1 Introduction

Hybrid rice (Oryza sativa L.) has been a milestone in the quest to enhance global rice productivity. Initiated in China during the 1970s, hybrid rice breeding has significantly increased grain yields by over 20% compared to traditional inbred varieties (Yuan, 1966; Shi, 1985; Deng et al., 1999; Li et al., 2007). This remarkable improvement has led to the widespread adoption of hybrid rice in regions such as Africa, Southern Asia, and America (Tan and Chen, 2015; Zhu, 2016). The ability to utilize heterosis, or hybrid vigor, through hybrid rice cultivation has been instrumental in meeting the growing food demands of an increasing global population (Murakami et al., 2018). The development and utilization of hybrid rice are crucial for ensuring food security and addressing the challenges posed by limited arable land and changing climatic conditions.

MS in plants refers to the inability of a plant to produce functional pollen, which is essential for fertilization and seed production. This trait is particularly valuable in hybrid breeding programs as it facilitates controlled cross-pollination. In rice, MS can be broadly categorized into two types, CMS and GMS. CMS is maternally inherited and results from mutations in the mitochondrial genome, while GMS is controlled by nuclear genes and can be influenced by environmental factors such as photoperiod and temperature (Li et al., 2007; Wang et al., 2013). There are several broadly utilized indica and japonica subtypes of CMS, mainly including WA (Wild Abortive), HL (Hong Lian), BT (Chinsurah Boro II ), and DT (Dian), each associated with specific genetic markers and restorer genes (Zhu et al., 2009; Tan and Chen, 2015; Zhu, 2016; Hu and Qian, 2022; Zang et al., 2024). Environment-sensitive genic male sterility (EGMS) can be further divided into photoperiod-sensitive (PGMS), temperature-sensitive (TGMS) and humidity-sensitive (HGMS) types, which are regulated by specific genetic loci (Xue et al., 2018; Chen et al., 2020; Sun et al., 2021).

The primary objective of this paper is to provide a comprehensive overview of the current status and future prospects of MS genes in hybrid rice. This includes the identification and functional analysis of key genes involved in MS, as well as the molecular mechanisms underlying this trait. By synthesizing recent advancements in genomics, proteomics, and biotechnology, this review aims to highlight the potential applications of MS genes in improving hybrid rice breeding programs. Additionally, we discuss the challenges and opportunities associated with the development of novel MS systems and their implications for global food security. Through this review, we seek to provide valuable insights for researchers, breeders, and policymakers involved in the advancement of hybrid rice technology.

2 Overview of MS in Rice

MS in rice is a crucial trait for hybrid rice breeding, enabling the production of high-yielding hybrid varieties. MS can be classified into three main types like CMS, GMS, and EGMS. Each type has distinct genetic and molecular mechanisms that contribute to their utility in hybrid rice breeding programs.

2.1 CMS

CMS is a maternally inherited trait caused by the interaction between mitochondrial and nuclear genomes. CMS results in the dysfunction of pollen and anther development, leading to MS. The CMS trait is controlled by specific mitochondrial genes, which can be counteracted by nuclear-encoded restorer of fertility (Rf) genes that restore male fertility (Chen and Liu, 2014; Li et al., 2007; Song et al., 2020; Tang et al., 2016; Sun et al., 2021). For instance, the WA352 gene in the WA type of CMS interacts with the mitochondrial copper chaperone COX11, leading to the production of reactive oxygen species and resulting in MS (Wang et al., 2018). The CMS system has been widely utilized in hybrid rice breeding due to its stable inheritance and the ability to produce high-yielding hybrids.

2.2 GMS

GMS is controlled by nuclear genes and does not involve the cytoplasmic genome. GMS can be induced by mutations in specific nuclear genes that are essential for pollen development. The GMS system is advantageous because it allows for the easy manipulation of MS through traditional breeding methods. Recent advancements have utilized CRISPR/Cas9 technology to create GMS mutants, such as the CYP703A3-deficient male-sterile mutant, which can be used in hybrid rice production (Song et al., 2020). GMS systems are characterized by their stable sterility and the ability to combine freely with other genetic backgrounds, making them valuable for hybrid breeding.

2.3 EGMS

EGMS includes PGMS, TGMS and HGMS. EGMS lines exhibit MS under specific environmental conditions, such as changes in day length or temperature or humidity, and revert to fertility under different conditions. This reversible sterility is controlled by nuclear genes and can involve epigenetic regulation by noncoding RNAs (Li et al., 2007; Chen and Liu, 2014; Tang et al., 2016; Toriyama, 2021). For example, the P/TGMS line Peiai64S (PA64S) shows MS under high temperatures and long daylight conditions, with miRNAs such as miR156, miR5488, and miR399 playing roles in regulating this sterility (Sun et al., 2021). The TGMS trait of AnS-1 was found to be caused by C-to-A mutation in TMS5, resulting in a premature stop codon in the RNase Z protein (Zhou et al., 2014; Yan et al., 2024). HGMS is a new type of EGMS discovered in rice recent year, it has defects in the structure of the pollen wall, and the pollen is prone to dehydration and inactivation in low humidity environments, but remains viable in high humidity environments (Xue et al., 2018; Chen et al., 2020) (Figure 1). EGMS systems are particularly useful for hybrid seed production as they allow for the easy multiplication of MS lines under appropriate environmental conditions.

Figure 1 NPhenotypes of humidity-sensitive genic male sterility 1 (hms1) mutants in different humidity conditions (Adopted from Xue et al., 2018) Image caption: (a) Whole-plant morphology of rice (Oryza sativa) Zhonghua11 (ZH11) and the hms1 mutant grown in a paddy field in Guangzhou, China (50%~90% relative humidity (RH)). (b) Seed setting of the ZH11 and hms1 plants grown in the paddy field. (c) Whole-plant morphology of ZH11 and hms1 plants grown in an artificial climate chamber (75% RH). (d) Seed setting of ZH11 and hms1 plants grown in an artificial climate chamber. (e) Seed setting of ZH11. (f-i) Seed setting of hms1 plants at 45% (f), 55% (g), 60% (h) and 75% RH (i). (j) Seed-setting rates of ZH11 and hms1 plants at different humidity percentages. Bars: (a, c) 20 cm; (b, d-i) 5 cm. Error bars indicate SD (n=5) (two-tailed Student’s t-test; **, P < 0.01) (Adopted from Xue et al., 2018)

2.4 Importance of MS in hybrid rice breeding

MS is a cornerstone of hybrid rice breeding, enabling the production of high-yielding hybrid varieties through the exploitation of heterosis or hybrid vigor. The use of CMS, GMS, and EGMS systems has significantly contributed to the success of hybrid rice breeding programs worldwide. CMS systems, such as the WA, HL, BT, DT types, have been extensively used for over 40 years, leading to substantial increases in rice productivity (Chen and Tan, 2015; Toriyama, 2021). GMS and EGMS systems offer additional flexibility and ease of use in breeding programs, allowing for the development of diverse hybrid combinations (Tang et al., 2016; Chen et al., 2020; Song et al., 2020). The integration of molecular and genetic insights into MS mechanisms continues to enhance the efficiency and effectiveness of hybrid rice breeding, ensuring food security and agricultural sustainability (Li et al., 2007; Chen and Liu, 2014; Sun et al., 2021; Jiang et al., 2022).

3 Identification of MS Genes

3.1 Traditional genetic approaches

Mutagenesis and phenotypic screening have been fundamental in identifying MS genes in rice. For instance, a GMS gene, ms-h (t), was induced by chemical mutagenesis and mapped to chromosome 9. This gene was found to have a pleiotropic effect on chalky endosperm, making it a valuable resource for understanding the biochemical mechanisms underlying MS and its related traits (Koh et al., 1999).

Genetic mapping and QTL analysis have been extensively used to locate MS genes. The TGMS gene tms3(t) was mapped using bulked segregant analysis (BSA), identifying several RAPD markers linked to the gene on chromosome 6 (Subudhi et al., 1997). Similarly, two fertility restorer loci for the WA-CMS system were mapped to chromosomes 1 and 10, providing crucial markers for marker-assisted selection in hybrid rice breeding programs (Suresh et al., 2012). Additionally, qCTR5 and qCTR12 for cold tolerance and anther length affecting MS were identified on chromosomes 5 and 12 respectively, with specific candidate genes linked to these QTLs (Shimono et al., 2016).

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 SaM genes in indica-japonica hybrids restores pollen viability, demonstrating their direct involvement in pollen abortion (Xie et al., 2017).

Environmental factors, particularly temperature, significantly impact the expression and function of MS genes. The TGMS system in rice, governed by genes like TMS5, is highly sensitive to temperature changes, with specific mutations leading to sterility at higher temperatures and fertility at lower temperatures (Zhou et al., 2016; Xu et al., 2020; Fang et al., 2022). This temperature-dependent regulation is crucial for the practical application of TGMS lines in hybrid rice breeding, allowing for controlled fertility based on environmental conditions. The functional analysis of MS genes in hybrid rice involves a combination of advanced genomic tools, detailed expression studies, and an understanding of the physiological and biochemical mechanisms underlying MS. These insights are essential for developing efficient hybrid breeding strategies and overcoming reproductive barriers in rice and other crops.

5 Case Studies of Key MS Genes in Rice

5.1 osnp1-1 mutant gene

To uncover new rice genes that regulate MS, Chang et al. (2016) generated an ethyl methanesulfonate-induced mutant library in Huanghuazhan (HHZ), a prominent indica cultivar in China, and screened for MS mutants. Among these, a mutant named osnp1-1 exhibited complete MS but maintained normal vegetative growth, inflorescence, and flower morphology (Figure 2A). The osnp1-1 mutant had small, whitish anthers (Figure 2B) devoid of pollen grains (Figure 2C). This MS phenotype was stable under varying day lengths (10 h~16 h) and temperature ranges (20 °C~38 °C). Additionally, approximately 87%~92% of spikelets displayed the extrusion of one or both stigmas (Figure 3D). Cross-pollination with wild-type (WT) HHZ under natural field conditions resulted in a seed set of 40% or higher. These characteristics suggested that osnp1-1 could be a valuable MS line for hybrid rice production. Back-crossing with WT HHZ produced fertile F1 progeny, and the F2 population showed a 3:1 segregation ratio of fertile to sterile plants (216:68), indicating that osnp1-1 is a single recessive mutation (Chang et al., 2016).

Figure 2 NPhenotypes of osnp1-1 and molecular identification of OsNP1 (Adopted from Chang et al., 2016) Image caption: This image shows in detail the changes in the osnp1-1 mutant at the morphological and molecular levels, and provides important information about the role of the OsNP1 gene in rice flower organ development (Adopted from Chang et al., 2016)

5.2 Dominant GMS gene

Dominant GMS plants are a type of germplasm resource that is extremely rare in nature, with only a few regulatory genes having been cloned. As early as 2001, the Academy of Agricultural Sciences in Sanming City, Fujian Province, discovered a natural mutant plant exhibiting dominant nuclear sterility in rice. This mutant exhibited stable and complete sterility, good exsertion of stigmas, and unaffected female fertility, demonstrating significant application potential. However, the gene regulating Sanming Dominant Genic Male Sterility (SDGMS) had remained uncloned.

Recently, Huazhong Agricultural University and Nanjing Agricultural University simultaneously reported on the cloning and functional studies of the same gene locus, designated SDGMS and OsRIP1, respectively. The gene encodes a typical ribosome-inactivating protein (RIP), which inhibits protein synthesis at the translational level. This inhibition results in a defense response in the anthers of the sterile plants during meiosis, triggering intense programmed cell death (PCD) in the tapetum, leading to male sterility (Li et al., 2023; Xu et al., 2023 ). This is the first cloned Dominant Genic Male Sterility (DGMS) gene in rice, providing new insights into the role of transposable elements in genome and phenotypic evolution. It also lays the foundation for further application of SDGMS germplasm resources. The utilization of DGMS genes promises to eliminate the need for emasculation in rice hybrid breeding and genetic research, thus significantly saving labor and resources on a large scale, and potentially giving rise to new breeding models.

5.3 EGMS genes

EGMS includes PGMS, TGMS and HGMS. These systems are highly valuable for hybrid rice breeding due to their sensitivity to environmental conditions. The PGMS mutant NK58S, identified in 1973, has been pivotal in the development of two-line hybrids. Key loci such as PMS1 and PMS3 encode long noncoding RNAs (Ding et al., 2012), while the TGMS locus TMS5 encodes an RNase Z (Fan and Zhang, 2017). The mapping of TGMS genes, such as tms3(t) on chromosome 6, further underscores the genetic complexity and potential for marker-assisted selection (MAS) breeding (Song et al., 2020). Furthermore, the identification of miRNAs such as miR156, miR5488, and miR399 in PA64S P/TGMS rice highlights the complex regulatory networks involving GMS genes (Sun et al., 2021).

The study previously reveals that tms5 carries a mutation in ribonuclease ZS1, the latest report demonstrate that TMS5 is a tRNA 2′,3′-cyclic phosphatase. The tms5 mutation leads to accumulation of 2’,3’-cyclic phosphate (cP)-ΔCCA-tRNAs (tRNAs without 3’ CCA ended with cP), which is exacerbated by high temperatures, and reduction in the abundance of mature tRNAs, particularly alanine tRNAs (tRNA-Alas) (Figure 3) (Yan et al., 2024).

Figure 3  Preventing the generation of cP-ΔCCA-tRNAs fully restores the male fertility of Zhu1S (Adopted from Yan et al., 2024) Image caption: a: Total levels of cP-ΔCCA-tRNAs in Zhu1, Zhu1S, and tms5 osvms1-1 grown in the field (2021, DAT, ~26.5 °C) by RcP-RNA-seq. B: Box plots show the global abundance of mature tRNAs in Zhu1, Zhu1S, and tms5 osvms1-1 grown in the field (2021, DAT, ~26.5 °C). c: Pollen fertility and plant morphology of tms5 osvms1-1, tms5 osvms1-2, and two independent complementation lines (OsVms1C-1 and OsVms1C-2) grown in the field (2021, DAT, ~26.5 °C). d: Statistical analysis of pollen fertility in c. e: Pollen fertility of tms5 osvms1-1, tms5 osvms1-2, and complementation line OsVms1C-1 at 22 °C, 25 °C and 30 °C. f: Statistical analysis of pollen fertility in e. Error bars indicate standard deviation (SD) (n=10 replicates). Significant differences were determined by oneway ANOVA with Tukey’s multiple comparisons test with different letters at P<0.05 (Adopted from Yan et al., 2024)

The HGMS lines were discovered more recently, and show fertility under high humidity and sterility under low humidity. Xue et al. (2018) demonstrate that deficiency of a triterpene pathway results in HGMS in rice, OsOSC12/OsPTS1 encodes a triterpene synthase, which affects the biosynthesis of C16 and C18 fatty acids in tryphine and regulates HGMS in rice. Another study reveals the molecular mechanism by which the rice HMS1 and HMS1I genes interact to regulate the synthesis of very-long-chain fatty acids and the formation of the oil layer in the pollen wall, thereby controlling HGMS (Chen et al., 2020).

5.4 Comparative analysis of gene function and regulation

Comparing the function and regulation of CMS, GMS, and EGMS genes reveals distinct mechanisms and regulatory pathways. CMS genes often involve mitochondrial-nuclear interactions, with nuclear restorer genes (Rf) playing a crucial role in fertility restoration (Mishra and Bohra, 2018). In contrast, GMS genes are primarily nuclear and can be manipulated through genetic engineering techniques such as CRISPR/Cas9 (Song et al., 2020). EGMS genes, regulated by environmental factors, involve complex epigenetic controls and noncoding RNAs, which add another layer of regulation (Tang et al., 2016). The integration of high-throughput sequencing and molecular mapping techniques has significantly advanced our understanding of these regulatory networks, providing new strategies for hybrid rice breeding (Fan and Zhang, 2017; Sun et al., 2021). By examining these case studies, we gain a comprehensive understanding of the diverse genetic and molecular mechanisms underlying male sterility in rice, paving the way for future innovations in hybrid rice breeding.

6 Application of MS Genes in Rice Breeding

6.1 Development of MS lines

The development of MS lines is a critical step in hybrid rice breeding. MS can be induced through various genetic and cytoplasmic mechanisms. For instance, the FA-CMS system, controlled by the mitochondrial gene FA182, has shown stable MS, which can be restored by a single nuclear gene, OsRf19. This system simplifies the breeding process and has demonstrated promising results in developing high-performing hybrids (Jiang et al., 2022) (Figure 4). Additionally, the CRISPR/Cas9-mediated editing of the TMS5 gene has accelerated the development of TGMS lines, which are crucial for hybrid seed production (Zhou et al., 2016).

Figure 4 NFunctional analysis of the CMS gene FA182 and the restorer gene OsRf19 (Adopted from Jiang et al., 2022) Image caption: The image provided contains five panels labeled A to E, each showing different aspects of the functional analysis of the CMS gene FA182 and the restorer gene OsRf19 in rice (Adopted from Jiang et al., 2022)

6.2 Hybrid seed production systems

The two-line system involves the use of EGMS lines that are sensitive to environmental conditions such as temperature or photoperiod or humidity. For example, the CRISPR/Cas9 system has been used to create GMS lines by editing the OsOPR7 gene, which can be restored to fertility by exogenous methyl jasmonate, thus establishing a two-line system for hybrid rice production (Pak et al., 2020). This system offers flexibility and efficiency in hybrid seed production. In addition, a third-generation hybrid rice system has succeeded in propagating and utilizing recessive nuclear MS lines using a transgenic construct-driven nongenetically modified (GM) system called seed production technology (Chang et al., 2016; Wu et al., 2016). Such a genetically engineered MS system has the ability to propagate nontransgenic MS seeds for hybrid rice seed (HRS) production and to overcome the intrinsic problems of the first two generations of hybrid rice systems (Wu et al., 2016; Zhang et al., 2018).

The three-line system utilizes CMS lines, maintainer lines, and restorer lines. The CMS/Rf system, such as the WA-, HL-, BT-, DT-CMS, is widely used, with fertility restoration controlled by specific nuclear genes. For instance, the mapping of fertility restorer loci on chromosomes 1 and 10 has provided valuable markers for MAS in hybrid rice breeding programs (Yao et al., 2004; Tan and Chen, 2015; Zhu, 2016). The integration of both cytoplasmic and nuclear genome variations has further informed the breeding strategies for hybrid rice (Wan et al., 2019).

6.3 Enhancing hybrid rice yield and stability

Enhancing the yield and stability of hybrid rice involves the continuous exploration and application of MS genes. The identification and characterization of GMS genes have deepened our understanding of the molecular mechanisms controlling anther and pollen development, facilitating the development of biotechnology-based MS systems (Zhou et al., 2016; Wan et al., 2019). The use of novel strategies, such as combining CMS and GMS genes, has led to the creation of third-generation hybrid rice technology (Wang and Deng, 2018; Song et al., 2020）, which offers stable sterility and improved hybrid seed production (Chang et al., 2016; Li et al., 2022).

6.4 Challenges and solutions in practical breeding

Despite the advancements, several challenges remain in the practical breeding of hybrid rice. The genetic complexity of MS and fertility restoration systems can complicate the breeding process. However, the development of single-gene inheritance systems, such as the FA-CMS/OsRf19 system, has simplified the breeding process and improved hybrid performance (Jiang et al., 2022). Additionally, the use of CRISPR/Cas9 technology has accelerated the development of sterile lines, addressing the time constraints associated with traditional breeding methods (Figure 5) (Chang et al., 2016; Abbas et al., 2021). The continuous exploration of genetic and biotechnological approaches will be essential to overcome these challenges and enhance the efficiency of hybrid rice breeding. By leveraging these advancements, the future prospects of hybrid rice breeding look promising, with the potential to significantly improve rice productivity and ensure global food security.

Figure 5 Genetic network of genic male sterility (GMS) genes for anther and pollen development including lipid and polysaccharide metabolism (Adopted from Abbas et al., 2021) Image caption: The diagram serves as a comprehensive visual representation of the intricate genetic and molecular processes involved in anther and pollen development in rice, particularly focusing on the role of GMS genes and their associated metabolic pathways (Adopted from Abbas et al., 2021)

7 Future Prospects and Research Directions

7.1 Emerging technologies in gene identification and functional analysis

The rapid advancement of biotechnological tools has significantly enhanced our ability to identify and functionally analyze MS genes in rice. Techniques such as CRISPR/Cas9 genome editing, RNA sequencing, and high-throughput phenotyping are revolutionizing the field. For instance, the use of small RNA, transcriptome, and degradome sequencing has revealed the involvement of miRNAs like miR156, miR5488, and miR399 in regulating male sterility in P/TGMS rice (Jiang et al., 2022). Additionally, the identification of single nucleotide polymorphisms (SNPs) in key regulatory genes, such as the R2R3 MYB TF gene in soybean, underscores the potential of SNP analysis in understanding MS mechanisms (Yu et al., 2021).

7.2 Integration of genomics and breeding strategies

Integrating genomics with traditional breeding strategies holds promise for developing superior hybrid rice varieties. The characterization of CMS and Rf genes, such as the FA-CMS and OsRf19, has simplified the breeding process and improved hybrid performance (Song et al., 2022). Moreover, mapping and genetic analysis of PGMS genes, like pms1 and pms2, have provided valuable insights into the genetic basis of MS, facilitating MAS in breeding programs (Wan et al., 2019; Jiang et al., 2022).

7.3 Addressing climate change and environmental variability

Climate change and environmental variability pose significant challenges to rice production. Understanding the genetic and molecular mechanisms underlying MS in response to environmental factors is crucial. Studies have shown that temperature or photoperiod or humidity can influence the expression of MS genes, as seen in the regulation of P/TGMS rice by miRNAs and their target genes (Jiang et al., 2022). Additionally, the identification of novel noncoding RNAs that produce small RNAs, such as osa-smR5864w, highlights the complex interaction between genetic networks and environmental conditions in controlling MS (Yu et al., 2021).

7.4 Ethical and biosafety considerations

The application of emerging biotechnologies in rice breeding raises important ethical and biosafety considerations. The development and deployment of genetically modified organisms (GMOs) must be carefully regulated to ensure environmental safety and public acceptance. The potential risks associated with the release of MS lines and their impact on biodiversity need thorough assessment. Furthermore, the ethical implications of using advanced genetic tools, such as CRISPR/Cas9, must be addressed to balance innovation with societal concerns. Ensuring transparent communication and stakeholder engagement will be key to navigating these challenges.

In conclusion, the future of hybrid rice breeding lies in the integration of cutting-edge technologies with traditional breeding practices, addressing environmental challenges, and adhering to ethical and biosafety standards. Continued research and collaboration will be essential to harness the full potential of MS genes in improving rice productivity and ensuring global food security.

8 Concluding Remarks

The findings from these studies have significant implications for hybrid rice improvement. The CMS/Rf of WA- HL-, BT-, DT-, and FA-CMS/OsRf19 systems offer a promising breeding strategy that can enhance the efficiency and stability of hybrid rice production. The identification of PGMS, TGMS and HGMS genes and their chromosomal locations facilitates the development of new EGMS lines, which are crucial for hybrid rice breeding programs. Understanding the regulatory mechanisms of miRNAs in P/TGMS rice provides new avenues for manipulating MS and improving hybrid rice varieties. Furthermore, the evolutionary trajectory of new CMS genes, such as WA352c, has been reconstructed, providing a model for the formation and evolution of these genes. The insights into the evolution of CMS genes can guide the development of new CMS lines with improved traits, thereby enhancing the genetic diversity and adaptability of hybrid rice.

The identification and functional analysis of MS genes have advanced our understanding of the genetic and molecular basis of MS in hybrid rice. These findings offer valuable tools and strategies for improving hybrid rice breeding programs. Future research should focus on further elucidating the molecular mechanisms underlying MS and exploring new genetic elements that can be utilized for hybrid rice improvement. Additionally, the integration of advanced biotechnological methods, such as CRISPR/Cas9, the third generation of genetic engineering GMS lines, can accelerate the development of new MS systems and enhance the efficiency of hybrid rice breeding. Continued efforts in this field will contribute to the sustainable production of hybrid rice and global food security.

Acknowledgments

We extend our sincere thanks to two anonymous peer reviewers for their invaluable feedback on the initial draft of this paper, whose critical evaluations and constructive suggestions have greatly contributed to the improvement of our manuscript.

Funding

This work was supported by the grants from the Central Leading Local Science and Technology Development Project (grant no. 202207AA110010) and the Key and Major Science and Technology Projects of Yunnan (grant nos. 202202AE09002102, 202402AE090026-04).

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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