3. Functional confirmation methods of GMS genes in crop plants

As described above, once the putative GMS gene has been cloned, it should be tested by using a series of experiments, including transgenic complementation, targeted mutagenesis, allelism test and allelic mutant sequencing, anther-specific expression analysis, phylogenetic analysis, and cytological observation (Figure 4).

#### 3.1 Transgenic complementation

sequence and tries to gain insight into the underlying function by selecting for mutations that disrupt the sequence and thereby its function. The reverse genetic approaches for GMS gene cloning include homology-based cloning, anther-specific

Homology-based cloning is a simple and straightforward method of GMS gene cloning, and it relies on the conservation in sequence and function of the reference GMS gene among different species, mainly through the sequence alignment and phylogenetic analysis of the related GMS genes. As a lot of GMS genes have been cloned in model plants and the genome sequencing information become available for most important crops [88], there are several GMS genes that have been identified through homology-based cloning approach, such as OsTDF1, OsACOS12, OsCER1, OsIG1, OsRAFTIN,TaMs26, and TaMs45 (Table 1). In Arabidopsis and rice, the molecular, genetic, and biochemical pathways regulating anther and pollen development have been extensively studied [89, 90], revealing the same number of developmental stages (14) and relatively conserved regulatory pathways in both species [91, 92]. The information about GMS obtained in Arabidopsis and rice provides opportunities to identify and utilize male sterility in economically important crops such as maize, barley, and wheat where GMS systems are not as well characterized [88, 93]. Based on this gene cloning strategy, about 62 putative maize GMS genes have been predicted and analyzed recently [3], and this will greatly enlarge the GMS gene number after functional confirmation via multiple methods (refer to

Given that most of GMS genes show anther-specific expression pattern, some putative GMS genes can be isolated by differential screening of the anther cDNA library, such as the GMS genes OsC6, OsG1, OsADF, and OsUAM3 in rice and TaRAFTIN in wheat (Table 1). OsC6, encoding a lipid transfer protein, was reported to be abundantly expressed in tapetal cells of the anther and played a crucial role in the development of lipidic orbicules and pollen exine during another development in rice [78, 94]. OsG1 was originally cloned from a rice anther cDNA library, encoding a β-1,3-glucanase and belonging to the defense-related subfamily A. OsG1 was essential for callose degradation in tetrad dissolution, and its silencing results in male sterility [79]. OsADF, encoding an anther development F-box protein, was obtained from a rice panicle cDNA clone and played a critical role in rice

tapetum cell development and pollen formation [80]. OsUAM3 (UDP-

structural protein that is essential for pollen development [75].

2.2.3 Other reverse genetic approaches

82

arabinopyranose mutase 3) was identified by screening the expression patterns of the OsUAM genes in various vegetative and reproductive tissues and found to be a unique gene required for pollen wall morphogenesis in reproductive development [81]. TaRAFTIN was identified from an anther cDNA library of hexaploid wheat and cloned by using the RACE-PCR method, encoding a sporophytically produced

Once a GMS gene is cloned and characterized, its interaction protein or targeted gene may be involved in male-sterility regulation, too. Therefore, some of the GMS genes could be isolated by other reverse genetic approaches, such as chromatin immunoprecipitation sequencing (ChIP-Seq), genome-wide expression analysis of

expression gene screening, and other methods.

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2.2.2 Anther-specific expression gene screening

2.2.1 Homology-based cloning

Section 3).

Transgenic complementation is an essential tool and an effective way to confirm the function of a putative GMS gene. Based on the difference of transformation receptors, it includes two ways. The first one is transformation of the male-sterile mutants from which the GMS gene has been cloned, and then observation of the malefertility phenotype in transgenic plant. For example, maize ZmMs7 gene was confirmed by the transformation of proZmMs7-ZmMs7 construct into maize HiII hybrid line.

Figure 4.

The functional confirmation approaches of GMS genes in crop plants.

The transgenic plants were then crossed with the ms7-6007 mutant, and transgenic plant in the ms7-6007 homozygous mutant background can rescue the male-sterility defect of ms7-6007 mutant and recovered the fertility phenotype (Table 1) [9]. The second one is transformation of the corresponding heterozygous male-sterility mutants with the orthologous GMS gene in model plants (such as Arabidopsis) and segregation analysis of complementation by the transgene. The putative GMS ortholog needs to be fused to a promoter to drive its expression in the Arabidopsis mutant, either by a constitutive, overexpression promoter, or via the Arabidopsis native gene-specific promoter. Although the first option is usually quicker, the results are not always satisfactory, due to the temporal and cell-specific regulation observed in some genes. For instance, anther and pollen transcription factors such as AtMs1 orthologs in rice (PTC1) and barley (HvMs1) did not recover Arabidopsis ms1 mutant fertility when driven by the CaMV35S overexpression promoter. However, once the rice and barley ortholog genes were fused to the Arabidopsis AtMs1 native promoter, fertility was restored in the ms1 homozygous mutant [35, 93].

allelic mutants, such as ms33-6019, ms33-6029, ms33-6024, ms33-6038, and ms33-6052 [20, 97]. Most of the cloned GMS genes have allelic mutants and are confirmed by allelic mutant sequencing (Table 1), so it is a usefully functional confirmation strategy besides the genetic complementation and targeted

Molecular Cloning of Genic Male-Sterility Genes and Their Applications for Plant Heterosis…

The anther-specific expression analysis is another important method for functional confirmation of putative GMS gene. In general, the expression pattern of GMS gene could be analyzed by using the following approaches: semiquantitative reverse transcription (RT)-PCR, quantitative real-time RT-PCR (qRT-PCR), northern blotting, promoter-GUS or GMS-GFP transgenic plant analysis, RNA in situ hybridization, and immunoblotting (or western blotting). For instance, the spatiotemporal expression pattern of Ms6021 was analyzed by qRT-PCR, RNA in situ hybridization, and western blotting, and the results indicated that Ms6021 is mainly expressed in the tapetum and microspore in maize [23]. The anther- and tapetum-specific expression pattern of rice PTC1 was analyzed by using RT-PCR, qRT-PCR, and PTC1pro-GUS transgenic rice anther staining [35]. Spatiotemporal expression pattern of TaMs2 was analyzed by using RT-PCR, RNA in situ hybridization, and TaMs2:GFP transgenic anther microscopy, indicating that TaMs2 is an anther-specific expression and dominant GMS gene [50].

In order to get more functional information of the putative GMS gene, phylogenetic analysis should be carried out for expounding the evolutionary relationship with other putative orthologs. The detailed method is as follows: protein sequences of the putative orthologs of the targeted GMS can be obtained from Gramene (http://www.gramene.org) or NCBI (https://www.ncbi.nlm.nih.gov/) and aligned using ClustalX program [98]. A phylogenetic tree can be generated using molecular evolutionary genetics analysis (MEGA6) program based on a Poisson model with the maximum likelihood method [99]. Support values are estimated by 1000 times of bootstrap replicates. For instance, by using phylogenetic analysis, maize Ms23 and its paralog bHLH122 fall in the same clade with two rice GMS proteins, TIP2 and EAT1. TIP2 is the rice ortholog of Ms23, whereas maize bHLH122 is the ortholog of rice EAT1. Maize bHLH51, rice TDR, and Arabidopsis AMS fall in the same clade, while maize Ms32, rice UDT1, and Arabidopsis DYT1 fall in the same clade [15]. These results not only confirm the function of Ms23 and Ms32 in regulating male sterility but also predict that their paralogs bHLH122 and bHLH51 may be involved in male sterility, and this needs to be confirmed by targeted mutagen-

As to the forward genetic cloning of the GMS gene, cytological observation is one of the phenotypic analyses of the male-sterile mutant. When the candidate GMS gene is cloned by reverse genetic approaches, cytological observation is one of the most important strategies for functional confirmation of the putative GMS gene. Cytological observation methods include light microscopy of transverse sections, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) of anther and pollen development. For instance, the functions of rice OsTGA10 and OsAGO2 in male sterility were confirmed by targeted mutagenesis of these genes

mutagenesis.

3.4 Anther-specific expression analysis

DOI: http://dx.doi.org/10.5772/intechopen.86976

3.5 Phylogenetic analysis

esis analysis and/or other strategies.

3.6 Cytological observation

85

#### 3.2 Targeted mutagenesis

Targeted mutagenesis of the putative GMS gene includes two ways: knockdown and knockout approaches. Knockdown strategy, such as RNA interference (RNAi) silencing, is very helpful to those genes in which null mutant is lethal. RNAi silencing is a useful technique to characterize gene function; however, this approach may not generate clear phenotypes due to the threshold level needed for effective silencing [61]. RNAi target genes generally have reduced expression rather than being fully silenced; thus enough transcript may remain to maintain wild-type function. This partial reduction in gene expression was seen in several GMS gene RNAi silencing [63, 73, 80, 81, 93], where pollen development was affected by the silencing and showed a partial male-sterility phenotype. In addition, RNAi silencing has been shown to be unreliable after successive generations [95].

Knockout strategies, such as zinc finger nucleases (ZFNs), customized homing endonucleases (meganucleases), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9 technology, have been shown to significantly increase the frequency and precision of genome editing. Especially, CRISPR-Cas9 has quickly become the technology of choice for genome editing and functional confirmation of GMS gene due to its simplicity, efficiency, and versatility [96]. For instance, rice OsLAP6/OsPKS1, maize ZmMs30, ZmMs33, and wheat TaMs45 gene are confirmed as GMS genes by using the CRISPR-Cas9 technology, respectively. Targeted mutagenesis of these genes leads to complete male-sterility phenotype [18, 20, 67, 76].

#### 3.3 Allelism test and allelic mutant sequencing

Allelism test (complementation test for functional allelism) is a test to determine whether two mutants are caused by the same gene. If there is more than one mutant of a specific GMS gene, allelism test should be carried out. A male-sterile (ms) homozygote is pollinated by a fertile heterozygote (+/ms) from the putative allelic line. If the progeny exhibits a fertile/ sterile segregation ratio of 1:1, the two mutants are allelic with each other. If all the progenies display male fertile, suggest that the two mutations complement each other and they are not allelic. Furthermore, the allelic mutant gene can be confirmed based on sequencing and alignment with each other. If different ms mutants come from the mutation of the same GMS gene, the GMS gene function in male-sterility will be confirmed. For instance, the function of maize ZmMs33 has been confirmed by allelism test and sequencing of several ms33

### Molecular Cloning of Genic Male-Sterility Genes and Their Applications for Plant Heterosis… DOI: http://dx.doi.org/10.5772/intechopen.86976

allelic mutants, such as ms33-6019, ms33-6029, ms33-6024, ms33-6038, and ms33-6052 [20, 97]. Most of the cloned GMS genes have allelic mutants and are confirmed by allelic mutant sequencing (Table 1), so it is a usefully functional confirmation strategy besides the genetic complementation and targeted mutagenesis.
