Rice Genomics and Genetics 2025, Vol.16, No.1, 1-13 http://cropscipublisher.com/index.php/rgg 4 3.2 Molecular and genomic approaches High-throughput sequencing technologies have revolutionized the identification of MS genes. For example, whole genome sequencing and de novo assembly of the mitochondrial genome identified the chimeric gene orf312, which is associated with the Tetep-CMS in rice. This gene was found to encode a peptide toxic to Escherichia coli and inhibited cell growth, highlighting its potential role in MS (Jin et al., 2021). Comparative genomics and transcriptomics have provided deeper insights into the genetic basis of MS. The identification of S5-interacting genes (SIG) regulating hybrid sterility in rice involved mapping four QTLs and analyzing their effects using near-isogenic lines (NIL). This study highlighted the complex genetic interactions underlying hybrid sterility and provided a basis for further fine-mapping and functional analysis (Rao et al., 2021). Additionally, the identification of a toxin-antidote system involving ORF2 and ORF3 in the qHMS7 locus demonstrated how selfish genetic elements can drive reproductive isolation and MS in inter-subspecific hybrid rice (Yu et al., 2018). By integrating traditional genetic approaches with advanced molecular and genomic techniques, significant progress has been made in identifying and understanding the functional mechanisms of MS genes in hybrid rice. These findings not only enhance our knowledge of plant reproductive biology but also have practical implications for improving hybrid rice breeding programs. 4 Functional Analysis of MS Genes 4.1 Gene cloning and characterization The CRISPR-Cas9 system has revolutionized the functional analysis of MS genes in hybrid rice. This genome editing tool allows for precise mutations in target genes, facilitating the study of their roles in MS. For instance, the TMS5 gene, a key player in TGMS, has been successfully edited using CRISPR-Cas9 to create new TGMS lines, significantly accelerating hybrid rice breeding (Zhou et al., 2016; Fang et al., 2022). Similarly, the SaF and SaM genes, which cause hybrid MS in indica-japonica hybrids, were knocked out using CRISPR-Cas9, resulting in hybrid-compatible lines (Xie et al., 2017). This demonstrates the utility of CRISPR-Cas9 in overcoming reproductive barriers and enhancing hybrid breeding programs. Complementation and knockout studies are essential for validating the function of MS genes. For example, the ZmMs33 gene in maize, which encodes a glycerol-3-phosphate acyltransferase, was identified through map-based cloning. Functional complementation experiments confirmed that ZmMs33 can rescue the male-sterile phenotype, while CRISPR-Cas9-induced knockouts validated its role in male fertility (Xie et al., 2018). Additionally, multiplex gene editing has been employed to simultaneously mutate multiple homologous genes, such as ZmTGA9-1/-2/-3, to study their collective impact on male fertility in maize (Li et al., 2019). 4.2 Expression analysis and regulation Understanding the temporal and spatial expression patterns of MS genes is crucial for elucidating their roles in pollen development. For instance, ZmMs33 is preferentially expressed in immature anthers during specific microspore stages and in root tissues at the fifth leaf growth stage, indicating its critical role in early pollen development (Xie et al., 2018). Similarly, the expression of TMS5 in rice is regulated by temperature, with specific mutations leading to complete MS at higher temperatures and restored fertility at lower temperatures (Fang et al., 2022). The regulatory networks and signaling pathways involving MS genes are complex and multifaceted. Proteomic analyses have revealed that mutations in tms5 result in significant changes in protein expression, affecting various biosynthetic and metabolic pathways (Fang et al., 2022). Additionally, transcription factors (TFs) such as ZmbHLH51 and ZmbHLH122 have been shown to interact and regulate the expression of other genes involved in male fertility, highlighting the intricate regulatory networks at play (Jiang et al., 2021). 4.3 Physiological and biochemical mechanisms MS genes play a pivotal role in pollen development and viability. For example, the ZmMs33 gene is essential for the proper formation of anthers and viable pollen grains in maize (Xie et al., 2018). The suppression of SaF and
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