3.4 Anther-specific expression analysis

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].

has been shown to be unreliable after successive generations [95].

3.3 Allelism test and allelic mutant sequencing

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

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

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

3.2 Targeted mutagenesis

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[18, 20, 67, 76].

84

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].
