Fertility restorer gene CaRf and PepperSNP50K provide a promising breeding system for hybrid pepper

Abstract

Cytoplasmic male sterility (CMS) is pivotal in plant breeding and widely employed in various crop hybrids, including pepper. However, the functional validation of the restorer of fertility (Rf) gene in pepper has been lacking until now. This study identifies and characterizes CaRf, a single dominant locus crucial for restoring CMS in the pepper strong recovery inbred line Zhangshugang. The CaRf gene encodes a mitochondria-targeted pentatricopeptide repeat protein, validated through the induction of male sterility upon its silencing in hybrid F₁ plants. To enhance pepper breeding efficiency, 176 important pepper breeding parent materials were resequenced, and a PepperSNP50K liquid-phase breeding chip was developed, comprising 51 172 markers. Integration of CaRf functional characterization and PepperSNP50K facilitated the development of a high-quality red pepper hybrid. These findings provide significant insights and practical strategies for advancing molecular-designed breeding in peppers.

Acknowledgements

This work was supported by the Construction of Innovative Provinces in Hunan Province (Grant No. 2021NK1006), the National Natural Science Foundation of China (Grant No. 32402571), the Hunan Provincial Natural Science Foundation of China (Grant No. 2024JJ6239), the China Postdoctoral Science Foundation (Grant No. 2023M741144), and the Postdoctoral Fellowship Program of CPSF (Grant No. GZC20230777).

Author contributions

T.B.Q. and Y.H.P. contributed to population generation, phenotypic analysis, data curation, marker development, and manuscript writing. C.Y., L.X.M., C.Q.Z., W.M.Q., L.J.Y., X.F.L., X.L.L., C.L., and D.J. participated in the phenotypic identification. Z.X.H., Y.Q.B., and C.J.W. participated in phenotypic analysis. L.Y.T., T.B.Q., L.F., T.S.X., and C.M.Y. contributed to the design and production of liquid-phase chips. M.W. and L.F. participated in field pepper breeding. W.Z.Y., Z.F., and X.C. participated in genotyping of breeding lines. D.X.Z., Z.X.X., and L.F. participated in the study design and project management supporting this research.

Data availability statement

The reference genome sequences used in this study are available at PepperGD (http://ted.bti.cornell.edu/cgi-bin/pepper/index). The raw resequencing and transcriptome sequencing data are deposited in the GSA (https://ngdc.cncb.ac.cn/gsa/) under project ID PRJCA027547 and PRJCA013331.

Conflict of interests

The authors have no relevant financial or non-financial interests to disclose.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Chen L, Liu YG. Male sterility and fertility restoration in crops. Annu Rev Plant Biol. 2014; 65:579-606

[2] Kubo T, Arakawa T, Honma Y. et al. What does the molecular genetics of different types of restorer-of-fertility genes imply? Plants (Basel). 2020; 9:361

[3] Tang H, Zheng X, Li C. et al. Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes. Cell Res. 2017; 27:130-46

[4] Xu FY, Yang XD, Zhao N. et al. Exploiting sterility and fertility variation in cytoplasmic male sterile vegetable crops. Hortic Res. 2022; 9:uhab039

[5] Jo YD, Ha Y, Lee JH. et al. Fine mapping of restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein. Theor Appl Genet. 2016; 129:2003-17

[6] Kim YJ, Zhang D. Molecular control of male fertility for crop hybrid breeding. Trends Plant Sci. 2018; 23:53-65

[7] Jiang H, Lu Q, Qiu S. et al. Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice. Proc Natl Acad Sci USA. 2022; 119:e2208759119

[8] Wang Z, Zou Y, Li X. et al. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell. 2006; 18:676-87

[9] Peng X, Wang K, Hu C. et al. The mitochondrial gene orfH79 plays a critical role in impairing both male gametophyte development and root growth in CMS-Honglian rice. BMC Plant Biol. 2010; 10:125

[10] Itabashi E, Kazama T, Toriyama K. Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm. Plant Cell Rep. 2009; 28:233-9

[11] Fujii S, Kazama T, Yamada M. et al. Discovery of global genomic re-organization based on comparison of two newly sequenced rice mitochondrial genomes with cytoplasmic male sterility-related genes. BMC Genomics. 2010; 11:209

[12] Fujii S, Komatsu S, Toriyama K. Retrograde regulation of nuclear gene expression in CW-CMS of rice. Plant Mol Biol. 2007; 63:405-17

[13] Luo D, Xu H, Liu Z. et al. A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice. Nat Genet. 2013; 45:573-7

[14] Okazaki M, Kazama T, Murata H. et al. Whole mitochondrial genome sequencing and transcriptional analysis to uncover an RT102-type cytoplasmic male sterility-associated candidate gene derived from Oryza rufipogon. Plant Cell Physiol.. 2013; 54:1560-8

[15] Igarashi K, Kazama T, Motomura K. et al. Whole genomic sequencing of RT98 mitochondria derived from Oryza rufipogon and northern blot analysis to uncover a cytoplasmic male sterility-associated gene. Plant Cell Physiol. 2013; 54:237-43

[16] Xie H, Peng X, Qian M. et al. The chimeric mitochondrial gene orf182 causes non-pollen-type abortion in Dongxiang cytoplasmic male-sterile rice. Plant J. 2018; 95:715-26

[17] Takatsuka A, Kazama T, Toriyama K.Cytoplasmic male sterility-associated mitochondrial gene orf312 derived from rice (Oryza sativa L.) cultivar Tadukan. Rice. 2021; 14:46

[18] Cui X, Wise RP, Schnable PS. The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science. 1996; 272:1334-6

[19] Qin X, Tian S, Zhang W. et al. The main restorer Rf3 of maize S type cytoplasmic male sterility encodes a PPR protein that functions in reduction of the transcripts of orf355. Mol Plant. 2021; 14:1961-4

[20] Jaqueth JS, Hou Z, Zheng P. et al. Fertility restoration of maize CMS-C altered by a single amino acid substitution within the Rf 4 bHLH transcription factor. Plant J. 2020; 101:101-11

[21] Lin Y, Yang H, Liu H. et al. A P-type pentatricopeptide repeat protein ZmRF5 promotes 5′ region partial cleavages of atp6c transcripts to restore the fertility of CMS-C maize by recruiting a splicing factor. Plant Biotechnol J. 2024; 22:1269-81

[22] Castillo A, Atienza SG, Martín AC. Fertility of CMS wheat is restored by two Rf loci located on a recombined acrocentric chromosome. J Exp Bot. 2014; 65:6667-77

[23] Ui H, Sameri M, Pourkheirandish M. et al. High-resolution genetic mapping and physical map construction for the fertility restorer Rfm1 locus in barley. Theor Appl Genet. 2015; 128:283-90

[24] Uyttewaal M, Arnal N, Quadrado M. et al. Characterization of Raphanus sativus pentatricopeptide repeat proteins encoded by the fertility restorer locus for Ogura cytoplasmic male sterility. Plant Cell. 2008; 20:3331-45

[25] Yin J, Guo W, Yang L. et al. Physical mapping of the Rf 1 fertility-restoring gene to a 100 kb region in cotton. Theor Appl Genet. 2006; 112:1318-25

[26] Matsuhira H, Kagami H, Kurata M. et al. Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.). Genetics. 2012; 192:1347-58

[27] Zhang H, Wu J, Dai Z. et al. Allelism analysis of BrRfp locus in different restorer lines and map-based cloning of a fertility restorer gene, BrRfp1, for pol CMS in Chinese cabbage (Brassica rapa L.). Theor Appl Genet. 2017; 130:539-47.

[28] Koizuka N, Imai R, Fujimoto H. et al. Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J. 2003; 34:407-15

[29] Kim S, Kim CW, Park M. et al. Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq. Theor Appl Genet. 2015; 128:2289-99

[30] Zhang Z, Zhu Y, Cao Y. et al. Fine mapping of the male fertility restoration gene CaRf 032 in Capsicum annuum L. Theor Appl Genet. 2020; 133:1177-87

[31] Kim S, Park M, Yeom SI. et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet. 2014; 46:270-8

[32] Qin C, Yu C, Shen Y. et al. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc Natl Acad Sci USA. 2014; 111:5135-40

[33] Wu L, Wang P, Wang Y. et al. Genome-wide correlation of 36 agronomic traits in the 287 pepper (Capsicum) accessions obtained from the SLAF-seq-based GWAS. Int J Mol Sci. 2019; 20:5675

[34] Cheng J, Chen Y, Hu Y. et al. Fine mapping of restorer-of-fertility gene based on high-density genetic mapping and collinearity analysis in pepper (Capsicum annuum L.). Theor Appl Genet. 2020; 133:889-902

[35] Zhang Z, An D, Yu H. et al. Fine mapping of Rf2, a minor restorer-of-fertility (Rf) gene for cytoplasmic male sterility in chili pepper G 164 (Capsicum annuum L.). Theor Appl Genet. 2022; 135:2699-709

[36] Wei B, Bosland PW, Zhang Z. et al. A predicted NEDD8 conjugating enzyme gene identified as a Capsicum candidate Rf gene using bulk segregant RNA sequencing. Hortic Res. 2020; 7:210

[37] Nie Z, Song Y, Wang H. et al. Fine mapping and gene analysis of restorer-of-fertility gene CaRf_HZ in pepper (Capsicum annuum L.). Int J Mol Sci. 2022; 23:7633

[38] Barkan A, Small I. Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol. 2014; 65:415-42

[39] Liao Y, Wang J, Zhu Z. et al. The 3D architecture of the pepper genome and its relationship to function and evolution. Nat Commun. 2022; 13:3479

[40] Liu F, Zhao J, Sun H. et al. Genomes of cultivated and wild Capsicum species provide insights into pepper domestication and population differentiation. Nat Commun. 2023; 14:5487

[41] Chen W, Wang X, Sun J. et al. Two telomere-to-telomere gapless genomes reveal insights into Capsicum evolution and capsaicinoid biosynthesis. Nat Commun. 2024; 15:4295

[42] Chen H, Xie W, He H. et al. A high-density SNP genotyping array for rice biology and molecular breeding. Mol Plant. 2014; 7:541-53

[43] Yu H, Xie W, Li J. et al. A whole-genome SNP array (RICE6K) for genomic breeding in rice. Plant Biotechnol J. 2014; 12:28-37

[44] Ganal MW, Durstewitz G, Polley A. et al. A large maize (Zea mays L.) SNP genotyping array: development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome. PLoS One. 2011; 6:e28334

[45] Guo Z, Yang Q, Huang F. et al. Development of high-resolution multiple-SNP arrays for genetic analyses and molecular breeding through genotyping by target sequencing and liquid chip. Plant Commun. 2021; 2:100230

[46] Guo J, Wang P, Cheng Q. et al. Proteomic analysis reveals strong mitochondrial involvement in cytoplasmic male sterility of pepper (Capsicum annuum L.). J Proteomics. 2017; 168:15-27

[47] Zhou X, Zheng W, Li Y. et al. I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction. Nat Protoc. 2022; 17:2326-53

[48] Su HG, Li B, Song XY. et al. Genome-wide analysis of the DYW subgroup PPR gene family and identification of GmPPR4 responses to drought stress. Int J Mol Sci. 2019; 20:5667

[49] Barchenger DW, Said JI, Zhang Y. et al. Genome-wide identification of Chile pepper pentatricopeptide repeat domains provides insight into fertility restoration. J Am Soc Hortic Res. 2018; 143:418-29

[50] Jo YD, Kim YM, Park MN. et al. Development and evaluation of broadly applicable markers for restorer-of-fertility in pepper. Mol Breed. 2010; 25:187-201

[51] Wang LH, Zhang BX, Lefebvre V. et al. QTL analysis of fertility restoration in cytoplasmic male sterile pepper. Theor Appl Genet. 2004; 109:1058-63

[52] Kang MC, Kang HJ, Jung SY. et al. The unstable restorer-of-fertility locus in pepper (Capsicum annuum. L) is delimited to a genomic region containing PPR genes. Theor Appl Genet. 2022; 135:1923-37

[53] Zhang Z, An D, Cao Y. et al. Development and application of KASP markers associated with restorer-of-fertility gene in Capsicum annuum L. Physiol Mol Biol Plants. 2021; 27:2757-65

[54] Peleman JD, van der Voort JR. Breeding by design. Trends Plant Sci. 2003; 8:330-4

[55] Flavell R. From genomics to crop breeding. Nat Biotechnol. 2010; 28:144-5

[56] Morrell PL, Buckler ES, Ross-Ibarra J. Crop genomics: advances and applications. Nat Rev Genet. 2011; 13:85-96

[57] Qu Y, Fernie AR, Liu J. et al. Doubled haploid technology and synthetic apomixis: recent advances and applications in future crop breeding. Mol Plant. 2024; 17:1005-18

[58] Kabade PG, Dixit S, Singh UM. et al. SpeedFlower: a comprehensive speed breeding protocol for indica and japonica rice. Plant Biotechnol J. 2024; 22:1051-66

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

[60] Allen GC, Flores-Vergara MA, Krasynanski S. et al. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc. 2006; 1:2320-5

[61] Tang B, Xie L, Li X. et al. Novel structural annotation and functional expression analysis of GTP_EFTU conserved genes in pepper based on the PacBio sequencing data. Hortic Plant J. 2021; 7:443-56

[62] Wan H, Yuan W, Ruan M. et al. Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L.). Biochem Biophys Res Commun. 2011; 416:24-30

[63] Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32:1792-7

[64] Letunic I, Bork P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016; 44:W242-5

[65] Tang H, Krishnakumar V, Zeng X. et al. JCVI: a versatile toolkit for comparative genomics analysis. iMeta. 2024; 3:e211

[66] Minamikawa T, Sriratana A, Williams DA. et al. Chloromethyl-X-rosamine (MitoTracker red) photosensitises mitochondria and induces apoptosis in intact human cells. J Cell Sci. 1999; 112:2419-30

[67] Xue W, Xing Y, Weng X. et al. Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat Genet. 2008; 40:761-7

[68] Nelson BK, Cai X, Nebenführ A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007; 51:1126-36

[69] Chen S, Zhou Y, Chen Y. et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018; 34:i884-90

[70] Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data. 2010.

[71] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12:357-60

[72] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:550

[73] Klopfenstein D V, Zhang L, Pedersen B S. et al.. GOATOOLS: A Python library for Gene Ontology analyses. Scientific reports. 2018; 8:1-17.

[74] Aramaki T, Blanc-Mathieu R, Endo H. et al. KofamKOALA: KEGG Ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics. 2020; 36:2251-2

[75] Yu G, Wang LG, Han Y. et al. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16:284-7

[76] Zhou Y, Deng Y, Liu D. et al. Promoting virus-induced gene silencing of pepper genes by a heterologous viral silencing suppressor. Plant Biotechnol J. 2021; 19:2398-400

[77] Kendig KI, Baheti S, Bockol MA. et al. Sentieon DNASeq variant calling workflow demonstrates strong computational performance and accuracy. Front Genet. 2019; 10:736

[78] Danecek P, Auton A, Abecasis G. et al. The variant call format and VCFtools. Bioinformatics. 2011; 27:2156-8

[79] Hill JT, Demarest BL, Bisgrove BW. et al. MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq. Genome Res. 2013; 23:687-97