FRUIT WEIGHT VARIATION IN TOMATO BREEDING LINES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1008344
• Project No.: GEO00780
• Project Start Date: Nov 1, 2015
• Project End Date: Oct 31, 2020

Project Director
van der Knaap, ES.

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
Horticulture

Non Technical Summary
SUMMARY:Factors that determine economic success of vegetable varieties are the weight and shape of the produce. Based on appearance, consumers select the type of produce which is influenced by the culinary purpose of the product. Additionally, producers require dimensions of the product that are compatible with mechanical harvesting and processing while growers prefer large produce as this often relates to higher yields and market value.This Hatch projectaims to identify and functionally investigatethe genesthat lead to tomato improvement including fruit quality.We will usecontemporary tomato breeding lines for crop improvement. The main focus will be on the fresh market types: beefsteak, plum and grape; as well as processing types.

Animal Health Component

0%

Research Effort Categories

Basic

10%

Applied

50%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	0120	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0120 - Land;

Field Of Science
1080 - Genetics;

Keywords

tomato

fruit weight

crop improvement

Goals / Objectives
REASONS FOR UNDERTAKING THE WORK:Factors that determine economic success of vegetable varieties are the weight and shape of the produce. Based on appearance, consumers select the type of produce which is influenced by the culinary purpose of the product. Additionally, producers require dimensions of the product that are compatible with mechanical harvesting and processing while growers prefer large produce as this often relates to higher yields and market value.Genes that control variation in tomato fruit morphology are prime targets of crop improvement using traditional breeding strategies and genome editing approaches. Knowledge gained from genetic studies that led to the cloning of these genes has offered insights into developmental plant processes that can be further manipulated for crop improvement. With more precision we can soon work towards fruit size increases and altered shapes as demanded by the different market classes. On the other hand, larger tomatoes don't always equate to improved fruit quality or plant characters. As we learn more about the effect of the fruit weight and shape alleles in different genetic backgrounds, we can select combinations of genes to ensure the appropriate fruit dimensions while also improving other plant characteristics and fruit quality.Our laboratory focuses on identifying novel genes that impact the fruit size and shape of tomato. Much of this research is funded through competitive funding sources. With this Hatch project, we aim to translate the findings to tangible crop improvement using contemporary tomato breeding lines for improvement. The main focus will be on the fresh market types: beefsteak, plum and grape; as well as processing types.OBJECTIVES: Determine the allele distribution of fruit weight and shape genes in contemporary tomato breeding germplasm.Exchange the natural fruit weight and shape alleles in relevant tomato breeding lines and evaluate fruit morphology and quality, plant horticultural traits, ripening, physiological disorders, and nutritional deficiencies.Employ genome editing using CRISPR-Cas9 technology to edit promoter and coding regions of known tomato fruit morphology genes. Evaluate the resulting plants for changes in fruit weight or shape.

Project Methods
PROCEDURES:I Determine fruit weight and shape gene allele distribution in modern tomato germplasm. Previously, we showed that the three known fruit weight genes are fixed in the modern tomato germplasm with the exception of the grape and cherry tomato types and a distribution of shape alleles that varies based on the market class (Blanca et al., 2015) (van der Knaap unpubl). With the discovery of additional genes, we will develop molecular markers and screen the same germplasm as was used in the previous study.II Exchange fruit weight and shape alleles in grape, beefsteak, plum and processing tomatoes. We will introgress the alternate alleles in the different genetic backgrounds. Over the course of 3-5 years, we will backcross these lines and evaluate fruit morphology, plant architecture and fruit quality traits of the newly developed lines every summer during this process. The breeding lines will also be evaluated by our tomato breeder collaborators.III Modify expression or the protein sequence of the fruit weight genes using CRISPR-Cas9 technology (Belhaj et al., 2015). The natural alleles provide insights into how fruit morphology can be further altered without creating extreme phenotypes. For example, overexpression using the strong 35S promoter may lead to unacceptable phenotypes whereas knock out expression of certain genes may result in lethality. The changes in promoter sequences as indicated by the natural alleles could further be exploited and lead to slight changes in expression that would lead to further crop improvement. Moreover, once the edits are made, the remaining transgene will be removed by backcrosses to wild type, thus leaving only a small mutation in the tomato genome (the region that was edited). For the first 2 years, we plan to create CRISPR-Cas9 constructs targeting the promoters of FW2.2, FW3.2 and SUN as well as the protein sequence of FW11.3. The constructs will be transformed in the relevant breeding lines and edited alleles will be identified by PCR and sequencing. To remove unintentional mutations resulting from the CRISP-Cas9 technology, the lines will be backcrossed to untransformed tomato. These crosses will also ensure the removal of the original transgene. In year 4-5, the resulting lines will be evaluated for changes in fruit morphology and for additional horticultural traits, including their potential as prebreeding germplasm for crop improvement.

Progress 11/01/15 to 10/31/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Swee accomplishments in report submitted for period ending 9/30/2020

Publications

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Plant breeders and plant geneticists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post doc Qiang Li has been trained on this project. Last year he was promoted to a faculty position in China. How have the results been disseminated to communities of interest?Qiang Li has presented his work at the Plant and Animal Genome meeting in Jan 2020. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Regarding aim 3, we accomplished in the last year of this project the analyses of the M9 promoter mutations in VIR1011. Unfortunately, the data were inconclusive because the fruit weight in the controls varied too much. Instead, we used the same CRISPR construct to target the fw3.2 promoter in the cultivated tomato M82. The preliminary data showed that the same mutation in LA1589 also increased fruit weight in M82. The replication of this experiment is currently underway in Blairsville GA and results are expected by August 2021.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Li, Q., M. Chakrabarti, N.K. Taitano, Y. Okazaki, K. Saito, A.M. Al-Abdallat, E. van der Knaap (2020) Differential expression of SlKLUH controlling fruit and seed weight is associated with changes in lipid metabolism and photosynthesis related genes. J Exp Botany 72, 1225-1244.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Li, Q., M. Sapkota, E. van der Knaap (2020) Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement. Hort Research 7, 36.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The world is experiencing rising demands for crop production, however, the current breeding effort and the rate of crop yield increase are likely unable to keep up with the ever-increasing food demands. Genetic diversity is the key source for crop improvement. CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) holds great promise for expanding genetic diversity and accelerating crop improvement. SlKLUH, also known as FW3.2, is a major positive regulator controlling fruit mass. Some orthologs are known to regulate organ size in other crops. However, limited genetic variations have restricted the use of KLUHs to improve yield. Here, we design a single gRNA targeting a cis-regulatory region including a natural genetic change, named M9 SNP, which is highly associated with the fruit weight, in the promoter of SlKLUH. A total of 21 and 13 mutant alleles were generated in wild type tomato LA1589 (S. pimpinellifolium) and semi-cultivated tomato VIR1011 (S. lycopersicum var. cerasiforme), respectively. These mutant alleles provide a range of fruit weight variation in both backgrounds. In LA1589 background, m2+4bp and m3+1bp homozygote significantly increase fruit weight by 10.7% ~ 15.7% and 8.7% ~16.3%, respectively. Chi-Square test indicated that plant homozygous for m2+4bp and m3+1bp showed significant difference in fruit number of each fruit weight quartile compared to LA1589. Notably, compared with LA1589, m2+4bp and m3+1bp showed a significant decrease or the rend of decrease in the proportion of smaller fruits in all replicates. The effects of mutant alleles on fruit weight in VIR1011 background need to be confirmed with two more biological replicates in 2020. This approach allows generate novel cis-regulatory alleles with a range of quantitative effects. Our findings also provide beneficial variants that can be introduced into elite tomato germplasm by precise genome editing, leading to fast-forward crop improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The post doc working on this project has been presenting his work at conferences. How have the results been disseminated to communities of interest?the results are currently written up for publication. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The world is experiencing rising demands for crop production, however, the current breeding effort and the rate of crop yield increase are likely unable to keep up with the ever-increasing food demands. Genetic diversity is the key source for crop improvement. CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) holds great promise for expanding genetic diversity and accelerating crop improvement. SlKLUH, also known as FW3.2, is a major positive regulator controlling fruit mass. Some orthologs are known to regulate organ size in other crops. However, limited genetic variations have restricted the use of KLUHs to improve yield. Here, we design a single gRNA targeting a cis-regulatory region including a natural genetic change, named M9 SNP, which is highly associated with the fruit weight, in the promoter of SlKLUH. A total of 21 and 13 mutant alleles were generated in wild type tomato LA1589 (S. pimpinellifolium) and semi-cultivated tomato VIR1011 (S. lycopersicum var. cerasiforme), respectively. These mutant alleles provide a range of fruit weight variation in both backgrounds. In LA1589 background, m2+4bp and m3+1bp homozygote significantly increase fruit weight by 10.7% ~ 15.7% and 8.7% ~16.3%, respectively. Chi-Square test indicated that plant homozygous for m2+4bp and m3+1bp showed significant difference in fruit number of each fruit weight quartile compared to LA1589. Notably, compared with LA1589, m2+4bp and m3+1bp showed a significant decrease or the rend of decrease in the proportion of smaller fruits in all replicates. The effects of mutant alleles on fruit weight in VIR1011 background need to be confirmed with two more biological replicates in 2020. This approach allows generate novel cis-regulatory alleles with a range of quantitative effects. Our findings also provide beneficial variants that can be introduced into elite tomato germplasm by precise genome editing, leading to fast-forward crop improvement.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: In vivo promoter engineering (IPE) to enable fruit weight variation for crop improvement

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Plant breeders and geneticists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post doc Qiang Li has been trained on this project How have the results been disseminated to communities of interest?Qiang Li has presented his work in a poster presentation at the UGA Plant Center retreat What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? For 2018, the major accomplishments were for objective 3. We created using CRISPR/Cas9 a range of promoter mutations in the wild-type LA1589 and semi-cultivated tomato VIR1011 carrying the wild type allele of fw3.2. To date, 23 and 13 mutant alleles were identified in LA1589 and VIR1011, respectively.Preliminary analysis in LA1589 indicated that larger fruit weight mutant alleles were obtained and there is quantitative variation in fruit weight among different homozygous mutants. Further analysis of fruit weight showed that different mutations impact fruit weight of the inflorescence position on plants and fruit position on inflorescence. In addition, 3 conserved motifs were identified in the promoters of SlKLUH homologues in other crops, providing clues for other crop yield improvement via CRISPR/Cas9. Therefore, this study is not only suitable to generate novel quantitative variation that can accelerate crop yield improvement, it can also provide insights into application of CRISPR/CAS9 for functional validating QTL in non-coding region in plants.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Modifying cis-regulatory elements to enable fruit weight variation for crop improvement. Qiang Li, UGA Plant Center retreat September 2018

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Qiang Li is working on thise project and he has attended the CROPS meeting in Huntsville Alabama in May 2017. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? For 2017, the major accomplishments are: Objective 3. We successfully set up tomato transformation in our lab.In brief, tomato seeds were germinated on 1/2 MSO medium after sterilization with 70% ethanol (1min) and 50% bleach (20min). After 7-8 days culture, the intermediate cotyledons were excised into small slices of approximately 0.5-1 cm and then the explants were cultured on 2ZI medium (MS+2mg/L zeatin+0.5mg/L IAA). After 2 days, the explants were co-cultivated with A. tumefaciens strain LBA4404 harboring the CRISPR/Cas9 plasmid for 15-20min and then the explants were transferred to 2ZIA medium (MS+2mg/L zeatin+0.5mg/L IAA+200uM acetosyringone). Following a 2d co-cultivation, the cotyledon segments were transferred to a selective regeneration medium (2ZIK, MS+2mg/L zeatin+0.5mg/L IAA+75mg/L Kanamycin+ 300mg/L Timentin ) for 2 weeks and then transfer explants to new 2Z selection medium with 0.1mg/L IAA, 75mg/L Kan and 300mg/L Timentin (2Z0.1IK medium) every 2 weeks. When shoots were approximately 1.5 cm tall, they were transferred to a selective rooting medium that contained 0.1mg/L IAA, 60mg/L Kanamycin and 300mg/L Timentin and only well-rooted plants were transferred to the greenhouse. About 80 seeds were sown on 03/09/2017, 146 explants were obatined on 03/16/2017. The first shoot was transferred to rooting medium on 05/04/2017. Total 18 transgenic lines with 10 mutant alleles were obtained. The transformation efficiency was 18/146=12.3%. For the CRISPR/Cas9 construct targeting the fw3.2 QTL region (M9 SNP), one single-guide RNA (sgRNA, GAGCAAGATCGGGAGAGCG) was designed using the CRISPR-P tool (http://crispr.hzau.edu.cn/CRISPR/). To create the mutations in the fw3.2 promoter with the goal to impact fruit size, we generated agRNA. ThePCR reaction was performed with a forward primer containing the gRNA sequence (tgtggtctcaATTGGAGCAAGATCGGGAGAGCGgttttagagctagaaatagcaag) and a reverse primer (tgtggtctcaAGCGTAATGCCAACTTTGTAC), using the plasmid pICH86966::AtU6p::gRNA_PDS (Addgene plasmid 46966) as template. The gRNA was cloned into the level 1 vectors pICH47751::AtU6p::sgRNA. Level 1 construct pICH47732-NOSpro::NPTII (Addgene plasmid 51144), pICH47742-35S:Cas9 (Addgene plasmid 49771) and pICH47751::AtU6p::sgRNA were then assembled in the binary Level 2 vector pAGM4723 (Addgene plasmid 48015) as described (Weber et al., 2011).The level2 vector pAGM4723-35S:Cas9-NOSpro::NPTII-AtU6p::sgRNA was transferred into Agrobacterium tumefaciens strain LBA4404 by electroporation. We have generated 9 promoter mutation alleles in the plants. We are selfing the lines to generatehomozygous lines.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: THE MOLECULAR and GENETIC BASIS of BLOSSOM END ROT IN TOMATO. Yasin Topcu and Esther van der Knaap. Plant Center retreat poster October 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Molecular genetic control of fruit weight in tomato. Esther van der Knaap, China Agricultural University, August 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Molecular genetic control of fruit weight in tomato. Esther van der Knaap, Plant Genomics in China XVIII, August 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Controlling plant morphology: the sizes and shapes of tomato fruits as a model. Esther van der Knaap, INRA Versailles, France. March 2017

Progress 11/01/15 to 09/30/16

Outputs
Target Audience:The target audience of this project are tomato breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post doc and staff are beingtrained in tomato transformation. How have the results been disseminated to communities of interest?Representatives of the laboratory have presented the data at the National Association of Plant Breeders meeting as well as several local venues. What do you plan to do during the next reporting period to accomplish the goals?Our major goals are to set up efficient tomato transformation protocol in the laboratory.

Impacts
What was accomplished under these goals? Objective 1: We sought to determine the allele distribution of the fruit weight and shape genes in contemporary tomato breeding germplasm. The fresh market plum types that were evaluated from the public breeding programs in North Carolina, New York and Florida are fixed for the large alleles of fw2.2, fw3.2 and fw11.3. They are mostly wild type for lc and entirely wild type for ovate, sun and fas. The globe/slicing types from Florida and North Carolina are fixed for the large alleles of fw2.2, fw3.2 and fw11.3 as well as the mutation in lc conferring increased locule number. These tomatoes are wild type for ovate, sun and fas. The grape fresh market type from North Carolina carries the mutations in fw3.2 and ovate, and is wild type for all other loci. The processing lines from Ohio, New York and the Heinz OH germplasm are fixed for the large alleles of fw2.2, fw3.2 and fw11.3. The lines are wild type for all fruit shape loci. Overall, the data show that the large fruited tomato germplasm carries the cultivated alleles of the three fruit weight genes. The flatter shape and large size of the globe/slicing germplasm may be due to the lc mutation whereas the shape of the small grape tomato is the result of a mutation in ovate. There is little variation in shape and size within each of the market classes at both the genetic and fruit morphological level. Objective 3: The main aim is to employ CRISPR Cas9 technology to edit promoter and coding regions of known tomato fruit morphology genes. Our main focus this year has been to establish a reliable tomato transformation protocol in the laboratory. Once this protocol has been developed, we will start to target the promoter and coding regions of genes of interest.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Genetic Approaches to Study Tomato Fruit Development. Zavalla University, Rosario, Argentina, Oct 13, 2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Genetic Approaches to Study Tomato Fruit Development. Seminar in Genetics UGA. Athens GA, USA, Jan 27, 2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Distribution of tomato size and shape alleles in modern germplasm. UGA Plant Center Retreat, Helen, GA, Oct 27-28. Alexis Ramos.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Distribution of tomato size and shape alleles in modern germplasm. National Association of Plant Breeders meeting in Raleigh, NC, Aug 12-18. Alexis Ramos.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Distribution of tomato size and shape alleles in modern germplasm. UGA Institute of Plant Breeding Genetics and Genomics retreat, Athens, GA, May 19-20. Alexis Ramos.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: The control of tomato fruit weight by fw2.2, fw3.2 and fw11.3 in near isogenic lines in the tomato wild relative background. International Plant & Animal Genome XXIV Conference in San Diego CA USA, Jan. 9-13. Xiaoxi Liu.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: MANTIS: A Reliable Microfluidic Reagent Dispenser for High-Throughput SNP Genotyping in tomato. UGA Plant Center Fall Retreat, Helen GA USA, Oct. 27-28. Eudald Illa Berenguer.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Genetic Fruit Weight Regulation in 3 Distinct Tomato (Solanum lycopersicum L.) Market Classes. International Plant & Animal Genome XXIV Conference in San Diego CA USA, Jan. 9-13. Eudald Illa Berenguer.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Illa-Berenguer, E., J. Van Houten, Z. Huang, E. van der Knaap. 2015. Rapid and reliable identification of tomato fruit weight and locule number loci by QTL-seq. Theor Appl Genet 128: 1329-1342.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Blanca, J., J. Montero-Pau, C. Sauvage, G. Bauchet, E. Illa, M.J. D�ez, D. Francis, M. Causse, E. van der Knaap, J. Ca�izares. 2015. Genomic variation in the tomato, from wild ancestors to contemporary breeding accessions. BMC Genomics 16:257.