Abstract
Owing to their superior agronomic performance, the hybrids of vegetable crops are currently applied extensively. However, effective hybrid production requires a laborious manual emasculation to ensure the purity of hybrid seeds in tomato because of the lack of an effective male sterility system. Here, we created two types of tomato nuclear male-sterile lines with different screening markers in a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Co-knockouts of male sterile 1035 (Ms1035) and glutathione S-transferase (GSTAA) created a male-sterile line marked by a green hypocotyl. The Ms1035 biallelic mutation was introduced into the woolly tomato background, resulting in the linkage of male sterility and a non-woolly phenotype. Two types of male-sterile lines were easily selected at the seedling stage by hypocotyl color or trichome density and further showed high seed purity during hybrid seed production. Our work established the procedure for a rapid transfer of the male-sterile phenotype to the parents of hybrids without extra-modification by the CRISPR/Cas9 system that can be practically applied to hybrid seed production in tomato. This method will be the basis and example for sterile parent creation of multiple crops for hybrid production with the CRISPR/Cas9 system.
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Acknowledgements
We thank Prof Qijun Chen (China Agricultural University, Beijing) for kindly providing the plant genome editing vector pKSE401 and pCBC1-DT1T2 plasmids.
Funding
This work was financially supported by NSFC (No. 31972430) and Research Collaborative Innovation of Yangling Demonstration Zone (2018CXY-09) to Xiaofeng Wang, “Light of the West” Talent Training and Introduction Program of Chinese Academy of Sciences (XAB2018AW16) to Yanfeng Zhang.
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J.L., S.W., H.W., Y.Z., and X.W. designed the studies. J.L., S.W., H.W., B.L., Y.C., and X.L. performed the experiments. J.L., S.W., H.W., Y.Z., and X.W. wrote the manuscript. All the authors gave final approval for submission of the manuscript.
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Supplementary information
Supplementary Fig. 1
Morphological analysis of Ms1035ms1035/WoDWo and CR-ms1035/Wo. a Two-month-old plants in the field, woollyand non-woolly phenotype plants from two dependent male-sterile lines. b Morphological comparison of Ms1035ms1035/WoDWo and CR-ms1035/Wo flowers; scale bar: 1 cm. c Morphology of Ms1035ms1035/WoDWo and CR-ms1035/Wo anther cones, red arrow indicates exerted stigmata; scale bar: 1 cm. (PNG 2262 kb)
Supplementary Fig. 2
CR-Ms035/Wo and CR-Ms035/gstaa can set normal fruits by artificial pollination. The red arrow indicates the fruit set by artificial pollination. (TIF 6068 kb) (PNG 2090 kb)
Supplementary Fig. 3
Analysis of hypocotyl color and genotype in Ms1035ms1035/F3Hf3h self-crossed progeny. a The second exon of F3H was targeted by the CRISPR/Cas9 system using single-guide RNAs (red arrows indicate target). Black arrows indicate forward (F) and reverse (R) primers used for PCR genotyping and sequencing. The target sequences in F3H are underlined, minus symbols represent deletions. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” indicating deletion of the given number of nucleotides. b Morphological comparison of wild-type (WT) and CR-ms1035/f3h hypocotyls; scale bar: 1 cm. c Editing analysis of F3H using restriction enzyme site Xba I loss. The two bands and three bands indicate WT and heterozygous, respectively. (PNG 4642 kb)
Supplementary Fig. 4
a Morphology of fruits and seeds in 6Q71, 5N21, CR-ms1035/Wo and CR-ms1035/gstaa during tomato seed production. b Germination rate of hybrid F1 seeds from 6Q71, 5N21, CR-ms1035/Wo and CR-ms1035/gstaa. Per plug, 128 seeds were sowing, and the germination rate was counted seven days after sowing. Three plugs were sown for each type of seed as three independent biological repeats. Statistically significant differences were test by Student’s t-test (P < 0.05). (PNG 1007 kb)
Supplementary Table 1.
Detection of mutations in potential off-target sites in T0 edited plants. (PDF 184 kb)
Supplementary Table 2.
Linkage between ms1035 and f3h in the T2 and T3 generations. (PDF 246 kb)
Supplementary Table 3.
Primers used for this study. (PDF 255 kb)
ESM 8
(DOCX 151 kb)
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Liu, J., Wang, S., Wang, H. et al. Rapid generation of tomato male-sterile lines with a marker use for hybrid seed production by CRISPR/Cas9 system. Mol Breeding 41, 25 (2021). https://doi.org/10.1007/s11032-021-01215-2
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DOI: https://doi.org/10.1007/s11032-021-01215-2