Fine mapping of the male fertility restoration gene CaRf032 in Capsicum annuum L.

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A novel strong candidate gene CA00g82510 for the male fertility restoration locus CaRf032 in Capsicum annuum was identified by genome re-sequencing and recombination analysis.


A single dominant locus (CaRf032) for fertility restoration of cytoplasmic male sterility was identified in the strong restorer inbred line IVF2014032 of chili pepper (Capsicum annuum L.). CaRf032 was localized within an 8.81-Mb candidate intervals on chromosome 6 using bulked segregant analysis based on high-throughput sequencing data. Subsequently, the candidate interval was genetically mapped and defined to a 249.41-kb region using an F2 population of 441 individuals generated by crossing the male-sterile line 77013A and the restorer line IVF2014032. To fine map CaRf032, eight newly developed KASP markers were used to genotype 23 recombinants screened from a larger F2 population of 2877 individuals. The CaRf032 locus was localized to a 148.05-kb region between the KASP markers S1402 and S1354, which was predicted to contain 22 open reading frames (ORFs). One ORF with an incomplete sequence was predicted to contain a PPR motif, and its physical position overlapped with the Rf candidate gene CaPPR6_46. The PPR ORF sequence before the gap showed 100% identity with the CA00g82510 locus of the CM334 reference genome. CA00g82510 encodes a protein of 583 amino acids, containing 14 PPR motifs, and shows significantly differential expression between the flower buds of the maintainer line 77013 and the restorer line IVF2014032. These results indicated that CA00g82510 is a strong candidate gene for CaRf032. Five KASP markers, which detected single-nucleotide polymorphisms in CA00g82510 of 77013 and IVF2014032, co-segregated with CaRf032 and showed 64.4% successful genotyping of 38 maintainer and 63 restorer lines.

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Some of the pepper accessions were kindly provided by the National Mid-term Genebank for Vegetables (Beijing, China), Dr. Alain Palloix (INRA, France), Dr. Paul Bosland (Chile Pepper Institute, New Mexico State University), and Dr. Qin Cheng (Zunyi Academy of Agricultural Sciences, China). We thank Dr. Huang Zejun and Dr. Shu Jinshuai for constructive advice on genetic mapping and candidate gene prediction, and Drs Paul Bosland and Derek Barchenger for friendly discussion and constructive comments on pepper Rf genes. We thank Robert McKenzie, PhD, from Liwen Bianji, Edanz Group China (, for editing the English text of a draft of this manuscript. This work was performed at the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing, China, and was supported by the National Key Research and Development Program of China (2016YFD0101700), the China Agricultural Research System (CARS-25), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Science (CAAS-ASTIP-IVF-CAAS).

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ZHZ designed the study and developed the KASP markers. YSZ carried out the genetic mapping and RT-PCR. YCC and HLY performed the BSA analysis. RQB and HZ performed the phenotypic investigation. BXZ and LHW participated in the design of the study and revision of the manuscript. All authors read and approved the final version of the manuscript.

Correspondence to Lihao Wang.

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Zhang, Z., Zhu, Y., Cao, Y. et al. Fine mapping of the male fertility restoration gene CaRf032 in Capsicum annuum L.. Theor Appl Genet (2020).

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