Brems1 mutation induced tapetum deficiency leading to male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Male sterile lines are ideal for hybrid seed production in Chinese cabbage. Herein, the complete male sterile mutants M5026 and M5073 were obtained through ethyl methanesulfonate (EMS) mutagenesis in the cabbage double haploid line ‘FT’. Observation of paraffin sections showed that M5026 lacked the tapetum and had excessive microsporocytes. Transmission electron microscopy (TEM) revealed abnormal exine formation in M5026 microspores. Genetic analysis revealed a single recessive nuclear gene caused the male sterility phenotype of M5026. Using Mutmap sequencing and Kompetitive allele-specific PCR (KASP) identification and gene cloning, BraA10g029920.3.5C, encoding EMS1 (Excess microsporocytes 1), a leucine-rich repeat receptor-like protein kinase (LRR-RLK), was identified as the candidate gene of M5026 and named Brems1. A nonsynonymous G-to-A mutation in an exon of the Brems1 gene in M5026 resulted in the substitution of glycine with arginine. Employing Mutmap and cloning approaches, a C-to-T SNP was identified within Brems1 of M5073, resulting in premature translation termination. Both BrEMS1 and Brems1 were subcellularly localized at the cell membrane. qRT-PCR analysis indicated Brems1 exhibited the highest expression level in flower buds, while no expression was detected in roots. Transcriptomic analysis revealed that mutation in Brems1 reduced the expression levels of genes associated with the tapetum, pollen tube, and LRR-RLK family. These results suggested that Brems1 plays a critical role in pollen development and contributes to elucidating the molecular mechanisms underlying tapetum development and male sterility in Chinese cabbage.