Whitening fruit by CRISPR/Cas9-mediated homoeolog-specific gene editing of MYB10-1B in strawberry (F. × ananassa)

Abstract

Fruit color is a key quality trait in strawberry breeding and cultivar development, as it directly influences consumer preference and marketability. Anthocyanins are the pigments responsible for the red coloration in strawberries, and the transcription factor MYB10 gene plays a crucial role in regulating the anthocyanin biosynthetic pathway. Our previous study identified a homoeolog-specific copy, MYB10-1B, located on chromosome 1B, as a key regulator of fruit color. The natural mutation in MYB10-1B, such as in the variety ‘Florida Pearl’ leads to the development of white fruit. Building on this discovery, we applied CRISPR/Cas9-mediated homoeolog-specific editing to target the functional dominant allele, MYB10-1B, in the cultivated octoploid strawberry ‘Florida Brilliance’, successfully altering the fruit color from red to white. Gene expression analysis in the edited lines revealed downregulation of MYB10-1B and key anthocyanin biosynthesis genes (CHS, DFR, and ANS). Furthermore, whole-genome resequencing results showed precise on-target mutations in MYB10-1B with minimal off-target effects. This study highlights the successful application of homoeolog-specific CRISPR/Cas9-mediated gene editing in polyploid species and provides a foundation for functional genomics and advanced breeding strategies in strawberries. Importantly, our findings demonstrate that specific targeting of the dominantly expressed homoeologous copy is essential for inducing phenotypic changes in polyploids. This underscores the importance of precise gene editing in octoploid strawberry to drive trait improvement.

Acknowledgements

We thank J. Zhou and Z. Liu at the University of Maryland for providing the gateway vectors for CRISPR/Cas9 and for their technical assistance in constructing the CRISPR/Cas9 vectors. This research is supported by grants from the United States Department of Agriculture National Institute of Food and Agriculture (NIFA) Specialty Crops Research Initiative (#2017-51181-B6833 and #2022-51181-38328-0). This work was also supported by ‘Cooperative Research Program of Agriculture Science and Technology Development’ (Project No. PJ017707), Rural Development Administration, Republic of Korea, and the 2025 RDA Fellowship Program of the National Institute of Horticultural and Herbal Science, Rural Development Administration, Republic of Korea. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00355164).

Authors contributions

M.B.L. and S.L. contributed to the conception and design of this study. M.B.L. created the CRISPR/Cas9 gene-edited lines and performed the majority of the experiments. Y.J.J. and K.S. conducted phenotyping of the gene-edited lines. Y.J.J. conducted qRT-PCR analysis and analyzed transcriptome data. H.H. and Y.O. analyzed amplicon sequencing data. H.H. analyzed whole-genome resequencing data. J.Y.K. contributed to Nested PCR and Sanger sequencing. M.B.L. wrote the draft manuscript. All authors contributed to manuscript preparation and approved the submitted version.

Data availability

Data supporting the results reported in the paper are available in the main text and supplementary data.

Conflicts of interest statement

The authors declare that there are no conflicts of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Bhat R, Geppert J, Funken E. et al. Consumers perceptions and preference for strawberries—a case study from Germany. Int J Fruit Sci. 2015; 15:405-24

[2] Chen Y, Liu L, Feng Q. et al. FvWRKY50 is an important gene that regulates both vegetative growth and reproductive growth in strawberry. Hortic Res. 2023; 10:115

[3] Gao Q, Luo H, Li Y. et al. Genetic modulation of RAP alters fruit coloration in both wild and cultivated strawberry. Plant Biotechnol J. 2020; 18:1550-61

[4] Hardigan MA, Feldmann MJ, Pincot DDA. et al. Blueprint for phasing and assembling the genomes of heterozygous poly-ploids: application to the octoploid genome of strawberry. bioRxiv. 2021.

[5] Edger PP, Poorten TJ, VanBuren R. et al. Origin and evo-lution of the octoploid strawberry genome. Nat Genet. 2019; 51:541-7

[6] Han H, Salinas N, Barbey CR. et al. A telomere-to-telomere phased genome of an octoploid strawberry reveals a recep-tor kinase conferring anthracnose resistance. Gigascience. 2025; 14:1-15

[7] Whitaker VM, Peres NA, Osorio LF. et al. ‘Florida Brilliance’ strawberry. HortScience. 2019; 54:2073-7

[8] Almeida JRM, D’Amico E, Preuss A. et al. Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria × ananassa). Arch Biochem Biophys. 2007; 465:61-71

[9] Wang H, Zhang H, Yang Y. et al. The control of red colour by a family of MYB transcription factors in octoploid strawberry ( Fragaria × ananassa) fruits. Plant Biotechnol J. 2020; 18:1169-84

[10] Castillejo C, Waurich V, Wagner H. et al. Allelic variation of MYB10 is the major force controlling natural variation in skin and flesh color in strawberry ( Fragaria spp.) fruit. Plant Cell. 2020; 32:3723-49

[11] Medina-Puche L, Cumplido-Laso G, Amil-Ruiz F. et al. MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of Fragaria × ananassa fruits. J Exp Bot. 2014; 65:401-17

[12] Hawkins C, Caruana J, Schiksnis E. et al. Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry. Sci Rep. 2016; 6:1-11

[13] Whitaker VM, Dalid C, Osorio LF. et al. Florida Pearl® ‘FL 16.78-109’ pineberry. HortScience. 2023; 58:143-6

[14] Hardigan MA, Lorant A, Pincot DDA. et al. Unraveling the com-plex hybrid ancestry and domestication history of cultivated strawberry. Mol Biol Evol. 2021; 38:2285-305

[15] Jiang F, Doudna JA. CRISPR-Cas 9 structures and mechanisms. Annu Rev Biophys. 2017; 46:505-29

[16] Martín-Pizarro C, Triviño JC, Posé D. Functional analysis of the TM6 MADS-box gene in the octoploid strawberry by CRISPR/Cas9-directed mutagenesis. J Exp Bot. 2019; 70: 885-95

[17] Xing S, Jia M, Wei L. et al. CRISPR/Cas9-introduced single and multiple mutagenesis in strawberry. J Genet Genomics. 2018; 45:685-7

[18] Wilson FM, Harrison K, Armitage AD. et al. CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry. Plant Methods. 2019; 15:1-13

[19] Kim J-H, Yoo C-M, Nguyen CD. et al. Optimization of shoot regeneration and Agrobacterium-mediated transformation in strawberry (Fragaria × ananassa). J Am Soc Hortic Sci. 2025; 150:10-18

[20] Bekalu ZE, Panting M, Bæksted Holme I. et al. Opportunities and challenges of in vitro tissue culture systems in the era of crop genome editing. Int J Mol Sci. 2023; 24:11920

[21] Jang YJ, Kim T, Lin M. et al. Genome-wide gene network uncover temporal and spatial changes of genes in auxin homeostasis during fruit development in strawberry (F. × ananassa). BMC Plant Biol. 2024; 24:876

[22] Song Y, Dimaio F, Wang RYR. et al. High-resolution compara-tive modeling with RosettaCM. Structure. 2013; 21:1735-42

[23] Park J, Bae S, Kim JS. Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics. 2015; 31:4014-6

[24] Gasiunas G, Barrangou R, Horvath P. et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleav-age for adaptive immunity in bacteria. Proc Natl Acad Sci. 2012;109:E2579-86

[25] Zhou J, Wang G, Liu Z. Efficient genome editing of wild strawberry genes, vector development and validation. Plant Biotechnol J. 2018; 16:1868-77

[26] Quevillon E, Silventoinen V, Pillai S. et al. InterProScan: pro-tein domains identifier. Nucleic Acids Res. 2005;33:W116-20

[27] Schaart JG, van de Wiel CCM, Smulders MJM. Genome editing of polyploid crops: prospects, achievements and bottlenecks. Transgenic Res. 2021; 30:337-51

[28] May D, Paldi K, Altpeter F. Targeted mutagenesis with sequence-specific nucleases for accelerated improvement of polyploid crops: progress, challenges, and prospects. Plant Genome. 2023; 16:e20298

[29] Vondracek K, Altpeter F, Liu T. et al. Advances in genomics and genome editing for improving strawberry (Fragaria × ananassa). Front Genet. 2024; 15:1382445

[30] Mao J, Wang Y, Wang B. et al. High-quality haplotype-resolved genome assembly of cultivated octoploid strawberry. Hortic Res. 2023;10:uhad002

[31] Lang J, Fu Y, Zhou Y. et al. Myb10-D confers PHS-3D resistance to pre-harvest sprouting by regulating NCED in ABA biosyn-thesis pathway of wheat. New Phytol. 2021; 230:1940-52

[32] Tuan PA, Bai S, Yaegaki H. et al. The crucial role of PpMYB10.1 in anthocyanin accumulation in peach and relationships between its allelic type and skin color phenotype. BMC Plant Biol. 2015; 15:1-14

[33] Lang J, Jiang H, Cheng M. et al. Variation of TaMyb10 and their function on grain color and pre-harvest sprouting resistance of wheat. Plant J. 2024; 118:1388-99

[34] Kang C, Darwish O, Geretz A. et al. Genome-scale transcrip-tomic insights into early-stage fruit development in woodland strawberry Fragaria vesca. Plant Cell. 2013; 25:1960-78

[35] Zhou J, Sittmann J, Guo L. et al. Gibberellin and auxin signal-ing genes RGA1 and ARF8 repress accessory fruit initiation in diploid strawberry. Plant Physiol. 2021; 185:1059-75

[36] Liu H, Ding Y, Zhou Y. et al. CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Mol Plant. 2017; 10: 530-2

[37] Zhang JH, Adikaram P, Pandey M. et al. Optimization of genome editing through CRISPR-Cas 9 engineering. Bioengi-neered. 2016; 7:166-74

[38] Xu X, Duan D, Chen SJ. CRISPR-Cas 9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment. Sci Rep. 2017; 7:1-9

[39] Doench JG, Fusi N, Sullender M. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016; 34:184-91

[40] Kim S, Bae T, Hwang J. et al. Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides. Genome Biol. 2017; 18:1-6

[41] Wang D, Zhang C, Wang B. et al. Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning. Nat Commun. 2019; 10:1-14

[42] Okafor IC, Singh D, Wang Y. et al. Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding. Nucleic Acids Res. 2019; 47:11880-8

[43] Lee MB, Shekasteband R, Hutton SF. et al. A mutant allele of the flowering promoting factor 1 gene at the tomato BRACHYTIC locus reduces plant height with high quality fruit. Plant Direct. 2022; 6:e422

[44] Li Q, Yu H, Li Q. High resolution melting analysis: a rapid and sensitive method for detection of mutations induced by CRISPR/Cas9-based genome editing in oysters. Aquaculture. 2023; 572:739533

[45] Li R, Ba Y, Song Y. et al. Rapid and sensitive screening and identification of CRISPR/Cas9 edited rice plants using quan-titative real-time PCR coupled with high resolution melting analysis. Food Control. 2020; 112:107088

[46] Roginsky J. Analyzing CRISPR editing results. Genet Eng Biotechnol News. 2018; 38:S24-6

[47] Hardigan MA, Feldmann MJ, Lorant A. et al. Genome synteny has been conserved among the octoploid progenitors of cul-tivated strawberry over millions of years of evolution. Front Plant Sci. 2020; 10:504896

[48] Huang M, Liu X, Zhou Y. et al. BLINK: a package for the next level of genome-wide association studies with both individu-als and markers in the millions. Gigascience. 2019; 8:1-12

[49] Zhang Z, Ersoz E, Lai CQ. et al. Mixed linear model approach adapted for genome-wide association studies. Nat Genet. 2010; 42:355-60

[50] Segura V, Vilhjálmsson BJ, Platt A. et al. An efficient multi-locus mixed-model approach for genome-wide association studies in structured populations. Nat Genet. 2012; 44: 825-30

[51] Liu X, Huang M, Fan B. et al. Iterative usage of fixed and ran-dom effect models for powerful and efficient genome-wide association studies. PLoS Genet. 2016; 12:e1005767

[52] Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versa-tile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014; 30: 1473-5

[53] Chen H, Nelson RS, Sherwood JL. Enhanced recovery of trans-formants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques. 1994; 16: 664-8

[54] Oh Y, Chandra S, Lee S. Development of subgenome-specific markers for FaRXf1 conferring resistance to bacterial angular leaf spot in allo-octoploid strawberry. Int J Fruit Sci. 2020; 20: S198-210

[55] Jang YJ, Oh Y, Verma S. et al. Updates on strawberry DNA test-ing and marker-assisted breeding at the University of Florida. Int J Fruit Sci. 2024; 24:219-28

[56] Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30:2114-20

[57] Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25: 1754-60

[58] Li H, Handsaker B, Wysoker A. et al. The sequence alignmen-t/map format and SAMtools. Bioinformatics. 2009; 25:2078-9

[59] Lee MB, Han H, Lee S. The role of WRKY transcription fac-tors, FaWRKY29 and FaWRKY64, for regulating Botrytis fruit rot resistance in strawberry (Fragaria × ananassa Duch.). BMC Plant Biol. 2023; 23:420