Mitogenomic comparison identifies rpl16 as a CMS driver via ROS dysregulation in pearl millet

Cytoplasmic male sterility (CMS) caused by mitogenomic variation underlies CMS lines essential for hybrid crop production. However, the role of CMS in pearl millet remains unclear. To clarify the function of CMS in pearl millet ( Pennisetum glaucum ), the mitochondrial genomes of the CMS line ‘23A’ (L23A) and its maintainer ‘23B’ (L23B) were sequenced and further assembled. With a mitochondrial genomic comparison, an rpl16 (ribosomal protein L16-like protein) gene in L23A that was absent in L23B was successfully identified. An analysis of RT-PCR and qRT-PCR validated that rpl16 was specifically expressed in the inflorescences of L23A. Overexpression of the rpl16 gene in rice truly induced pollen abortion, further leading to a reduced grain number. When compared to L23B at earlier stages of millet anther development, a higher ROS level and premature tapetal degradation (PCD) in L23A were observed through a reactive oxygen species (ROS) staining and anther paraffin section. The protein interaction between RPL16 and NADH dehydrogenase subunit 1 (NDUFV1) may indicate a potential role for the rpl16 gene in disrupting anther ROS homeostasis in the CMS line, suggesting an important role of CMS in hybrid millet breeding