As a major food crop, common wheat (Triticum aestivum L., AABBDD) plays a vital role in ensuring the food security of human beings. Development of wheat semi-dwarf varieties during the 1950s and 1960s improved lodging resistance and permitted significant increases in yield that led to a stable global food supply and eliminated the threat of hunger. These events became known as the “Green Revolution”. However, with arable land currently decreasing due to urbanization, environmental degradation, and rising population levels, there is an emerging need to further increase wheat yield. Heterosis, a common biological phenomenon widely used in maize (Zea mays L.) and rice (Oryza Sativa L.), offers a way to increase wheat yield ^[1][2][1,2]. Despite decades of research, the use of male sterility for the production of hybrid wheat has achieved only limited success. Wilson and Ross (1962) introduced the nucleus of common wheat into the cytoplasm of Triticum timopheevii to develop a common wheat T-type male-sterile line, and fertility restorer lines (T-type restorer lines) were obtained by breeding and selection [3]. Thus, a three-parent hybrid system was devised. Subsequently, new types of sterility systems, e.g., K- and V-type cytoplasmic male sterility systems, a photo-thermal sterility system, and chemical male sterilizing agents were described. However, critical biological and economic problems hindered each system, and hybrid wheat as a commercial enterprise never exceeded 0.2% of the total production ^[4][5][4,5].

In 1931, Bovicini first discovered the phenomenon of nuclear male sterile in wheat, and, to date, five nuclear male sterility loci have been identified, i.e., Ms1, Ms2, Ms3, Ms4, and Ms5 ^{[6][7][8][9][10]}[6,7,8,9,10]. Sterility alleles ms1 and ms5 are recessive and located on chromosome 4BS and 3AL, respectively ^[11][12][11,12], whereas Ms2, Ms3, and Ms4 are dominant. Ms2 and Ms4 are located on chromosomes 4DS, whereas Ms3 is on chromosome 5AS ^{[7][8][10][13][14]}[7,8,10,13,14]. Many sterile alleles of the Ms1 gene have been described. These presumably arose as natural mutants, such as Pugsley’s male sterile (ms1a, ^[6][15][6,15]) and LZ (ms1g, [16]); as X-ray-induced mutants, such as Probus male sterile (ms1b, [17]) and Cornerstone (ms1c, [18]); or as EMS induced mutants, including FS2 (ms1d), FS3 (ms1e), and FS24 (ms1f) ^[10][11][10,11]. Recently, 12 isomorphic variants of Ms1 gene, i.e., ms1d.1, ms1d.2, ms1h, ms1i, ms1j, ms1k, ms1l, ms1m, ms1n, ms1o, ms1p, and ms1q, from the spring wheat variety Ningchun 4 were identified following EMS treatment [19]. Many scientists believe that nuclear male-sterile lines are ideal lines for the production of hybrid varieties, but a lack of stable male-sterile maintainers prohibits their widespread application. Although a “two-line system” [20] and an “XYZ hybrid” system using the nuclear male-sterile mutant “Cornerstone” were proposed, neither proved successful ^[21][22][21,22]. Huang et al. (1988, 1991) discovered a nuclear male-sterile mutant in the offspring of a cross of wheat varieties 72,180 and Xiaoyan 6 and created a blue-aleurone nuclear male-sterile maintainer system (BM system) by introducing a 4E chromosome from Agropyron elongatum containing a dominant blue endosperm gene and a fertility restorer gene ^[23][24][23,24]. The seeds of the wheat 4E monomer alien addition line are light blue. This line can produce seeds normally by selfing, and the offspring can separate about 64% non-blue seeds (nuclear male sterile), 16% light blue seeds, 16% medium blue seeds, and 4% deep blue seeds (fertile). All plants grown from non-blue seeds were male sterile, whose fertility could be restored well in F₁ by almost any variety of bread wheat. The light blue seed is used as a maintainer for male sterility. Zhou et al. (2006) also introduced the same 4E chromosome into Lanzhou Mutant 257A (ms1g) and established a recessive nuclear male-sterile 4E-ms system [25]. This solved the difficulties in obtaining stable maintainer lines and producing homozygous male-sterile seeds in large quantities. It was a significant step forward in the applied research of wheat nuclear male sterility.

2. Discussion

Application of nuclear male sterility is one way to study heterosis in wheat. Although there are many reports on nuclear male sterility in wheat, only 5 loci Ms1-Ms5 have been identified and physically mapped. Sterility-causing alleles of Ms1 and Ms5 are recessive, and those of Ms2, Ms3, and Ms4 are dominant. Three of them have been isolated and cloned. The dominant Ms2 sterility allele in Taigu contains an originally unexpressed “orphan” gene in its promoter region, which is activated and specifically expressed in anthers causing male sterility [13]. Tucker et al. (2017) and Wang et al. (2017) cloned the wildtype allele of Ms1 and found that it encodes a lipid transport protein expressed only in microspores ^[19][26][19,26]. Pallotta et al. (2019) demonstrated that Ms5 encodes a lipid transfer protein anchored by glycosylphosphatidylinositol, which is required for normal development of the pollen outer wall [12].

Here, we studied the wheat nuclear male-sterile line 15 Fan 03 and compared it with Mian 07-374 and generated an F₂ population. The sterility gene was mapped between the SSR markers Ms4BS42 and Ms4BS199 physically positioned at 10.61 and 17.18 Mb, respectively. Sterility was caused by deletion of a chromosome 4BS fragment, detectable by chromosomal FISH. Based on IWGSCWGA v1.0, the deleted region included TraesCS4B01G017900 identified as the Ms1 allele positioned at 13.126 Mb. Among reported Ms1 mutants, ms1a (Pugsley’s mutant) and ms1c (Cornerstone) mutants are terminal deletions of chromosome 4BS ^[18][27][18,27]; ms1b (Probus mutant) and ms1g (LZ, [25]) are interstitial deletions ^[17][19][17,19]; and ms1d (FS2), ms1e (FS3), and ms1f (FS24) are nucleotide variants [10]. Nucleotide variant alleles in a series of EMS-induced sterile mutants in variety Ningchun 4 were named ms1h, ms1i, ms1j, ms1k, ms1l, ms1m, ms1n, ms1o, ms1p, and ms1q [19]. At the same time, a nucleotide variant allele in a Qual 2000 TILLING population was named ms1h. This allele has been re-designated as ms1r (RA McIntosh, personal communication).

The flowering phenotype of sterile plants in 15 Fan 03 differs from that in the fertile plants of Mian 07-374, as the glume remains open for a longer time at a wider angle, and at the tri-nuclear stage, the pollen grains degrade and the pollen activity is lost. However, genetically, the sterile line 15 Fan 03 was similar to ms1b and ms1g mutants in possessing interstitial deletions of 4BS chromosome segments bearing the Ms1 locus.

Wheat nuclear male-sterile lines have many advantages. For example, they are easily restored by most common wheat varieties, with significant heterosis, and without the adverse effect of exogenous cytoplasm. Furthermore, it is easy to select excellent hybrid combinations. The main problem with this type of male-sterile line is that it is difficult to obtain a stable male-sterile maintainer line; that is, it is difficult to produce a large number of homozygous male-sterile seeds. However, using the blue-aleurone nuclear male-sterile maintainer system (BM system), we can easily obtain wheat male-sterile lines (white grains, such as 15 Fan 03) and maintainer lines (light blue grains). We obtained 11 combinations of BM systems with modern main varieties. Moreover, white and blue seeds can be separated at the seed stage, so it is easier to obtain pure male-sterile lines for the production of hybrid wheat ^[23][24][23,24]. This solves the difficulties in obtaining stable male sterile maintainer lines and producing a large number of homozygous male sterile seeds. Moreover, the easy restoration of these lines by common wheat varieties has opened up a new direction for the application of and research into wheat nuclear male sterility.

Because the genome of common wheat is large, complex, and heterogeneous, many chromosomal variations are preserved in the species. These chromosomal abnormalities may also alter the plant phenotype. For example, the absence of the end of chromosome 5AL causes reduced plant height [28]. In this study, a deleted chromosomal fragment was found in sterile plants, and the positions of some markers were in opposite orientations in genetic and physical maps (Figure 16), indicating that chromosomal inversion might have occurred in the parental material. The upstream position of the deletion was between TraesCS4B02G015400LC and marker MS41, whereas the downstream position was between TraesCS4B021300 and TraesCS4B02G021600. Using this information and the wheat660K SNP chip data, three CAPS markers were developed and validated on the F₂ pooled DNA and heterozygous F₂ individuals, as well as 12 widely grown modern wheat varieties. These CAPS markers can be used to genotype individuals in segregating populations at an early stage of growth. Since an SNP marker is usually developed from a sequence polymorphism between different cultivars, it is background genotype specific. We are currently exploring other types of molecular markers that might be more suitable for marker-assisted selection of ms1s.

Figure 1. Linkage maps for locus Ms1. Green sectors portray the deleted chromosome 4BS segment, and the putative inverted segment is indicated.