Molecular characterization of the Rs-Rf 1 gene and molecular marker-assisted development of elite radish (Raphanus sativus L.) CMS lines with a functional marker for fertility restoration

Abstract

Cytoplasmic male sterility (CMS) is a maternally inherited trait that is widely utilized in hybrid seed production. In this study, the Rs-Rf 1 gene and its allele, the Rs-rf 1 gene, were isolated from a radish CMS restorer line and maintainer, respectively. The Rs-Rf 1 gene was identified as a member of the pentatricopeptide repeat family. The results of reverse transcription (RT)-PCR and quantitative RT-PCR analyses of the floral buds at different development stages were used to characterize the expression features of Rs-Rf 1 and orf138, a unique transcribed gene in the mitochondrion for its role in determining male sterility in the cytoplasm. orf138 was up-regulated in the CMS line and F1 hybrid at three bud developmental stages, including meiosis and the tetroid and microspore stages, but it was not expressed in the maintainer and restorer line, suggesting that the Rs-Rf 1 gene was not able to block orf138 transcription. As in the F1 plant, the transcription of Rs-Rf 1 was gradually up-regulated in the restorer line with progressive stages of bud development, but it was not transcribed in the CMS line and maintainer. Different restriction sites for SspI between the allele of Rs-Rf 1 and Rs-rf 1 were also identified, and a guideline for using the functional marker to distinguish the genotypes of individuals with Rs-Rf 1 was developed. Due to its co-dominant character, this marker could be used to discriminate the heterozygous and homozygous fertility-restoring allele. Using this functional marker, we established a system of molecular marker-assisted development of elite CMS lines in radish. This system will contribute greatly to candidate maintainer identification and accelerate the process of elite CMS line development in late-bolting radish breeding programs.