Fine-mapping and characterization of a novel fruit shape locus in tomato

(Solanum lycopersicum L.)

*Edgar Sierra-Orozco, Reza Shekasteband, Samuel Hutton; University of Florida, Gulf Coast Research and Education Center;

Eudald Illa-Berenguer, Esther van der Knaap; University of Georgia, Department of Horticulture

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Size Shape

Wild ancestor

Very small, weighs a few grams

Round

Modern tomato

Weighs up to 1 Kg (nearly 1000-fold increase)

round, oblate (flat), long, pear-shaped, torpedo-shaped, bell pepper-shaped, etc.

Source: Tanksley, 2004.Source: Grandillo et al., 1999; van der Knaap et al., 2002; Tanksley, 2004

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INTRODUCTION RESULTS CONCLUSIONS

The tomato fruit…

~ 30 QTLs account for most of the variation in fruit shape and size (Tanksley, 2004).

Source: Rodriguez et al., 2011

Explained to a large extent by: SUN, OVATE, LC and/or FAS (Rodriguez et al.,

2013; Azzi et al., 2015).

Mutation(s) in six loci seem to have been essential in the transition from wild to modern tomato (fw2.2).

Shape

Size

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The genetics of fruit shape and size in the domestication process

INTRODUCTION RESULTS CONCLUSIONS

• There is still some fruit variation

• The Flat vs the Globe tomatoes

• Variation in:• Fruit shape

• Fruit size

• Fruit quality

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Flat and globe shapes in fresh-market tomatoes (large-fruited)

INTRODUCTION RESULTS CONCLUSIONS

HYBRIDS are developed in commercial fresh-market tomatoes (Scott, 2004; Scott et al., 2006;

Gardner, 1990; Gardner, 1992; Gardner, 2000; Panthee and Gardner, 2011)

Shape Flats Globes

Quality*

Size

*Cracking, bursting and weather check

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The dilemma of flats and globes

INTRODUCTION RESULTS CONCLUSIONS

What are the genetics of flat vs globe in fresh market tomatoes?

• Variation of major fruit shape and size genes is believed to be smaller• Preliminary work showed that FW2.2,

FW3.2, FW11.3, LC and FAS are fixed in several inbred lines of the breeding program of UF

• Knowledge of the location of the gene(s) may improve cultivar development efforts.

https://psmag.com/environment/why-you-should-be-scared-of-someone-stealing-your-genome-58082

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INTRODUCTION RESULTS CONCLUSIONS

• To fine-map a locus that underlies segregation of fruit shape in fresh-market tomato germplasm

• To characterize its effects on fruit shape, size, and other plant morphology traits

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Objectives

INTRODUCTION RESULTS CONCLUSIONS

RESULTS

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INTRODUCTION RESULTS CONCLUSIONS

1. Mapping of the fruit shape locus

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INTRODUCTION RESULTS CONCLUSIONS

A single locus in Chromosome 12 appeared significantly associated with shape

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CIM analysis of the SNP array

INTRODUCTION RESULTS CONCLUSIONS

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Flanking SNPs at 714,799 and 903,522 bp

(Genome Build SL3.0)

CIM analysis of the SNP array, Chromosome 12

INTRODUCTION RESULTS CONCLUSIONS

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2.5 Mbp

CIM of the upper arm of Chr. 12

INTRODUCTION RESULTS CONCLUSIONS

1 Mbp

Recombinant F4 and F5 for the locus in the upper arm of chr. 12, 1 Mbp region

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No. Plants M1 M2 FS M4 M5 Shape

F4

6 G/G G/G F/F F/F F/F Flat3 G/G G/G G/G F/G F/G Globe

11 G/G G/G G/G G/G F/G Globe18 F/G F/G G/G G/G G/G Globe8 F/F F/F G/G G/G G/G Globe8 F/F F/F F/F G/G G/G Flat

F5

10 F/F F/F G/G Flat55 F/F G/G G/G Globe1 F/F F/G G/G Flat1 G/G G/G F/G Globe1 G/G F/G F/F Flat

11 G/G G/G F/F Globe55 G/G F/F F/F Flat

400 Kbp region

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Germplasm surveyINTRODUCTION RESULTS CONCLUSIONS

2. Characterization of fruit and plant morphology

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INTRODUCTION RESULTS CONCLUSIONS

Fruit characterization

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Globe (G/G)Heterozygous (F/G)Flat (F/F)

INTRODUCTION RESULTS CONCLUSIONS

Average weight of fruits

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F/F

F/G

G/G

↑ 36% weight

↑13% weight

INTRODUCTION RESULTS CONCLUSIONS

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Basic measurements

F/FF/G

G/GF/F

F/G

G/G

↑4.9% height

↑21.9% height

Height Width

↑7.6% width

↑2.6% width

G/G

F/F

INTRODUCTION RESULTS CONCLUSIONS

Fruit shoulders

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G/G

F/F

INTRODUCTION RESULTS CONCLUSIONS

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Blockiness descriptors

G/G

F/F

INTRODUCTION RESULTS CONCLUSIONS

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Effects on plant biomass

F/F

F/G

G/G

↓ 20.3% weight

↓5.1% weight

INTRODUCTION RESULTS CONCLUSIONS

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Effects on fruit pedicel

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F/FF/G

G/GF/F

F/G

G/G

↑12.1% length

↑88% length↓22.8% width

↓2.8% width

INTRODUCTION RESULTS CONCLUSIONS

• The fruit shape locus was narrowed to a 400 Kb area in the upper arm of chromosome 12.

• There is a strong candidate mutation that segregates completely with shape even in other breeding lines.

• The locus affects several fruit traits, some of the main are fruit size, fruit shape index (width/height), fruit shoulders, fruit height and proximal blockiness.

• The locus also affects plant biomass and fruit pedicel.

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Conclusions

INTRODUCTION RESULTS CONCLUSIONS

Acknowledgements

• My advisor Dr. Samuel Hutton

• Tomato Breeding lab team at the GCREC• Lab staff: Tim, Nate, Judith, Dolly, Jose, Keri, Kazuyo

• Lab Grads: Rebecca, Jessica and Jazmin

• Committee members: Dr. Tong Geon Lee, Dr. Shinsuke Agehara, Dr. Esther van der Knaap

• COMEXUS Fulbright-Garcia Robles scholarship

• Dr. Reza Shekasteband and Dr. Gill Upinder

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Literature

Azzi L., Deluche C., Gevaudant F., Frangne N., Delmas F., Hernould M., Chevalier C. (2015). Fruit growth-related genes in tomato. Journal of Experimental Botany, Vol. 66, No. 4: 1075-1086. doi:10.1093/jxb/eru527

Brewer M.T., Lang L., Fujimura K., Dujmovic N., Gray S., and van der Knaap E. (2006). Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species. Plant Physiology, 141: 15-25.

Causse, M., Friguet, C., Coiret, C., Lépicier, M., Navez, B., Lee, M., . . . Grandillo, S. (2010). Consumer Preferences for Fresh Tomato at the European Scale: A Common Segmentation on Taste and Firmness. Journal of Food Science, 75(9).

Fulton M., Chunwongse J., and Tanksley S. 1995. Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Molecular Biology Reporter, 13(3), 207-209. Gardner R., (1990). ‘Mountain Delight’ tomato; NC 8288 tomato breeding line. HortScience 25(8): 989-990.

Gardner R., (1990). ‘Mountain Delight’ tomato; NC 8288 tomato breeding line. HortScience 25(8): 989-990.Gardner R. (1992). ‘Mountain Spring’ tomato; NC 8276 and NC 84173 tomato breeding lines. HortScience 27(11): 1233-1234.Gardner R. (2000). ‘Carolina Gold’, a hybrid tomato, and its parents, NC 1Y and NC 2Y. HortScience 35(5): 966-967.Grandillo S., Ku H.M., and Tanksley S.D. (1999). Identifying the loci responsible for natural variation in fruit size and shape in tomato. Theor Appl Genet, 99: 978-987.Huang Z., van Houten J., Gonzalez G., Xiao H., and van der Knaap E. (2013). Genome-wide identification, phylogeny, and expression analysis of SUN, OFP and YABBY gene family in

tomato. Mol Genet Genomics, 288: 111-129. doi:10.1007/s00438-013-0733-0Panthee D., and Gardner R. (2011). ‘Mountain Majesty’: A tomato spotted wilt virus-resistant fresh-market hybrid tomato and its parents NC 714 and NC 1CS. HortScience 46(9):

1321-1323.R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL. https://www.R-project.org/.Rodriguez G., Muños S., Anderson C., Sim S., Michel A., Causse M., McSpadden B., Francis D., and van der Knaap E. 2011. Distribution of SUN, OVATE, LC and FAS in the tomato

germplasm and the relationship to fruit shape diversity. Plant Physiology. Vol. 156.Rodriguez G.R., Kim H.J., van der Knaap E. (2013). Mapping of two suppressors of OVATE (sov) loci in tomato. Heredity, 111: 256-264. doi:10.1038/hdy.2013.45Scott JW. (2004). Fla. 7946 tomato breeding line resistant to Fusarium oxysporum f.sp. lycopersici races 1, 2 and 3. HortScience 39(2): 440-441.Scott JW., Olson SM., Bryan HH., Bartz JA., Maynard DN., and Stoffella PJ. (2006). ‘Solar Fire’ Hybrid tomato: Fla. 7776 tomato breeding line. HortScience 41(6): 1504-1505.Tanksley S.D. (2004). The genetic, developmental, and molecular basis of fruit size and shape variation in tomato. The Plant Cell, Vol. 16, S181-189. van der Knaap E., Lippman Z.B., Tanskley S.D. (2002). Extremely elongated tomato fruit controlled by four quantitative trait loci with epistatic interactions. Theor Appl Genet, 104:

241-247. doi:10.1007/s00122-001-0776-1Wang, J., H. Li, L. Zhang, and L. Meng. (2016). Users’ Manual of QTL IciMapping. The Quantitative Genetics Group, Institute of Crop Science, Chinese Academy of Agricultural

Sciences (CAAS), Beijing 100081, China, and Genetic Resources Program, International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico25

Materials and Methods

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INTRODUCTION M&M RESULTS CONCLUSIONS

• Mapping:• Progeny from Fla. 8111B (globe) x Fla. 8000 (flat)

• Characterization:• NILs from three different backgrounds:

• Fla. 8735 x Fla. 8059

• Fla. 7776 x Fla. 7946

• Fla. 8022 x Fla. 8021• The first, globe, recurrent parent, the second the flat

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INTRODUCTION M&M RESULTS CONCLUSIONS

Plant Material

1. Illumina Infinium SolCAP SNP array (7,720 SNPs)

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Composite Interval Mapping Analysis (CIM) (QTL ICiMapping 4.1, Wang et al., 2016)

INTRODUCTION M&M RESULTS CONCLUSIONS

Mapping of the fruit shape locus

2. Additional sequencing-based markers

Genotype a large population of segregating F2 plants

3. Markers for fine mapping

Germplasm survey using the marker at the locus

• 3 different genetic backgrounds

• Both parents and F1 set in a CRBD with 4 reps of 15 plants each• 4 weekly harvests of 8 plants

• Traits to measure:• Fruit size

• Several fruit shape traits (Tomato Analyzer 2.2, Brewer et al., 2006)

• Plant biomass

• Fruit pedicel (length and width)

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INTRODUCTION M&M RESULTS CONCLUSIONS

Characterization

Statistical Analysis• Linear Mixed Effects Model

• Fixed effect: Genotype• Random effect: Block

• post hoc analysis by Tukey’s HSD (P≤0.05)

• R software

Fruit shape index

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INTRODUCTION M&M RESULTS CONCLUSIONS

• Flats are more top heavy

• Even though fruits are asymmetric both horizontally and vertically, the locus only affected the horizontal asymmetry and not the vertical asymmetry of the fruit

• Globes fit better a circular shape whereas globes fit better a rectangular fit

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INTRODUCTION M&M RESULTS CONCLUSIONS

Other fruit shape traits affected by the locus