Acknowledgements

which produces the hybrid seeds that are sold in the marketplace. A few modifications have been implemented to make the RHS system more practical and manage-

technology combines the relative simplicity and convenience of a systemic herbicide spray methodology with targeted protein expression to create an inducible male sterility system for industrial production of maize hybrid seeds in an environmentally independent manner [102]. The Barnase/Barstar system has been used successfully for the commercial production of canola hybrids (Brassica napus) in Canada [4]. However, most of the BMS systems have not been used in commercial

environment-friendly BMS systems and/or the regulatory acceptance of using BMS

In this chapter, we focus on the molecular cloning, functional confirmation, and application value assessment of GMS genes as well as their application in hybrid seed production via several BMS systems in cereal crops, such as rice, maize, and wheat. With the rapid development of the next-generation sequencing technology, more genome information of cereal crops are available, leading to plenty GMS genes cloned and characterized in crop plants. As shown in Table 1, there are more than 70 GMS genes cloned in cereal crops, and most of them (57/73) are identified by using forward genetic approach, including 38 genes isolated by map-based cloning,

able in the field [101]. Most recently, the second-generation RHS (RHS2)

hybrid seed production, maybe because of lacking the cost-effective and

15 genes identified by T-DNA/Transposon tagging and 4 genes isolated by

for cloning of the orthologs in wheat and barley through reverse genetic approaches. For example, the functions of TaMs26 and TaMs45, the wheat

PTC1, was analyzed by using RNAi silencing in barley [93].

homologs of maize Ms26 and Ms45, were confirmed via a custom-designed homing endonuclease and CRISPR-Cas9-targeted mutagenesis in wheat, respectively [76, 77], while the role of HvMs1, the barley homolog of Arabidopsis Ms1 and rice

Although there are a lot of GMS genes identified in cereal crops up to now, less than 10 GMS genes are assessed for the value in heterosis utilization and hybrid seed production (Table 1). For example, ZmMs7, ZmMs30, and ZmMs33 are tested in maize MCS system [9, 18, 21]; ZmMs26, Zmms44, and ZmMs45 are tested in maize SPT (or SPT-like) system [16, 22, 62]; OsNP1 is tested in rice SPT-like system [69]; and TaMs1 is tested in wheat SPT-like system [49]. All these BMS systems belong to transgenic construct-driven non-transgenic product strategies, leading to potential application of these systems in commercial hybrid seed production, especially in the countries and/or regions with strict regulatory policy. These systems have many advantages, such as non-transgenic final products, environment-friendly without application of herbicide in hybrid seed production fields, and deregulated by the regulatory authority in some countries, whereas they are limited by using

MutMap method. Whereas the rest of GMS genes are identified via reverse genetic approach, including 7 genes isolated through homology-based cloning, 5 genes identified by anther-specific expression gene screening and 4 genes cloned by other reverse genetic methods. Among them, there are 49 GMS genes cloned in rice, 17 GMS genes in maize, 6 GMS genes in wheat, and 1 GMS gene in barley. From these data, we conclude that the forward genetic approaches, especially map-based cloning, are the most popular method for GMS gene cloning in crops; most GMS genes have been cloned in rice and maize, whereas only a few GMS genes are cloned in wheat and barley. Consider that the conserved role of GMS genes in different species and the sequence information of GMS genes in rice and maize can be used

systems among different countries.

Synthetic Biology - New Interdisciplinary Science

6. Conclusions

92

The research in our lab was supported by the National Transgenic Major Program of China (2018ZX0800922B, 2018ZX0801006B), the National Natural Science Foundation of China (31771875, 31871702), the National Key Research and Development Program of China (2018YFD0100806, 2017YFD0102001, 2017YFD0101201), the Fundamental Research Funds for the Central Universities of China (06500060), and the "Ten Thousand Plan"—National High-Level Talents Special Support Plan (For Xiangyuan Wan).

#### Conflict of interest

The authors declared that they have no conflict of interest.

#### Abbreviations



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