Abstract

In this chapter, we summarize the strategies about molecular cloning and functional confirmation of plant genic male-sterility (GMS) genes and their applications for hybrid breeding and seed production via biotechnology-based male-sterility (BMS) systems in crop plants. The main content includes four sections: (1) GMS gene cloning strategies, including forward genetic approaches (e.g., map-based cloning, T-DNA or transposon tagging, and MutMap method) and reverse genetic approaches (e.g., homology-based cloning, anther-specific expression gene screening, and other reverse genetic methods); (2) functional confirmation methods of GMS genes, including transgenic complementation, targeted mutagenesis, allelic mutant test and sequencing, anther spatiotemporal expression analysis, and cytological observation; (3) application value assessment of GMS genes and mutants, such as genetic stability analysis of male sterility controlled by GMS genes under different genetic backgrounds and multiple environments, and genetic effects driven by GMS genes on plant heterosis and analysis of potential linkage with bad traits; (4) development and application of BMS systems based on GMS genes and/or their mutants, including transgenic construct-driven non-transgenic seed systems (e.g., seed production technology (SPT) and multi-control sterility (MCS)), and transgenic male-sterility systems (e.g., roundup hybridization systems (RHS1 and RHS2) and Barnase/Barstar system). Finally, we summarize and provide our perspectives on the studies of GMS genes and development of cost-effective and environment-friendly BMS systems in crop plants.

Keywords: gene cloning, genic male sterility (GMS), biotechnology-based male sterility (BMS), heterosis application, genetically modified plants

### 1. Introduction

The demand for food supply is increasing exponentially with the human population continuously growing. According to a report, the world population is predicted to increase by 34% by 2050 [1], whereas the area of land for agriculture practices is decreasing consistently over the last few decades because of urbanization and land degradation. Therefore, it is necessary to increase the food production per unit area as cultivated lands are limited [2].

Hybrid vigor (or heterosis) is the superior performance of the heterozygous hybrid progeny over both homozygous parents. Most crops show hybrid vigor, such as maize, rice, wheat, sorghum, rapeseed, and sunflower, but commercial production of hybrids is only feasible if a reliable and cost-effective pollination control system is available. In cereal crops, maize is a monoecious and diclinous species, which makes it very successful in heterosis utilization with relatively feasible emasculation. The emasculation, namely, the physical removal of the male floral structure, usually includes manual and mechanical detasseling. However, emasculation is not only time-consuming, labor-intensive, and expensive but also detrimental to plant growth, thus reducing the yield of hybrid seed. At the same time, it is unfeasible for the crops that have small, bisexual flowers, such as rice, wheat, and barley. Therefore, it is an ideal alternative to use male-sterile line for pollination control in these cereal crops [3, 4].

Male sterility (MS) refers to cases in which viable male gametes (i.e., pollen) are not produced, while female gametes are fully fertile. Male sterility can be generated by either cytoplasmic or nuclear genes. Cytoplasmic male sterility (CMS) is caused by mitochondrial genes together with nuclear genes and has been used in commercial hybrid production in crops (such as maize, rice, and oilseed rape), but this method suffers from the poor genetic diversity, increased disease susceptibility, and unreliable restoration of CMS lines [5]. Genic male sterility (GMS) is caused by nuclear genes alone, and the use of GMS can overcome these drawbacks, but it is difficult to obtain a pure and large-scale increase of male-sterile female lines through self-pollination. Fortunately, with the rapid development of GMS gene isolation methods, plant-transformation techniques, and other new biotechnologies, many efforts have been made to identify and utilize GMS genes and ultimately develop more efficient biotechnology-based male sterility (BMS) systems in crop plants [3, 4, 6].

In this chapter, we systemically described the molecular cloning methods, functional confirmation approaches, application value assessment of GMS genes, as well as the strategies and comprehensive evaluation of BMS systems based on elite GMS genes in cereal crops (Figures 1 and 2). This will provide a guideline and shed light on GMS gene cloning and application in hybrid seed breeding and production via BMS systems in major cereal crops.

2. Molecular cloning strategies of GMS genes in crop plants

hybrid combinations with male lines Chang 7-2 and Dedan5M in two locations, respectively.

strategies.

77

Figure 2.

There are basically two ways to clone GMS genes in crops: forward and reverse genetics. Forward genetic approaches require the cloning of sequences underlying the male-sterile phenotype, such as map-based cloning, T-DNA or transposon tagging, and MutMap method (Figure 3), whereas reverse genetic strategies seek to identify and select mutations in a known sequence, such as homology-based cloning, anther-specific expression gene screening, and other reverse genetic cloning

A typical example of maize ZmMs30 gene: from map-based cloning and functional confirmation to application value assessment (adapted from Ref. [18]). (A) Map-based cloning of ZmMs30 gene; (B) gene structure and mutation site comparison between WT and ms30-6028 mutant; (C) predictive protein sequence comparison of ZmMs30 between WT and ms30-6028 mutant; (D) transgenic complementation and CRISPR-Ca9-targeted mutagenesis confirmation of ZmMs30 gene in maize; (E and F) cytological observation of anther and pollen development in WT and ms30-6028 mutant at stage 11 (S11) by using transverse section light microscopy, SEM (E) and TEM (F); (G) heterosis comparison of WT and ms30-6028 mutant by using two representative

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

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

#### Figure 1.

The technology workflow of cloning methods and application strategies of GMS genes in crop plants.

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

#### Figure 2.

Hybrid vigor (or heterosis) is the superior performance of the heterozygous hybrid progeny over both homozygous parents. Most crops show hybrid vigor, such as maize, rice, wheat, sorghum, rapeseed, and sunflower, but commercial production of hybrids is only feasible if a reliable and cost-effective pollination control system is available. In cereal crops, maize is a monoecious and diclinous species, which makes it very successful in heterosis utilization with relatively feasible emasculation. The emasculation, namely, the physical removal of the male floral structure, usually includes manual and mechanical detasseling. However, emasculation is not only time-consuming, labor-intensive, and expensive but also detrimental to plant growth, thus reducing the yield of hybrid seed. At the same time, it is unfeasible for the crops that have small, bisexual flowers, such as rice, wheat, and barley. Therefore, it is an ideal alternative to use male-sterile line for pollination

Male sterility (MS) refers to cases in which viable male gametes (i.e., pollen) are not produced, while female gametes are fully fertile. Male sterility can be generated by either cytoplasmic or nuclear genes. Cytoplasmic male sterility (CMS) is caused by mitochondrial genes together with nuclear genes and has been used in commercial hybrid production in crops (such as maize, rice, and oilseed rape), but this method suffers from the poor genetic diversity, increased disease susceptibility, and unreliable restoration of CMS lines [5]. Genic male sterility (GMS) is caused by nuclear genes alone, and the use of GMS can overcome these drawbacks, but it is difficult to obtain a pure and large-scale increase of male-sterile female lines through self-pollination. Fortunately, with the rapid development of GMS gene isolation methods, plant-transformation techniques, and other new biotechnologies, many efforts have been made to identify and utilize GMS genes and ultimately develop more efficient biotechnology-based male sterility (BMS) systems in crop

In this chapter, we systemically described the molecular cloning methods, functional confirmation approaches, application value assessment of GMS genes, as well as the strategies and comprehensive evaluation of BMS systems based on elite GMS genes in cereal crops (Figures 1 and 2). This will provide a guideline and shed light on GMS gene cloning and application in hybrid seed breeding and production via

The technology workflow of cloning methods and application strategies of GMS genes in crop plants.

control in these cereal crops [3, 4].

Synthetic Biology - New Interdisciplinary Science

BMS systems in major cereal crops.

plants [3, 4, 6].

Figure 1.

76

A typical example of maize ZmMs30 gene: from map-based cloning and functional confirmation to application value assessment (adapted from Ref. [18]). (A) Map-based cloning of ZmMs30 gene; (B) gene structure and mutation site comparison between WT and ms30-6028 mutant; (C) predictive protein sequence comparison of ZmMs30 between WT and ms30-6028 mutant; (D) transgenic complementation and CRISPR-Ca9-targeted mutagenesis confirmation of ZmMs30 gene in maize; (E and F) cytological observation of anther and pollen development in WT and ms30-6028 mutant at stage 11 (S11) by using transverse section light microscopy, SEM (E) and TEM (F); (G) heterosis comparison of WT and ms30-6028 mutant by using two representative hybrid combinations with male lines Chang 7-2 and Dedan5M in two locations, respectively.

#### 2. Molecular cloning strategies of GMS genes in crop plants

There are basically two ways to clone GMS genes in crops: forward and reverse genetics. Forward genetic approaches require the cloning of sequences underlying the male-sterile phenotype, such as map-based cloning, T-DNA or transposon tagging, and MutMap method (Figure 3), whereas reverse genetic strategies seek to identify and select mutations in a known sequence, such as homology-based cloning, anther-specific expression gene screening, and other reverse genetic cloning strategies.

No. Cloning strategy GMS

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

genes

ZmMs10/ APV1

ZmMs22/ MSCA1

IPE1/ ZmMs20

TIP2/ bHLH142

PDA1/ OsABCG15

79

Crops Functional

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

1 Map-based cloning ZmMs7 Maize 1, 3, 4, 5 MCS system [9]

confirmation methods\*

ZmMs8 Maize 4 No data [10] ZmMs9 Maize 3, 4 No data [11]

ZmMs23 Maize 3, 4, 5 No data [15] ZmMs26 Maize 1, 2, 3, 4 SPT system [16, 17]

ZmMs32 Maize 3, 4, 5 No data [19] ZmMs33 Maize 1, 2, 3, 4, 5 MCS, MAS [20, 21] Zmms44 Maize 4 Maize SPT-like [22] ZmMs6021 Maize 1, 3, 4, 5 No data [23] IG1 Maize 3, 4 No data [24] MAC1 Maize 2, 3, 4 No data [25]

ZmMs30 Maize 1, 2, 3, 4 MCS, sterility stability,

Maize 3, 4 No data [12]

Maize 1, 3, 4, 5 No data [13, 14]

Maize 3, 4 No data [26, 27]

Rice 1, 4, 5 No data [30, 31]

Rice 3, 4, 5 No data [46]

MTR1 Rice 1, 4 No data [47] OsERS1 Rice 1, 4 No data [48] TaMs1 Wheat 1, 2, 3, 4 Wheat SPT-like [49] TaMs2 Wheat 1, 2, 3, 4 No data [50, 51] TaMs5 Wheat 1, 2, 3, 4 No data [52]

MIL1 Rice 1, 4 No data [28] MIL2 Rice 3, 4 No data [29]

TIP3 Rice 1, 2, 3, 4 No data [32] CYP704B2 Rice 1, 4, 5 No data [33] CYP703A3 Rice 4, 5 No data [34] PTC1 Rice 1, 4, 5 No data [35] TDR Rice 1, 4, 5 No data [36] OsNP1 Rice 1, 4, 5 No data [37] OsGPAT3 Rice 1, 3, 4, 5 No data [38] DPW Rice 1, 4, 5 No data [39] DPW2 Rice 1, 3, 4 No data [40] OsDEX1 Rice 1, 3, 4, 5 No data [41] CSA Rice 1, 4 No data [42] OsABCG26 Rice 1, 2, 4, 5 No data [43] OsPKS2 Rice 1, 4, 5 No data [44] EAT1 Rice 1, 3, 4, 5 No data [45]

Application value evaluation

and heterosis analysis

References

[18]

#### Figure 3.

The forward genetic approaches of GMS gene cloning in crop plants.

#### 2.1 Forward genetic approaches

#### 2.1.1 Map-based cloning

Map-based cloning or positional cloning is a classical forward genetic strategy of GMS gene cloning. Map-based cloning strategy relies on linkage disequilibrium between markers and the gene of interest, that is, as distances between the gene of interest and the analyzed markers decrease, so does the frequency of recombination. In general, the procedure of map-based cloning method includes the following steps (Figure 3A). First is the construction of segregating population of F2 or BC1F1 by crossing male-sterile mutant with a distant male-fertile line followed by selfpollination or backcrossing with the male-sterility mutant line. Second is primary mapping of GMS gene based on bulked segregant analysis (BSA) and molecular marker (such as SSR, SNP, and INDELs) linkage analysis using the F2 or BC1F1 segregating populations which have high levels of linkage disequilibrium. Third is fine mapping of GMS gene by developing more polymorphic markers and enlarging the segregating population. Finally, the GMS gene will be narrowed down to a small interval on the targeted chromosome. Under the help of bioinformatic analysis, the putative GMS gene will be identified.

So far, there are at least 38 GMS genes in major cereal crops that have been cloned via map-based cloning strategy, including 20, 15, and 3 GMS genes reported in rice, maize, and wheat, respectively (Table 1). As the genome sequence information of more crop plants is available, there will be more GMS genes isolated by map-based cloning strategy in crop plants.

#### 2.1.2 T-DNA or transposon tagging

T-DNA or transposon tagging are efficient and straightforward approaches for GMS gene cloning based on the T-DNA or transposon insertion male-sterile mutants (Figure 3B), PCR, and bioinformatic analysis. If the male-sterile mutant comes from a T-DNA or transposon insertion, rapid identification of the GMS gene is at least theoretically possible by locating the sequence tag and analyzing its neighboring sequences by using thermal asymmetric interlaced (TAIL) PCR,


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

2.1 Forward genetic approaches

Synthetic Biology - New Interdisciplinary Science

The forward genetic approaches of GMS gene cloning in crop plants.

putative GMS gene will be identified.

map-based cloning strategy in crop plants.

2.1.2 T-DNA or transposon tagging

78

Map-based cloning or positional cloning is a classical forward genetic strategy of

GMS gene cloning. Map-based cloning strategy relies on linkage disequilibrium between markers and the gene of interest, that is, as distances between the gene of interest and the analyzed markers decrease, so does the frequency of recombination. In general, the procedure of map-based cloning method includes the following steps (Figure 3A). First is the construction of segregating population of F2 or BC1F1 by crossing male-sterile mutant with a distant male-fertile line followed by selfpollination or backcrossing with the male-sterility mutant line. Second is primary mapping of GMS gene based on bulked segregant analysis (BSA) and molecular marker (such as SSR, SNP, and INDELs) linkage analysis using the F2 or BC1F1 segregating populations which have high levels of linkage disequilibrium. Third is fine mapping of GMS gene by developing more polymorphic markers and enlarging the segregating population. Finally, the GMS gene will be narrowed down to a small interval on the targeted chromosome. Under the help of bioinformatic analysis, the

So far, there are at least 38 GMS genes in major cereal crops that have been cloned via map-based cloning strategy, including 20, 15, and 3 GMS genes reported in rice, maize, and wheat, respectively (Table 1). As the genome sequence information of more crop plants is available, there will be more GMS genes isolated by

T-DNA or transposon tagging are efficient and straightforward approaches for

GMS gene cloning based on the T-DNA or transposon insertion male-sterile mutants (Figure 3B), PCR, and bioinformatic analysis. If the male-sterile mutant comes from a T-DNA or transposon insertion, rapid identification of the GMS gene is at least theoretically possible by locating the sequence tag and analyzing its neighboring sequences by using thermal asymmetric interlaced (TAIL) PCR,

2.1.1 Map-based cloning

Figure 3.


inverse PCR, and genomic PCR methods. There are at least 10 GMS genes cloned by using T-DNA tagging in rice, such as API5, bHLH142, OsGT1, UDT1, RIP1, WDA1, OsCP1, GSL5, DTM1, and DTC1 (Table 1). For instance, rice UDT1 (Undeveloped Tapetum1) gene was isolated from a T-DNA insertional rice male-sterile mutant by using T-DNA tagging method [7]. The flanking region of the inserted T-DNA in mutant line was amplified by TAIL–PCR. Sequence analysis of that region revealed that T-DNA was inserted into a gene located on chromosome 7. BLAST analysis indicated that the most similar proteins are the Brassica napus bHLH transcription factor CAD54298 and the Arabidopsis bHLH protein AMS (At2g16910), each of which shares 32% overall identity with the rice protein. These results indicated that

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

In addition, there are at least two and three GMS genes cloned via transposon

MutMap method is based on whole-genome sequencing of pooled DNA from a segregating population of plants that show a useful phenotype [86], for example,

(Figure 3C). In classic MutMap method, a GMS mutant is crossed directly to the original wild-type line; the resulting F1 is self-pollinated to obtain F2 progeny segregating for the GMS mutant and wild-type phenotypes. DNA of F2 displaying the mutant phenotype is bulked and subjected to whole-genome sequencing followed by alignment to the reference sequence. SNPs with sequence reads composed only of mutant sequences (SNP index of 1; SNP index is defined as the ratio between the number of reads of a mutant SNP and the total number of reads corresponding to the SNP) are closely linked to the causal SNP for the mutant phenotype [86]. The MutMap method was used for rice GMS gene cloning, e.g., OsLAP6/OsPKS1 [67]. Recently, a modified MutMap method was developed in rice male-sterility gene (OsMs55/MER3) cloning [87]. Different from the original MutMap method that aligns the mutant pool DNA sequence with the assembled WT genome, the modified MutMap method was to align the re-sequencing data of the mutant pool DNA and WT DNA with the Nipponbare reference genome. The resulting SNPs of mutant/Nipponbare and WT/Nipponbare were further compared to determine the candidate mutant gene. This modified method does not need an assembled WT genome as reference and thus is more cost-effective and widely applicable. The modified MutMap method was used for GMS gene cloning in rice and wheat, such as OsABCG26 [68], OsNP1 [69], and TaMs1 [70] (Table 1). As the next-generation sequencing technology advances and cost of sequencing decreases rapidly, the MutMap method will be applicable for more crop plants except for rice

Reverse genetic approach means from gene to phenotype and relies upon sequence information as retrieved from genome, cDNA library, and/or expressed sequence tag (EST) sequencing. The scientist starts with the selection of a specific

tagging in maize (Ms45 and OCL4) and rice (OsGAMYB, MSP1, and CAP1), respectively (Table 1). For example, maize Ms45 gene is isolated by an activator transposon tagging, the tassel-specific transcription of Ms45 gene is shown by RNA hybridization analysis, and genetic transformation of ms45 mutant with a copy of

male sterility resulted from ethyl methanesulfonate (EMS) mutagenesis

UDT1 encodes a putative bHLH transcription factor in rice [7].

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

Ms45 gene can restore the fertility phenotype in maize [8].

2.1.3 MutMap method

and wheat.

81

2.2 Reverse genetic approaches

\*Notes: (1) functional complementation, (2) knockout by using CRISPR-Cas9 or knockdown by using RNAi, (3) allelism test and allelic mutant sequencing, (4) anther-specific expression analysis, (5) phylogenetic analysis and orthologous analysis with known GMS gene, (6) cytological observation

OsTGA10 Rice 2, 3, 4, 5, 6 No data [84] OsAGO2 Rice 2, 3, 4, 6 No data [85]

#### Table 1.

Cloning, functional confirmation, and application value evaluation of GMS genes in crops.

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

inverse PCR, and genomic PCR methods. There are at least 10 GMS genes cloned by using T-DNA tagging in rice, such as API5, bHLH142, OsGT1, UDT1, RIP1, WDA1, OsCP1, GSL5, DTM1, and DTC1 (Table 1). For instance, rice UDT1 (Undeveloped Tapetum1) gene was isolated from a T-DNA insertional rice male-sterile mutant by using T-DNA tagging method [7]. The flanking region of the inserted T-DNA in mutant line was amplified by TAIL–PCR. Sequence analysis of that region revealed that T-DNA was inserted into a gene located on chromosome 7. BLAST analysis indicated that the most similar proteins are the Brassica napus bHLH transcription factor CAD54298 and the Arabidopsis bHLH protein AMS (At2g16910), each of which shares 32% overall identity with the rice protein. These results indicated that UDT1 encodes a putative bHLH transcription factor in rice [7].

In addition, there are at least two and three GMS genes cloned via transposon tagging in maize (Ms45 and OCL4) and rice (OsGAMYB, MSP1, and CAP1), respectively (Table 1). For example, maize Ms45 gene is isolated by an activator transposon tagging, the tassel-specific transcription of Ms45 gene is shown by RNA hybridization analysis, and genetic transformation of ms45 mutant with a copy of Ms45 gene can restore the fertility phenotype in maize [8].

#### 2.1.3 MutMap method

No. Cloning strategy GMS

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4 MutMap cloning OsLAP6/

5 Homology-based cloning

6 Anther-specific expression gene screening

7 Other reverse genetic cloning

GMS gene, (6) cytological observation

Table 1.

80

OsPKS1

genes

Crops Functional

2 T-DNA tagging bHLH142 Rice 1, 4, 5 No data [31]

3 Transposon tagging ZmMs45 Maize 1, 2, 3 STP system [8, 61, 62]

confirmation methods\*

OsAPI5 Rice 1, 3, 4 No data [53] OsGT1 Rice 3, 4 No data [54] UDT1 Rice 3, 4, 5 No data [7] RIP1 Rice 2, 3, 4 No data [55] WDA1 Rice 2, 3, 4, 5 No data [56] OsCP1 Rice 4, No data [57] GSL5 Rice 2, 3, 4, 5 No data [58] DTM1 Rice 3, 4 No data [59] DTC1 Rice 3, 4 No data [60]

OCL4 Maize 2, 3, 4 No data [63] OsGAMYB Rice 1, 3, 4, 5 No data [64] MSP1 Rice 1, 4, 5 No data [65] CAP1 Rice 1, 4, 5 No data [66]

OsABCG26 Rice 1, 2, 4, 5 No data [68] OsNP1 Rice 1, 2, 3, 4, 5 Rice SPT-like [69] TaMs1 Wheat 1, 2, 3, 4 No data [70]

OsTDF1 Rice 1, 2, 4, 5, 6 No data [71] OsACOS12 Rice 1, 3, 4, 5, 6 No data [72] OsIG1 Rice 2, 3, 4, 5, 6 No data [24, 73] OsCER1 Rice 2, 3, 4, 5, 6 No data [74] OsRAFTIN Rice 2, 4, 5, 6 No data [75] TaMs45 Wheat 1, 2, 3, 4, 5 No data [76] TaMs26 Wheat 1, 2, 4, 5, 6 No data [77]

OsC6 Rice 2, 4, 5, 6 No data [78] OsG1 Rice 2, 4, 6 No data [79] OsADF Rice 2, 4, 6 No data [80] OsUAM3 Rice 2, 4, 6 No data [81] TaRAFTIN Wheat 2, 4, 5, 6 No data [75]

OsSTRL2 Rice 2, 4, 5, 6 No data [82] OsFTIP7 Rice 2, 3, 4 No data [83] OsTGA10 Rice 2, 3, 4, 5, 6 No data [84] OsAGO2 Rice 2, 3, 4, 6 No data [85]

\*Notes: (1) functional complementation, (2) knockout by using CRISPR-Cas9 or knockdown by using RNAi, (3) allelism test and allelic mutant sequencing, (4) anther-specific expression analysis, (5) phylogenetic analysis and orthologous analysis with known

Cloning, functional confirmation, and application value evaluation of GMS genes in crops.

Rice 2, 3, 4, 5 No data [67]

Application value evaluation

References

MutMap method is based on whole-genome sequencing of pooled DNA from a segregating population of plants that show a useful phenotype [86], for example, male sterility resulted from ethyl methanesulfonate (EMS) mutagenesis (Figure 3C). In classic MutMap method, a GMS mutant is crossed directly to the original wild-type line; the resulting F1 is self-pollinated to obtain F2 progeny segregating for the GMS mutant and wild-type phenotypes. DNA of F2 displaying the mutant phenotype is bulked and subjected to whole-genome sequencing followed by alignment to the reference sequence. SNPs with sequence reads composed only of mutant sequences (SNP index of 1; SNP index is defined as the ratio between the number of reads of a mutant SNP and the total number of reads corresponding to the SNP) are closely linked to the causal SNP for the mutant phenotype [86]. The MutMap method was used for rice GMS gene cloning, e.g., OsLAP6/OsPKS1 [67]. Recently, a modified MutMap method was developed in rice male-sterility gene (OsMs55/MER3) cloning [87]. Different from the original MutMap method that aligns the mutant pool DNA sequence with the assembled WT genome, the modified MutMap method was to align the re-sequencing data of the mutant pool DNA and WT DNA with the Nipponbare reference genome. The resulting SNPs of mutant/Nipponbare and WT/Nipponbare were further compared to determine the candidate mutant gene. This modified method does not need an assembled WT genome as reference and thus is more cost-effective and widely applicable. The modified MutMap method was used for GMS gene cloning in rice and wheat, such as OsABCG26 [68], OsNP1 [69], and TaMs1 [70] (Table 1). As the next-generation sequencing technology advances and cost of sequencing decreases rapidly, the MutMap method will be applicable for more crop plants except for rice and wheat.

#### 2.2 Reverse genetic approaches

Reverse genetic approach means from gene to phenotype and relies upon sequence information as retrieved from genome, cDNA library, and/or expressed sequence tag (EST) sequencing. The scientist starts with the selection of a specific 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 expression gene screening, and other methods.

a specific gene family, targeted mutagenesis of candidate GMS genes, etc. For example, OsTGA10 encoding a bZIP transcription factor was identified as a target of the MADS box protein OsMADS8 by using the ChIP-seq technique, and mutation of OsTGA10 resulted in male sterility [84]. OsSTRL2 was identified based on the genome-wide expression analysis of rice STR-like (OsSTRL) gene family and its anther-specific expression pattern [82]. OsFTIP7 was identified as GMS gene through targeted mutagenesis of the rice genes encoding multiple C2 domain and transmembrane region proteins (MCTPs) using the clustered regularly interspaced short palindromic repeats (CRISPR)—CRISPR-associated nuclease 9 (Cas9) technology and targeted mutation of OsFTIP7 lead to complete male-sterility phenotype

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

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).

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.

in rice [83].

Figure 4.

83

The functional confirmation approaches of GMS genes in crop plants.

3.1 Transgenic complementation

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

### 2.2.1 Homology-based cloning

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 Section 3).

#### 2.2.2 Anther-specific expression gene screening

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

#### 2.2.3 Other reverse genetic approaches

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

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

a specific gene family, targeted mutagenesis of candidate GMS genes, etc. For example, OsTGA10 encoding a bZIP transcription factor was identified as a target of the MADS box protein OsMADS8 by using the ChIP-seq technique, and mutation of OsTGA10 resulted in male sterility [84]. OsSTRL2 was identified based on the genome-wide expression analysis of rice STR-like (OsSTRL) gene family and its anther-specific expression pattern [82]. OsFTIP7 was identified as GMS gene through targeted mutagenesis of the rice genes encoding multiple C2 domain and transmembrane region proteins (MCTPs) using the clustered regularly interspaced short palindromic repeats (CRISPR)—CRISPR-associated nuclease 9 (Cas9) technology and targeted mutation of OsFTIP7 lead to complete male-sterility phenotype in rice [83].
