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The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum)

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Abstract

Key message

The seedless mutant tn-1 in chili pepper is caused by a mutation in CaCKI1 (CA12g21620), which encodes histidine kinase involving female gametophyte development. An amino acid insertion in the receiver domain of CaCKI1 may be the mutation responsible for tn-1 .

Abstract

Seedlessness is a desirable trait in fruit crops because the removal of seeds is a troublesome step for consumers and processing industries. However, little knowledge is available to develop seedless chili peppers. In a previous study, a chili pepper mutant tn-1, which stably produces seedless fruits, was isolated. In this study, we report characterization of tn-1 and identification of the causative gene. Although pollen germination was normal, confocal laser microscopy observations revealed deficiency in embryo sac development in tn-1. By marker analysis, the tn-1 locus was narrowed down to a 313 kb region on chromosome 12. Further analysis combined with mapping-by-sequencing identified CA12g21620, which encodes histidine kinase as a candidate gene. Phylogenetic analysis revealed CA12g21620 was the homolog of Arabidopsis CKI1 (Cytokinin Independent 1), which plays an important role in female gametophyte development, and CA12g21620 was designated as CaCKI1. Sequence analysis revealed that tn-1 has a 3-bp insertion in the 6th exon resulting in one lysine (K) residue insertion in receiver domain of CaCKI1, and the sequence nearby the insertion is widely conserved among CKI1 orthologs in various plants. This suggested that one K residue insertion may reduce the phosphorylation relay downstream of CaCKI1 and impair normal development of female gametophyte, resulting in seedless fruits production in tn-1. Furthermore, we demonstrated that virus-induced gene silencing of CaCKI1 reduced normally developed female gametophyte in chili pepper. This study describes the significant role of CaCKI1 in seed development in chili pepper and the possibility of developing seedless cultivars using its mutation.

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Acknowledgements

Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. We would like to express our gratitude to Dr. Yukiko Yasui for providing a valuable opportunity to initiate the CLSM observations. TRV1 (Addgene plasmid # 148968) and TRV2(Addgene plasmid # 148969) vectors were gifts from Savithramma Dinesh-Kumar. The NMH seeds were kindly provided by Dr. Sota Koeda (Kindai University).

Funding

This study was supported in part by a Grant-in-Aid for Scientific Research (B) (21H02187).

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  1. Akira Yamazaki

    Present address: Faculty of Agriculture, Kindai University, Naka Machi, Nara, 631-8505, Japan

Authors and Affiliations

  1. Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-Ku, Kyoto, 606-8502, Japan

    Takahiro Maki, Hirokazu Kusaka, Yuki Matsumoto, Akira Yamazaki, Sho Ohno, Motoaki Doi & Yoshiyuki Tanaka

  2. Graduate School of Biostudies, Kyoto University, Sakyo-Ku, Kyoto, 606-8501, Japan

    Shohei Yamaoka

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  1. Takahiro Maki
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Contributions

YT designed the research and performed mapping-by-sequencing analysis. TM conducted identification of the tn-1 gene, sequence analysis of CaCKI1, gene expression analysis, and microscopy analysis of ovule structures. YM participated in genetic analysis. HK and AY conducted the pollen germination tests. SY participated in CLSM observations on ovule structures. SO assisted with the development of DNA markers using genome resequencing data. MD assisted with the cultivation of plant materials and contributed in the interpretation of CaCKI1 function. TM wrote the draft of the manuscript, and YT edited it. All authors read and approved the final manuscript.

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Correspondence to Yoshiyuki Tanaka.

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Supplementary Information

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Figure S1 Cross-section of fruits obtained from artificial pollination tests (JPG 146 KB)

122_2023_4342_MOESM2_ESM.jpeg

Figure S2 Flow of the mapping-by-sequencing approach for identification of the causative mutation in the tn-1 mutant (JPEG 579 KB)

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Figure S3 ΔSNP-index plots across the whole genome to identify candidate a mutation of tn-1. ΔSNP-index was calculated as: ΔSNP-index = tn-1-bulk SNP-index – WT-bulk SNP index (SNP-index indicates the ratio of tn-1 type reads per total reads). Red plots show 7 SNPs with high ΔSNP-index (>0.7) (JPEG 548 KB)

122_2023_4342_MOESM4_ESM.jpg

Figure S4 Comparison of CaCKI1 gene structures. YM, tn-1, CM334 (annotated in CM334 v.1.55) and Zunla (annotated in Zunla v.2.0) from top (JPG 58 KB)

Figure S5 Alignment of deduced amino acid sequences of CaCKI1 and its homologues (JPG 396 KB)

122_2023_4342_MOESM6_ESM.jpg

Figure S6 Predicted 3D structure of the receiver domain of CaCKI1. A green arrow indicates the 1 amino acid (K) insertion in tn-1. Yellow arrows indicate different structures between WT and tn-1 (JPG 98 KB)

Figure S7 CLSM observation on ovules derived from TRV2-CaAN2-CaCKI1 plants. Bar = 50 μm (JPG 147 KB)

Figure S8 Cross-section of fruits in CaCKI1 silenced plants. Bar =1 cm (JPG 184 KB)

Supplementary file9 (PDF 72 KB)

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Maki, T., Kusaka, H., Matsumoto, Y. et al. The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum). Theor Appl Genet 136, 85 (2023). https://doi.org/10.1007/s00122-023-04342-w

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