tomato breeding programs (North Carolina State University, 2022; University of Florida 2022). Here, we first evaluate the effects of the CRISPR-Cas9-driven brachytic and jointless pedicels on horticultural traits, including yield, in the large-fruited fresh-market tomato (also called round tomato or beefsteak tomato) variety Fla. 8059 using ground culture (i.e., without the support of stakes and ties). If field performance demonstrates the positive and/or negative effects of those mutated genes, breeders and geneticists can re-evaluate their br and j2 tomatoes, which can be further adapted to suit individual breeding aims.

Plant material
Fla. 8059 used in this study is an elite tomato germplasm that was registered and stored by the University of Florida (UF)/Institute of Food and Agricultural Sciences (IFAS) tomato breeding program (UF/IFAS, 2022). It is one of the two parental lines of the commercial hybrid (F 1 ) 'Tasti-Lee F1', currently in the US market (Bejo, Seeds, Oceano, CA; Scott et al. 2008). Fla. 8059 is a jointed pedicel (J2/J2), tall (wild-type) plant height (BR/BR), determinate (sp/sp; Pnueli et al. 1998) large-fruited fresh-market tomato (Scott et al. 2008). Targeted mutagenesis of J2 using the CRISPR-Cas9 system for Fla. 8059 was performed as previously described (Soyk et al. 2017). To generate a br/br:j2/j2 double mutant, the br/br mutant Fla. 8059 ) was crossed with the j2/j2 mutant in the T.G.L. laboratory. Potential double mutants were validated by whole genome sequencing as described in our previous study   (Fig. 1) and a greenhouse evaluation of crossing.

Phenotypic data collection
Field trials were conducted in three successive growing seasons during 2020 and 2021 in an open field at the UF's Gulf Coast Research and Education Center (Wimauma, FL, USA), where the Fla. 8059 was originally bred, as described in our previous study (Lee and Hutton 2021) with modifications in tying practice: plants were not tied on vertical stakes in raised plastic beds (71.12 cm in width) throughout the growing season (Fig. 2a). Given that current Florida field-grown fresh-market tomatoes are grown on raised plastic beds, US, all plants in the field trial were also grown on the raised plastic beds. All plants were evaluated for brachytic ( Fig. 2a; Lee et al. 2018 and jointless pedicel (Fig. 2b) phenotypes before harvest. During the spring of 2020, seed sowing in the greenhouse (S), seedling transplanting to the field (T), and fruit harvest (H) were performed on 22 January, 4 March, and 26 May, respectively. For the second growing cycle during the fall of 2020, S, T, and H were performed on 3 August, 16 September, and 15 December. Finally, during the spring of 2021, S was performed on 15 January, T on 1 March, and H on 3 June. In each growing season, the experiment was established as described in our previous study (Lee and Hutton 2021). Days to first flower data were collected and fruit collection and yield evaluations were performed as described in our previous study (Lee and Hutton 2021). The determination of fruits laying on the raised plastic bed ( Fig. 2c) was performed at the time of harvest by the same individual throughout the three seasons. The statistical significance level ( * p < 0.05, ** p < 0.01, *** p < 0.001) of the mean values for any two genotypes was based on the results of one-way analysis of variance (ANOVA) in conjunction with a two-tailed Tukey's HSD multiple comparison test, Welch's test, or two-tailed t-test. Error bars in the graph indicate the 95% confidence intervals (CIs).

Results and discussion
In order to be useful to the industry, a modified cultivar would have no negative effect on yield. To evaluate the effects of br and j2, the wild-type and mutants were grown in three successive seasons. The results showed that there were no significant differences in total fruit yield among genotypes in all seasons for which this study was conducted (Fig. 3a). Furthermore, there were no differences in the average weight of medium-sized fruits (between 5.715 cm and 6.428 cm in diameter; U.S. Department of Agriculture 2005) among all genotypes tested (Fig. 3d). Importantly, a clear positive impact (i.e., increase in yield at p < 0.05) of the j2 mutation on the extra-large fruit yield (fruit size > 6.985 cm in diameter) was found in a comparison between the br mutant and the br j2 double mutant across three seasons (Fig. 3e). Consequently, the extra-large fruit yield of the br j2 double mutant was not different from that of the wild-type in at least two seasons (2020 fall and 2021 spring). A reduction in the extra-large fruit yield ( * p < 0.05) compared to that of the wild-type was observed for the br The mutant (j2/j2, left panel) and wild-type jointed (J2/J2, right) pedicel phenotype. The joint is indicated by a red arrow. c Determination of fruits laying on the raised plastic bed. IN and OUT indicate fruits laying on the raised plastic bed and out of the plastic bed, respectively. An arrow indicates the limit of the plastic mutant across two seasons in 2020. However, in spring 2021, a wider variation across plant samples from the wild-type Fla. 8059 was observed, which resulted in CI error bar for the wild-type Fla. 8059 overlapping those of all other genotypes. Fruit size, especially large fruit size, is a particularly important trait for fresh-market tomato production, especially in the US, based on market demand and production systems (Florida Tomato Committee 2022). Thus, there is a demand for fruits classified as larger than medium in size, including extra-large fruits (U.S. Department of Agriculture 2005) (Florida Tomato Committee 2022). The presence of the genetic association between high extra-large-sized fruit yield of br-mediated shortened tomato and other genes was first investigated using sister lines derived from conventional crossing/selection methods, suggesting artificial selection contributes to commercially acceptable yield potential in br tomatoes . Therefore, the genetic association between the high extra-large-sized fruit yield of the br mutant and the j2 mutation found here can potentially influence breeding practices. The genetic and physiological mechanisms by which the j2 allele alters the yield of large fruits in the br-mediated shortened tomato should be investigated in future experiments. It is worth noting that fruit quality data from those CRISPR-Cas9-driven tomatoes are needed to determine if the br j2 double mutant can be rapidly incorporated into tomato improvement programs as fruit defects are often more severe in j2 tomatoes than in J2 tomatoes (Scott 2010). Current phenotyping challenges with regard to fruit quality have been discussed (Lee and Hutton 2021). For traits other than those mentioned above, the effect of the br, j2, or br and j2 mutations was unclear during yield evaluation (i.e., the results of ANOVA were inconsistent across three seasons) (Figs. 3b, 3c). Finally, none of the fruit yield traits evaluated in this study were negatively affected by the j2/j2. Our results showed that both the br/br:J2/J2 and br/br:j2/ j2 genotypes flowered faster than the wild-type in the spring of 2020, meaning that br affected flowering time (Fig. 4). However, such an effect was not consistently significant in the fall trial. Additionally, the proportion of colored fruits regardless of fruit size tended to be lower in genotypes with br/br than in BR/BR (Fig. 5). Data from more seasons is needed to determine if the fruit coloring effect is consistent across seasons. Furthermore, no negative correlation (i.e., less colored fruit) was observed between the br/br background and the j2 mutation across the three seasons.
There were significant differences in the proportion of fruits laying on the raised plastic bed across the three seasons between the genotypes with br/br and those without it. The genotypes with br/br showed a higher proportion of fruits laying on the bed compared to BR/BR genotypes, as expected from plants with shorter internode length. This result was supported by ANOVA and other statistical methods used to determine the significance of the difference between two genotypic means when the JOINTLESS2 gene was fixed for either of the homozygous alleles (J2/J2 or j2/ j2) (Fig. 6). A higher proportion of fruits laying on the bed in the genotypes with br/br indicates that the earliest fruit clusters were likely remained within the area of the raised plastic bed at the time of harvest. The elimination of staking and tying can significantly modify canopy microenvironments (e.g., light capture and humidity) and disease dynamics. Additionally, a higher concentration of fruit setting near the Fig. 4 Evaluation of days to first flower across two seasons. Letters A through D below each x-axis indicate the four different plant genotypes: A non-brachytic jointed pedicel Fla. 8059 tomato (wild-type); B CRISPR-Cas9-driven brachytic mutant; C CRISPR-Cas9-driven jointless2 mutant; and D brachytic jointless2 double mutant derived from a cross of B and C. All CRISPR-Cas9-driven mutations occur in the Fla. 8059 background. The n value represents the total number of plants for each genotype evaluated during each season; ns indicates that no significant difference (ANOVA, p > 0.05) was found between any genotypes. Statistically significant differences ( * p < 0.05 based on ANOVA in conjunction with a two-tailed Tukey's HSD multiple comparison test) are indicated between genotypes. Error bars indicate 95% confidence intervals All CRISPR-Cas9-driven mutations are occurred in a Fla. 8059 background. The n value represents the total number of plants for each genotype evaluated during each season; ns indicates no statistical difference (ANOVA, p > 0.05) was found between any genotypes. Statistically significant difference ( * p < 0.05, ** p < 0.01 based on ANOVA in conjunction with a two-tailed Tukey's HSD multiple comparison test) is indicated between genotypes. The Welch's test (p = 0.0530) was used for the comparison between A and B in 2021 spring trial. Additionally, a p value 0.0451 was obtained for the same comparison by the two-tailed t-test. Error bars indicate 95% confidence intervals Fig. 6 Evaluation of the proportion of fruits placed on the raised plastic bed across three successive growing seasons in 2020 and 2021. Letters A through D below each x-axis indicate four different plant genotypes: A non-brachytic jointed pedicel Fla. 8059 tomato (wildtype); B CRISPR-Cas9-driven brachytic mutant; C CRISPR-Cas9driven jointless2 mutant; and D brachytic jointless2 double mutant derived from a cross of B and C. All CRISPR-Cas9-driven mutations occur in the Fla. 8059 background. The n value represents the total number of plants for each genotype evaluated during each season; ns indicates that no significant difference (ANOVA, p > 0.05) was found between any genotypes. Statistically significant differences ( * p < 0.05 based on ANOVA in conjunction with a two-tailed Tukey's HSD multiple comparison test) are indicated between genotypes. The Welch's test was used for the comparison between A and B in the 2020 spring trial (p = 0.0156) and the comparison between C and D in the 2021 spring trial (p = 0.0609). Additionally, a p value of 0.0414 was obtained for the comparison between C and D in the spring of 2021 by the two-tailed t-test. Error bars indicate 95% confidence intervals 1 3 soil in plants could lead to greater disease incidence. Therefore, the current raised plastic bed practice will likely still be necessary with ground cultivation, particularly under the highly humid conditions in the southeastern US. Comparisons of the proportion of fruits laying on the raised plastic bed between the br and wild-type plants could contribute to tomato growers' adoption of new management practices.

Conclusions
This is the first study that shows the positive correlation observed between the high extra-large-sized fruit yield of the brachytic-mediated shortened tomato and the mutant allele of the JOINTLESS2 gene in a large-fruited freshmarket tomato variety. Given that we used CRISPR-Cas9driven gene-edited tomatoes, our data are more conclusive than those from traditional trait evaluation using breeding materials alone.