BrSKS13, a multiple-allele-inherited male sterility-related gene in Chinese cabbage (Brassica rapa L. ssp. pekinensis), affects pollen development and pollination/fertilization process

Abstract

Multiple-allele-inherited male sterility (MAMS) is important in Chinese cabbage (Brassica rapa L. ssp. pekinensis) breeding, but the molecular mechanisms leading to male sterility are poorly understood. In this study, we cloned a novel gene, BrSKS13, that is differentially expressed in fertile and sterile flower buds of Chinese cabbage. BrSKS13 is most similar to Arabidopsis thaliana AT3G13400 (SKS13) and encodes a predicted 61.87 kDa protein with three cupredoxin superfamily conserved domains in the multicopper oxidase family. Semi-quantitative reverse-transcription PCR (sqRT-PCR) showed that expression of BrSKS13 is higher in fertile buds than in sterile buds. Quantitative RT-PCR (qRT-PCR) and in situ hybridization showed that BrSKS13 is highly expressed in fertile anthers, peaking at pollen-maturation stage VI, but is weakly expressed in other tissues and floral organs. Expression patterns of BrSKS13 promoter::GUS reporter fusions in Arabidopsis showed that the BrSKS13 promoter drives expression of the GUS gene only in anthers. The relative expression of Brsks13 in fertile buds was higher than in sterile buds for all other MAMS lines of Chinese cabbage examined. These results suggest that BrSKS13 affects pollen development. In situ hybridization analysis of flower stigmas at different times after pollination showed that BrSKS13 expression was first observed in stigmas and immature seeds at 1 h after pollination, and the signal intensity in seeds increased with increasing maturity. Compared to Col-0, A. thaliana sks13 mutant plants have shorter and fewer siliques, shriveled pollen grains, pollen tube abnormalities, and reduced seed number. The phenotype of sks13 mutant was recovered by over-expressing BrSKS13. Our results suggest that BrSKS13 affects pollen development and the pollination/fertilization process, and will enable further study of the genetic mechanisms underlying MAMS in Chinese cabbage.

Keywords: Brassica rapa L. ssp. pekinensis; In situ hybridization; Multiple allele-inherited male sterility; Over-expression; Pollen development.