Researchers Identify Key Gene for Restoring Fertility in Hybrid Peppers

Cytoplasmic male sterility (CMS) systems have revolutionized hybrid seed production by eliminating the need for manual pollen removal. However, success depends on the ability to restore fertility in hybrid offspring using nuclear-encoded restorer genes (Rf). While CMS-Rf systems have been well studied in crops like rice and maize, similar progress in pepper has been hampered by the absence of functionally validated Rf genes and scalable genotyping tools. This has constrained breeding efficiency and slowed the development of superior pepper hybrids. Due to these limitations, there is a pressing need to identify effective Rf and pair them with high-throughput molecular tools to modernize pepper breeding programs. Because of these challenges, an in-depth investigation into pepper fertility restoration and genotyping tools is urgently needed.

A research team from Hunan Agricultural University has made a significant breakthrough in hybrid pepper breeding by identifying a novel restorer gene, CaRf, and launching a powerful genotyping platform known as PepperSNP50K. Published (DOI: 10.1093/hr/uhae223) on October 1, 2024, in Horticulture Research, the study combines cutting-edge genetic mapping, transcriptome analysis, and molecular validation to tackle longstanding bottlenecks in CMS-based breeding. The team's work not only provides the first functional validation of a pepper Rf gene but also delivers a versatile SNP chip capable of accelerating precision breeding across diverse pepper varieties.

The scientists began by examining fertility traits in the CMS line 9704A and the restorer line Zhangshugang. While the sterile 9704A failed to produce viable pollen, Zhangshugang restored fertility in hybrid offspring. Using bulked-segregant analysis and fine mapping, the team pinpointed CaRf, a dominant restorer gene, on chromosome 6. This gene, Caz06g28920, encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria-an organelle linked to CMS. Functional validation through gene silencing (VIGS) in F1 hybrids caused male sterility, confirming CaRf's critical role.

To support breeding at scale, the researchers developed PepperSNP50K, a 51,172-marker SNP chip derived from resequencing 176 diverse pepper lines. The chip offers uniform genome coverage, high polymorphism, and accurate genotype calls, making it ideal for identifying Rf and tracking genetic backgrounds. Its performance was demonstrated by creating elite hybrid lines through marker-assisted backcrossing and doubled haploid (DH) technology. Notably, the restorer line QN49R and sterile line XY21A, bred using this system, showed strong agronomic performance and commercial potential. Together, the validated CaRf gene and PepperSNP50K chip form a robust, scalable platform for next-generation pepper hybrid development.

This is a milestone for pepper genetics. For the first time, we have functionally verified a restorer gene in pepper and paired it with a high-throughput genotyping tool. CaRf allows us to control fertility restoration with precision, and the PepperSNP50K chip ensures that we can do so rapidly, efficiently, and at scale. It's the kind of integrated solution breeders have been waiting for-one that transforms complex breeding challenges into streamlined, data-driven strategies."

Dr. Feng Liu, co-corresponding author and professor at Hunan Agricultural University

The combined use of CaRf, PepperSNP50K, and DH technology represents a new breeding paradigm: fast, precise, and trait-targeted. This system enables breeders to develop male-sterile and restorer lines more quickly, improving the efficiency of three-line hybrid production. It also facilitates genome-wide selection for disease resistance, fruit quality, and other key traits. Looking forward, the team plans to build a pepper "speed breeding" facility inspired by rice platforms, aiming to compress breeding cycles to under two years. With PepperSNP50K at its core, this integrated approach offers a powerful model for advancing molecular design breeding in peppers-and sets the stage for broader applications in horticultural crops.