3.5 Phylogenetic analysis

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 mutagenesis analysis and/or other strategies.

### 3.6 Cytological observation

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

with antisense and CRISPR-Cas9 systems and cytological characterization of mutants by transverse section observation and TEM analysis of anthers at different stages [84, 85]. The functions of wheat TaMs26 in anther and pollen wall development in bread wheat were tested by targeted mutagenesis of all the three homologs and cytological analysis using SEM method [77]. Cytological observation is helpful to confirm the function mechanism of the putative GMS genes in the cellular level.

of the F2 populations are investigated, and anthers of three sterile individuals in each F2 population are collected and stained with 1% I2-KI solution to examine male-sterility status of pollen grains. If the segregation ratio of fertility to sterility in all crosses shows 3:1 as expected, we can say that the male sterility is genetically stable in different genetic backgrounds and various environments. Otherwise, if the ratio is not always 3:1 and confirmed by the molecular marker-assisted selection results, we can say that the male sterility is unstable in different genetic backgrounds and/or various environments. For instance, the male-sterility stability of maize ms30-6028 mutant was analyzed by crossing with 329 maize inbred lines and

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

observation of the segregation ratio of fertility to sterility in F2 populations, suggesting that ms30-6028 is a stable male-sterility mutant under diverse genetic

Secondly, the effects of ms mutant on heterosis should be analyzed by comparing the yield and related agronomy traits between F1 hybrid plants produced by using ms mutant and wild type (WT) as female parents and crossing with the same inbred line (Figure 5B). For instance, to test whether ms30-6028 gene affects maize heterosis and grain yield, ms30-6028 mutant and its homozygous WT line were used as female parents and crossed with 30 maize inbred lines, respectively. The harvested F1 hybrids and their corresponding parental lines were grown according to the planting model of maize field production in two different locations. Eighteen agronomic traits such as plot yield, whole growth period, plant height, ear height, and hundred-kernel weight were investigated to compare the differences of heterosis and field production performance of 30 pairs of hybrid combinations using ZmMs30 and ms30-6028 homozygous plants as female parents, respectively. The results indicated that ms30-6028 mutation has no obvious negative effects on maize heterosis and field production, suggesting that ZmMs30 gene and its mutant

4.2 Heterosis comparison between ms mutant and wild type

ms30-6028 are applicable for hybrid maize breeding and hybrid seed

4.3 Analysis of potential linkage with disadvantage genes and traits

disadvantage genes based on bioinformatic analysis.

Furthermore, other than the male-sterility stability analysis and heterosis comparison described above, the potential linkage with bad traits of ms locus should be analyzed. There are at least two ways to get this target: one is phenotypic observation of the hybrid plants that come from the homozygous ms mutant used as female parent, while those of the fertile sibling used as control. If the field production performances of the hybrid plants between ms mutant and WT are similar with each other, we can say that the ms mutation is not linked with disadvantage traits and thus can be applicable in hybrid seed breeding and production. For instance, maize Ms44 hybrids showed no yield penalty in any of the tested environments, indicating that it is desirable for commercially viable products [22]. The other is sequencing of the putative genes near the ms locus and screening for the potential

5. Application potential analyses of BMS systems by using GMS genes in

As described above, cloning and characterization of plant GMS genes have contributed significantly to our understanding of the molecular mechanisms of

backgrounds [18].

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

production [18].

crop plants

87
