The area of soybean (Glycine max) cultivation in the world have expanded by more than 900% since the 1960s in North and South America, due to its significant roles in animal feeding and human nutrition (http://www.fao.org/faostat/en/#home). However, soybean yield per unit area has not changed significantly compared with rice, wheat and maize, suggesting the lack of a true Green Revolution in soybean breeding (Liu, Zhang, et al. 2020). Soybean yield is determined by both node number and the number of pods per node, therefore the yield increase cannot be achieved in soybean by simply adoption of the shorter varieties. There are several options to increase soybean yields and hybrid breeding hold the greatest potential to boost yield.
Soybean is an autogamous legume species and male sterility lines are prerequisites for commercially available hybrid breeding and large quantities of seed production. An earlier heterosis test demonstrated that significant yield increase could be achieved in soybean; the heterozygous F1 plants of 248 combinations yielded 20% more than their parental lines among the 1123 combinations that were tested (Sun, et al. 1999; Palmer, et al. 2001). However, hybrid breeding in soybean has received limited attention in contrast to maize and rice. Male-sterile female lines with cytoplasmic male sterility (CMS) or genic-controlled photoperiod/ thermo-sensitive male sterility (GMS) have been extensively used for many years in maize and rice (Chen and Liu 2014; Wan, et al. 2019). Hybrid rice in China covers 50%-60% of the total rice cultivation fields, which contributed greatly to rice yield and ensure food security (Kim and Zhang 2018; Liao, et al. 2021).
Male sterility lines are available in soybean, and the first soybean CMS line was reported under the US patents No. 4545146 in 1985 (Davis). Since then, no further information of any other CMS line has been reported. Considerable research on soybean CMS lines has been conducted in China since the early 80s of the last century (Sun, et al. 1994; Palmer, et al. 2001). To date, more than 40 hybrid soybean varieties have been bred and approved in China after several generations of researchers with more than 40 years of efforts, and more than 30 invention patents and technical standards have been authorized for the use of new technologies and methods for soybean hybrid breeding (Sun, et al. 2021). However, CMS genes and underlying molecular mechanisms are still unknown in soybean, which has restricted the development of commercial varieties.
With the explosion in genomic resources and the rapid development of molecular biology and technology, the biotechnology-based male-sterility (BMS) systems for hybrid breeding have been established in maize, rice and other crops and vegetables (Chang, et al. 2016; Wu, et al. 2016; Singh, et al. 2019). The BMS systems utilize nuclear male sterility to propagate the pure nuclear male sterile seeds in a large scale, which not only make the climate change not a threat to the pure hybrid seed production anymore but also unlock the potential for breeding superior hybrids through expanding the parental germplasm pool. In soybean, the nuclear male sterile mutants ms4, ms1, ms6 and ms3 have been cloned in the recent years (Thu, et al. 2019; Fang, et al. 2021; Jiang, et al. 2021; Nadeem, et al. 2021; Yu, et al. 2021; Hou, et al. 2022). To speed up the large-scale commercial cultivation of hybrid soybean, it is time to consider where to put the investments, should we continue to count on the three-line hybrid system and looking for the ideal maintainer and restorer lines, or we can rely on the BMS systems to realize the commercialization of hybrid soybean. In this review, we try to cover recent advances in cytoplasmic-nuclear and nuclear male sterility systems in soybean to see whether the technological breakthroughs will make us to succeed in hybrid soybean production.
Male Sterility In Plant
Plant male sterility (MS) refers to the phenomenon that the stamen develops unnormal, losing the ability to produce the functionally active male gametes for fertilization. According to their phenotypic characteristics, Kaul (1988) divide MS into three categories: including structural, sporogenous and functional. Structural MS indicates that the stamen is either completely lack or abnormally formed, which results in the absence of pollen. Sporogenous MS indicates that the stamen is essentially morphological normal, but can’t produce functional microspores or pollen due to the failure of early microsporogenesis and late microgametogenesis. Functional MS indicates that the viable pollen is produced, but either can’t be released from the anther due to the absence of dehiscence or is unable to geminate on the stigma and to initiate fertilization.
On the other scheme, according to the origin of inheritance, two types of MS are distinguished: cytoplasmic male sterility (CMS) and nuclear or genic male sterility (GMS). CMS is co-controlled by the nuclear and cytoplasmic genes, while GMS is controlled by nuclear gene only. CMS is widely spread in the higher plants and more than 300 species possess CMS were reported up to now (Liu, et al. 2001). The CMS is resulted from the incompatibility between nuclear and mitochondrial gene products and there are several ways to create CMS, like wide/inter-specific hybridization, protoplasmic fusion, induced mutations and genetic engineering (Bohra, et al. 2016). GMS is derived from the changes in the structure and function of nuclear gene, which majority are caused by natural variation and can also be achieved by physical and chemical mutagenesis. Mostly, the fertility of a GMS line is controlled by a recessive gene, and seldomly is influenced by a dominant gene.
Cytoplasmic Male Sterility (Cms) System
CMS/Rf (Restorer-of-fertility) system, also known as the three-line hybrid system, comprises a cytoplasmic male sterile line, a maintainer line and a restorer line. The sterile line contains cytoplasmic male sterile gene, while lacks nuclear restorer gene, which is characterized by sterile pollen and unable to produce progeny by self-inbreeding. The maintainer line excludes the nuclear restorer gene but contains fertile cytoplasmic gene. However, the restorer line preserves a functionally nuclear gene and with or without a fertile cytoplasmic gene. The pollens of both the maintainer and the restorer line are fertile, so they can propagate by self-pollination. When sterile line is used as the female parent, it can receive pollen from either the maintainer or the restorer line and to produce hybrid progeny. The maintainer line is used to cross with the male sterile line to reproduce the male sterile line, while the restorer line is used to cross with the male sterile line to produce hybrid progenies with heterosis to realize yield increasing.
The CMS/Rf system has been exploited for hybrid seed production in plenty of crops such as maize, rice, wheat, rape, soybean, sorghum, carrot, sugar beet, sunflower, cotton, pepper and petunia (Garcia, et al. 2019). Although this system has already been applied in soybean successfully, the yield increasing was far away from that of rice and maize. One of the main reasons is the limited number of identified CMS lines, which heavily restricts the utilization of three-line system in soybean hybrid seed production. In order to solve this problem, a variety of cytoplasmic genes producing MS phenotypes along with their corresponding nuclear-encoded restorer-of-fertility genes need to be identified urgently.
Genic Male Sterility (Gms) System
GMS is controlled by nuclear genes without the influence of cytoplasmic genome that are either insensitive or sensitive to environmental conditions, called genetically stable GMS and environment-sensitive genic male sterility (EGMS), respectively. In the case of EGMS, male fertility is often impressionable to different environmental conditions, including photoperiod (PGMS), temperature (TGMS), photoperiod and temperature (PTGMS), and humidity (HGMS) (Chen and Liu 2014; Xue, et al. 2018; Abbas, et al. 2021). EGMS is regarded as an efficient genetic tool to develop two-line hybrids, since the need of a maintainer line can be eliminated, and the male sterile line can be propagated by self-pollination under specific conditions (Garcia, et al. 2019). In this system, almost every conventional inbred line can restore the fertility of male-sterile line, and no negative effects related to sterility-inducing cytoplasm have been observed. Furthermore, genes of this system can be easily transferred to other genetic backgrounds (Yu, et al. 2015). EGMS has long been exploited to efficiently produce hybrid rice seeds within a two-line system composed of one male-sterile line and one maintainer line. For instance, the first PGMS line Nongken 58S, discovered in japonica rice in 1973, is completely male sterile when grown under long-day conditions but male fertile when grown under short-day conditions (Shi 1985). While TGMS line Annong S-1, discovered in indica rice in 1988, is completely male sterile when grown at high temperatures but male fertile at low temperatures (Deng, et al. 1999).
Similar to CMS system, the use of genetically stable GMS in plant breeding and hybrid seed production also involves three different lines, including a male sterile line, a maintainer line, and a restorer line. However, the reproduction of male sterile line in GMS system presents a difficulty that the segregation obtained in the cross with the maintainer line calls for an additional step of identification and removal of 50% heterozygotes for hybrid seed production. Fortunately, the discovery BMS systems has overcome this drawback, which including but not limited to seed production technology (SPT) (Wu, et al. 2016), and genome-editing technology (Song, et al. 2021).
The SPT platform consists of a transgenic maintainer line capable of maintaining and propagating non-transgenic nuclear male-sterile female lines for hybrid seed production. The recessive genetic male-sterile mutant ms45, defective formation of outer pollen wall, was first used to establish the SPT system in maize (Wu, et al. 2016). Up to now, four fertility related genes, including rice Oryza sativa No Pollen 1 (OsNP1) (Chang, et al. 2016), maize MS44 (Fox, et al. 2017), MS7 (Zhang, et al. 2018), and MS30 (An, et al. 2019) were successfully used in developing SPT systems, individually. With the development of genome-editing technologies and the knowledge of the molecular genetics of male fertility genes, male sterile line can be created by CRISPR/Cas9 genome-editing technology, and no longer limited to the natural stable GMS mutants. Zhou et al. (2016) developed new ‘transgene clean’ commercial TGMS lines in rice by knocking out TMS5 via CRISPR/Cas9. Whereafter, Li et al. (2017) produced TGMS maize by targeted mutation of the maize homolog of rice TMS5 (called ZmTMS5) using the CRISPR/Cas9 editing system. In addition, two rice reverse PGMS lines in japonica cultivars 9522 and JY5B were also generated by editing Carbon Starved Anther (CSA) gene using CRISPR/Cas9 (Li, et al. 2016). Furthermore, Qi et al. (2020) integrated CRISPR/Cas9 and SPT systems to create Zmms26 male sterility line in maize by co-transforming two independent vectors. In soybean, Chen et al. (2021) used targeted editing of AMS (ABORTED MICROSPORES) homologs in soybean by CRISPR/Cas9 technology for the first time to generate stable male-sterile lines. Since GmAMS1 functions not only in the formation of the pollen wall but also in the controlling the degradation of the soybean tapetum, targeted editing of GmAMS1 resulted in a male-sterile phenotype. The recent advances in genomics and the emergence of multiple biotechnological methods have revolutionized the field of rice breeding. However, identifying more male-sterility genes and elucidating the molecular mechanisms of male sterility in crops are prerequisites for hybrid crop breeding and heterosis exploitation.
Cms System And Hybrid Breeding In Soybean
Studies on soybean CMS started relatively late. The phenomenon of CMS was firstly observed by Davis (1985), while no further information about this mutant is available. The similar work of identification CMS line with wide cross between RNTED (Ru Nan Tian E Dan, Glycine max) and 5090035 (Glycine soja) was performed in China since 1983 by Sun et al. (1993). It was not until 1985 that they found the high pollen abortion rate in (RNTED X 5090035) F1 at different environments. In addition, they conducted reciprocal crossed in 1987 and uncovered that the pollen abortion rate of (RNTED X 5090035) was much higher than that of (5090035 X RNTED). All these results confirmed they found a real CMS line and named it with CMS-RN (Sun, et al. 1993).
Hereafter, another five CMS types were developed and reported in soybean (Table 1). Peng et al. (1994) proposed that ZD8319, also known as Zhongdou 19 (Sun, et al. 2003), processes CMS features, hereafter the relevant CMS derivative lines were distributed CMS-ZD type, which composed of Zhongyou89B, M, ZA, W931A, W933A, W936A, W945A, W948A and FuCMS1A-FuCMS3A (Zhang, et al. 1999a; Sun, et al. 2003). In addition, Zhao et al. (1998) confirmed that a Glycine max named XXT contained cytoplasmic male sterile gene, so this kind of CMS was allocated as CMS-XX (Sun, et al. 2003). However, no further study, such as cytological and function analysis, has been performed for this CMS type. Gai et al. (1995) discovered the cytoplasmic-nuclear male sterility phenomenon in the F1 of (N8855 X N2899, two Glycine max cultivars). Ding et al. (Ding, et al. 2002) further developed a CMS line NJCMS1A (maintainer line NJCMS1B) by back-crossing with the recurrent parent N2899 for another four times of successive generations. Since N8855 contributing the cytoplasmic gene, this new variety are called CMS-N8855 type. Subsequently, NJCMS3A (maintainer line NJCMS3B) derived from the cross between N21566 and N21249, exhibited different cytological characteristics when compared with NJCMS1A and NJCMS2A (N8855 X N1628, CMS-N8855), which was considered owning a new CMS type, and was named CMS-N21566 type here after (Zhao and Gai 2006). Furthermore, Nie et al. (2017) developed a CMS line NJCMS4A from a cross of (N23661 X N23658). The mitochondrial markers and genome sequences analyses suggested that N23661 male sterile cytoplasm is distinguished from that of CMS-RN, CMS-ZD, CMS-N8855 and CMS-N21566 types, so CMS-N23661 was named as a new CMS type (Nie, et al. 2017).
Table 1
Characterized cytoplasmic male sterility type in soybean
Type of CMS | Soybean germplasms contributing male sterile cytoplasm | CMS line | Fertility restorer locus/gene | Reference |
CMS-RN | RNTED (Ru Nan Tian E Dan/167) | OA (maintainer line OB), YA (maintainer line YB), JLCMS9A (maintainer line JLCMS9B), JLCMS4A, JLCMS82A (maintainer line JLCMS82B), JLCMS89A (maintainer line JLCMS89B) | Rf1, Chr. 16, between dCAPS-1 and BARCSOYSSR_16_1076 Rf3, Chr. 9, Glyma.09G171200 | (Wang, et al. 2010), (Dong, et al. 2012), (Guo, et al. 2022), (Sun, et al. 2022) |
CMS-ZD | ZD8319 | Zhongyou89B, M, ZA, W931A, W933A, W936A, W945, W948A, FuCMS1A, FuCMS2A, FuCM3SA, FuCMS4A, FuCMS5A, FuCMS6A, FuCMS7A, FuCMS8A, FuCMS9A, FuCMS10A, FuCMS11A, FuCMS12A, SXCMS1A | Rf, Chr. 16, between BARCSOYSSR_16_1064 and BARCSOYSSR_16_1082 | (Zhao, et al. 1998), (Wang, et al. 2010), (Dong, et al. 2012) |
CMS-XX | XXT | - | - | (Zhao, et al. 1998), (Sun, et al. 2003) |
CMS-N8855 | N8855 | NJCMS1A, NJCMS2A | Rf, Chr. 16, GmPPR576 (Glyma.16G161900) | (Ding, et al. 2002), (Wang, et al. 2021) |
CMS-N21566 | N21566 | NJCMS3A (maintainer line MLCMS3B) | - | (Zhao and Gai 2006), |
CMS-N23661 | N23661 | NJCMS4A | - | (Nie, et al. 2017) |
Note: ‘_’ represents not reported. |
To date, only CMS system has been constructed and developed in soybean. The CMS-RN was the earliest discovered CMS type and was also the first three-line system used for hybrid breeding in soybean. Since the nuclear gene were replaced by the wild soybean 5090035, OA CMS line displayed the sprawling and shattering characteristics, which restricts its utilization for breeding (Zhang, et al. 1999a). Then Sun et al. (2001) used OA BC3 as the female parent to cross with the Glycine max and to create a new CMS line YA (maintainer line YB) in 1995, which brings the possibility to realize three-line system hybrid breeding in soybean. The world's first commercially approved soybean hybrid HybSoy 1 was bred in China in 2002 through continuous backcrossing (JLCMS9A X Jihui 1) (Zhao, et al. 2004). Taking advantage of this type of cytoplasm, more than five hundred pairs of CMS-RN lines and homologous maintainers have been bred, among which more than forty CMS lines with high sterility rate, high combining ability, and high outcrossing rate (called three-high CMS line) has been bred (Sun, et al. 2021). Totally, 42 commercialized soybean hybrid varieties have been developed in China (Table 2). Among the six different CMS types, CMS-RN was the most widely used and totally 34 varieties were generated for spring soybean region in northern China (Table 2). In addition, the CMS-ZD type is widely used in Huanghuai summer soybean region of China, and more than 50 pairs of CMS lines and homologous maintainer lines have been cultivated, but no three-high CMS line has been cultivated yet (Sun, et al. 2021). The hybrid soybean variety, namely HybSoy 1 (Zhao, et al. 2004) and Zayoudou No.1 (Zayoudou 1) (Zhang, et al. 2007), elevated the grain yield of soybean up to nearly 20% (Table 2).
Table 2
Hybrid soybean production with CMS system in China
No. | Name | Year of variety approval | Female parent | Male parent | Type of CMS | Yield increase compared with CK (%) | CK variety | Oil (%) | Protein (%) | Oil and protein (%) |
1 | HybSoy 1 | 2002 | JLCMS9A | JLR1 | CMS-RN | 20.8 | Jilin 30 | 21.09 | 39.19 | 60.28 |
2 | Zayoudou 1 | 2004 | W931A | WR016 | CMS-ZD | 19.1 | Zhongdou 20 | 18.96 | 43.56 | 62.52 |
3 | HybSoy 2 | 2006 | JLCMS47A | JLR2 | CMS-RN | 14.3 | Jilin 30 | 20.54 | 40.75 | 61.29 |
4 | HybSoy 3 | 2009 | JLCMS8A | JLR9 | CMS-RN | 2.8 | Heinong 38 | 20.84 | 40.54 | 61.38 |
5 | Fuzajiaodou 1 | 2010 | FuCMS5A | Fuhui 6 | CMS-ZD | 13.3 | Zhongdou 20 | 19.1 | 44.65 | 63.75 |
6 | Zayoudou 2 | 2010 | W931A | WR99071 | CMS-ZD | 0.7 | Zhongdou 20 | 18.8 | 44.12 | 62.92 |
7 | HybSoy 4 | 2010 | JLCMS47A | JLR83 | CMS-RN | 6.1 | Heinong 38 | 19.57 | 40.48 | 60.05 |
8 | HybSoy 5 | 2011 | JLCMS84A | JLR1 | CMS-RN | 19.7 | Jiunong 21 | 22.25 | 38.79 | 61.04 |
9 | Jiyu 606 | 2013 | JLCMS47A | JLR100 | CMS-RN | 13.5 | Jiyu 47 | 21.51 | 40.11 | 61.62 |
10 | Jiyu 607 | 2013 | JLCMS14A | JLR83 | CMS-RN | 5.1 | Jiyu 47 | 22.22 | 39.3 | 61.52 |
11 | Jindou 48 | 2014 | PZMS-1-1 | ZH-21-B-5 | CMS-RN | 15.2 | Jindou 19 | 19.89 | 36.99 | 56.88 |
12 | Jiyu 608 | 2014 | JLCMS84A | JLR113 | CMS-RN | 3.8 | Jiyu 47 | 23.04 | 37.22 | 60.26 |
13 | Jiyu 609 | 2015 | JLCMS103A | JLR102 | CMS-RN | 2.2 | Suinong 28 | 22.54 | 37.52 | 60.06 |
14 | Fuzajiaodou 2 | 2016 | FuCMS5A | Fuhui 9 | CMS-ZD | 7.3 | Zhonghuang 13 | 18.99 | 46.86 | 65.85 |
15 | Zayoudou 3 | 2016 | W018A | M0901 | CMS-ZD | 6.4 | Zhonghuang 13 | 18.81 | 46.06 | 64.87 |
16 | Jiyu 610 | 2016 | JLCMS128A | JLR98 | CMS-RN | 9.4 | Suinong 28 | 21.15 | 37.32 | 58.47 |
17 | Jiyu 611 | 2016 | JLCMS147A | JLR113 | CMS-RN | 15 | Jiyu 47 | 21.47 | 38.67 | 60.14 |
18 | Jiyu 612 | 2017 | JLCMS57A | JLR9 | CMS-RN | 4.4 | Jiyu 72 | 20.92 | 42.07 | 62.99 |
19 | Youshidou-A-5 | 2019 | JLSXCMS1 | Zhong 119 − 99 | CMS-RN | 12.1 | Jindou 19 | 20.96 | 42.1 | 63.06 |
20 | Jiyu 626 | 2019 | JLCMS230A | JLR9 | CMS-RN | 11.4 | Jiyu 72 | 20.19 | 40.68 | 60.87 |
21 | Jiyu 627 | 2019 | JLCMS197A | JLR98 | CMS-RN | 7.8 | Jiyu 86 | 20.74 | 40.97 | 61.71 |
22 | Jiyu 635 | 2019 | JLCMS34A | JLR300 | CMS-RN | 14.8 | Hejiao 02–69 | 22.71 | 36.27 | 58.98 |
23 | Jiyu 639 | 2019 | JLCMS191A | JLR403 | CMS-RN | 13.1 | Hejiao 02–69 | 23.78 | 37.04 | 60.82 |
24 | Jiaji 1 | 2019 | JLCMS178A | JLR124 | CMS-RN | 16 | Hejiao 02–69 | 22.15 | 40.46 | 62.61 |
Table 2 (Continued) |
No. | Name | Year of variety approval | Female parent | Male parent | Type of CMS | Yield increase compared with CK (%) | CK variety | Oil (%) | Protein (%) | Oil and protein (%) |
25 | Fudou 123 | 2020 | FuCMS5A | Fuhui 5 | CMS-ZD | 4.9 | Zhonghuang 13 | 18.15 | 44.96 | 63.11 |
26 | Wanzadou 5 | 2020 | W1101A | R1312 | CMS-ZD | 6.1 | Zhonghuang 13 | 19.34 | 42.53 | 61.87 |
27 | Jiyu 637 | 2020 | JLCMS210A | JLR209 | CMS-RN | 8.3 | Jiyu 86 | 20.19 | 41.7 | 61.89 |
28 | Jiyu 641 | 2020 | JLCMS191A | JLR158 | CMS-RN | 3.3 | Hejiao 02–69 | 22.43 | 38.58 | 61.01 |
29 | Jiyu 643 | 2020 | JLCMS212A | JLR346 | CMS-RN | 11.2 | Hejiao 02–69 | 21.69 | 38.14 | 59.83 |
30 | Jiyu 647 | 2020 | JLCMS5A | JLR2 | CMS-RN | 18.3 | Hejiao 02–69 | 19.12 | 42.52 | 61.64 |
31 | Jinong H1 | 2020 | JLCMS254A | JLR192 | CMS-RN | 18.2 | Hejiao 02–69 | 21.93 | 38.17 | 60.1 |
32 | Jiyu 633 | 2020 | JLCMS204A | JLR230 | CMS-RN | 14 | Hefeng 50 | 20.44 | 42.78 | 63.22 |
33 | Jindou 51 | 2021 | NJCMS3A | C-19 | CMS-N21566 | 12.3 | Jindou 19 | 20.82 | 40.81 | 61.63 |
34 | Jindou 52 | 2021 | H3A | Laopinzhong 4 | CMS-RN | 11.9 | Fendou 78 | 19.92 | 43.64 | 63.56 |
35 | Jiyu 653 | 2021 | JLCMS242A | JLR300 | CMS-RN | 9.6 | Hejiao 02–69 | 22.89 | 37.68 | 60.57 |
36 | Jiyu 654 | 2021 | JLCMS234A | JLR13 | CMS-RN | 9.2 | Hejiao 02–69 | 22.75 | 36.64 | 59.39 |
37 | Jiyu 660 | 2021 | JLCMS204A | JLR419 | CMS-RN | 8 | Jiyu 86 | 21.42 | 41.39 | 62.81 |
38 | Jiyu 645 | 2021 | JLCMS234A | JLR9 | CMS-RN | 11.6 | Suinong 26 | 20.12 | 44.77 | 64.89 |
39 | Jinong H2 | 2021 | JLCMS212A | JLR414 | CMS-RN | 9.6 | Hejiao 02–69 | 22.91 | 38.86 | 61.77 |
40 | Jiyu 649 | 2022 | JLCMS209A | JLR158 | CMS-RN | 12.5 | Hejiao 02–69 | 22.04 | 37.53 | 59.57 |
41 | Jiyu 667 | 2022 | JLCMS164A | JLR227 | CMS-RN | 6.4 | Jiyu 86 | 21.08 | 39.76 | 60.84 |
42 | Jiyu 668 | 2022 | JLCMS247A | JLR227 | CMS-RN | 12.7 | Hejiao 02–69 | 22.94 | 35.39 | 58.33 |
CK represents control. |
However, the cytological observation and functional analysis are far away from the applied research. Pollen abortion in male sterile lines may occur at the whole stage of reproductive process, and the patterns of abortions vary greatly. There are few cytological studies on soybean CMS lines, and the researches mainly focus on CMS-N8855, CMS-N21566 and CMS-ZD types. Ding et al. (2001) reported that pollen abortion of NJCMS1A (CMS-N8855) occurred at the stage of binucleate pollen. In addition, Fan (2003) observed that the pollen abortion of NJCMS1A occurred at the stages of the microspore mother cells (MMCs), tetrads, uninucleate microspores and binucleate pollens, but most occurred at the early binucleate pollen stage. However, the microspore abortion of another CMS-N8855 type line NJCMS2A was mainly happened at the late uninucleate stage (Fan 2003). In addition, Zhao and Gai (2006) found that the microspore abortion of NJCMS3A (CMS-N21566) was mainly at the middle uninucleate stage, which was also confirmed by (Fan 2003). Furthermore, Ren (2005) found that the pollen morphology of CMS line W931A (Zhongyou 89B X W206, CMS-ZD) was different from that of maintainer line W931B. The normal pollen grains from W931B were full, the surface of which was clearly visible, and the pollen apertures were easy to be identified. However, the surface of pollen grains from CMS line W931A was blurred and shriveled, the pollen apertures could not be distinguished, and the volume was even smaller than W931B.
The three-line system dependents on the ability of the fertile restorer gene (Rf) in the restorer line to restore the CMS line’s fertility. With the development of the next-generation sequencing and CRISPR/Cas9 technologies, more and more Rf gene have been identified in soybean, which will speed up the development three-line system in soybean. A set of works have been conducted for identifying Rf gene of CMS line, especially for CMS-RN tape (Guo, et al. 2022). To further narrow the candidate region for Rf1 gene for CMS-RN, Guo et al. (2022) constructed a F2 population by crossing JLCMS204A with JLR230 (restorer line), and linked the gene between the marker dCAPS-1 and BARCSOYSSR_16_1076. In addition, Glyma.09G171200, encoding a member a pentatricopeptide repeat (PRR) protein, was confirmed as the candidate gene of another Rf3 gene for CMS-RN (Sun, et al. 2022). In addition, the Rf gene of CMS-ZD type was linked to the marker BARCSOYSSR_16_1064 and BARCSOYSSR_16_1082 on Chr. 16 (Dong, et al. 2012). Furthermore, another PPR gene (GmPPR576, Glyma.16G161900) was identified as the candidate Rf gene of CMS-N8855 type by Wang et al. (Wang, et al. 2021). Three Rf genes for CMS-RN, CMS-ZD and CMS-N8855 were distributed on Chr. 16 with a close region (Dong, et al. 2012; Wang, et al. 2021; Guo, et al. 2022), whether they were controlled by the same gene need to be further verified.
In addition, due to lacks of systematically cytological observation and the inconsistent cytological phenotype even for the same CMS type, for example the CMS-N8855 line, whether the different CMS types are really distinguished from each other should be confirmed (Ding, et al. 2001; Fan 2003). Furthermore, an unusual phenomenon also happened that the same maintainer and restorer line can maintain and restore different CMS type, viz. YA (CMS-RN) and ZA (CMS-ZD) (Zhao, et al. 1998). Considering the contradictions, we could not rule out the possibility that the six classified CMS types may not completely different from each other.
Gms System In Soybean
The first report of GMS line in soybean was published in 1928, the mutant st1 was both male and female sterile causing by abnormal chromosome association, which was controlled by a single recessive gene (Owen 1928). To date, approximately 30 GMS lines have been identified in soybean (Table 3). According to the phenotypic characteristics, fs1fs2 (Johns and Palmer 1982) and ft (Singh and Jha 1978) belong to the structural MS, the others belong to sporogenous MS, and no functional MS has been reported in soybean.
Table 3
The main GMS locus in soybean
No. | GMS name | Gene | Chr. | Marker | MS type | Phenotypic characteristic | Genetic characteristic | Mutation type | Reference |
1 | ms3 | Glyma.02G107600 | 2 | - | Photesensitive male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Chaudhari and Davis 1977), (Palmer, et al. 1980), (Hou, et al. 2022) |
2 | 88-428-BY | - | - | - | Photesensitive male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Wei 1991), (Wang, et al. 2004) |
3 | ms8 | - | 7 | Sat_389, telomere | Temperature-sensitive male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Palmer 2000), (Frasch, et al. 2011) |
4 | ms9 | - | 3 | Satt521, Satt237 | Temperature-sensitive male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Palmer 2000), (Cervantes-Martinez, et al. 2007) |
5 | msp | - | 2 | GMES4176, Sat_069 (Satt172) | Temperature-sensitive male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Stelly and Palmer 1980), (Frasch, et al. 2011) |
6 | st1 | - | - | - | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Owen 1928), (Hao, et al. 2019) |
7 | st2 | - | 11 | BARCSOYSSR_11_122, BARCSOYSSR_11_137 | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Hadley and Starnes 1964), (Speth, et al. 2015) |
8 | st3 | - | - | - | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Hadley and Starnes 1964) |
9 | st4 | - | 1 | Satt436, Satt468 | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Palmer 1974), (Speth, et al. 2015) |
10 | st5 | - | 13 | Satt030, Satt146 | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Palmer and Kaul 1983), (Speth, et al. 2015) |
11 | st6 | - | 14 | BARCSOYSSR_14_84, BARCSOYSSR_14_109 | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Skorupska and Palmer 1990), (Speth, et al. 2015) |
12 | st7 | - | 2 | Satg001, telomere | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Jin, et al. 1997), (Speth, et al. 2015) |
Table 3 (Continued) |
No. | GMS name | Gene | Chr. | Marker | MS type | Phenotypic characteristic | Genetic characteristic | Mutation type | Reference |
13 | st8 | - | 16 | E107, Satt132 | Male sterile, female sterile | sporogenous | Sing-recessive gene | Natural variation | (Palmer and Horner 2000), (Kato and Palmer 2003) |
14 | NJS-1H | - | - | - | Male sterile, partial female sterile | sporogenous | Sing-recessive gene | Chemical mutagenesis | (Li, et al. 2010) |
15 | D8804-7 | - | - | - | Partial male sterile, partial female sterile | sporogenous | Sing-recessive gene | Introduction of exogenous DNA | (Zhao, et al. 1995) |
16 | fs1fs2 | - | - | - | Partial male sterile, partial female sterile | structural | Duplicate-recessive gene | Natural variation | (Johns and Palmer 1982) |
17 | ms1 | Glyma.13G114200 | 13 | - | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Brim and Young 1971), (Palmer, et al. 1978), (Albertsen and Palmer 1979), (Fang, et al. 2021), (Jiang, et al. 2021), (Nadeem, et al. 2021) |
18 | ms2 | - | 10 | Sat_190, Scaa001 | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Graybosch, et al. 1984), (Graybosch and Palmer 1985), (Cervantes-Martinez, et al. 2007) |
19 | ms4 | Glyma.02G243200 | 2 | - | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Palmer 1979), (Delannay and Palmer 1982), (Thu, et al. 2019) |
Table 3 (Continued) |
No. | GMS name | Gene | Chr. | Marker | MS type | Phenotypic characteristic | Genetic characteristic | Mutation type | Reference |
20 | ms5 | - | - | - | Male sterile, female fertile | sporogenous | Sing-recessive gene | Physical mutagenesis | (Buss 1983) |
21 | ms6 | Glyma.13G066600 | 13 | - | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Skorupska and Palmer 1989), (Ilarslan, et al. 1999), (Yu, et al. 2021) |
22 | ms7 | - | - | - | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Palmer 2000) |
23 | ms12 | Glyma.10G117000 | 10 | centromere | Male sterile, female fertile | sporogenous | Sing-recessive gene | Chemical mutagenesis | (Zhang 2019) |
24 | NJ89-1 | - | | | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Ma, et al. 1993), (Yang, et al. 2003) |
25 | msMOS | - | 2 | Satt157, Satt698 | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Jin, et al. 1997), (Cervantes-Martinez, et al. 2009) |
26 | msNJ | - | 10 | BARCSOYSSR_10_794, BARCSOYSSR_10_819 | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Nie, et al. 2019) |
27 | N7241S | - | - | - | Male sterile, female fertile | sporogenous | Single-dominant gene | Natural variation | (Tuanjie and Shouping 2005) |
28 | Wh921 | - | - | - | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Zhang, et al. 1999b) |
29 | mst-M | - | 13 | W1, Satt516 | Male sterile, female fertile | sporogenous | Sing-recessive gene | Natural variation | (Zhao, et al. 2019) |
30 | ft | - | - | - | Male sterile, female fertile | structural | Sing-recessive gene | Physical mutagenesis | (Singh and Jha 1978) |
Note: This table is modified and updated from the recent review (Li, et al. 2019), ‘_’ represents not reported. |
Two PGMS including ms3 (Chaudhari and Davis 1977) and 88-428-BY (Wei 1991) and three TGMS including ms8 (Palmer 2000), ms9 (Palmer 2000) and msp (Stelly and Palmer 1980)have already been reported. In addition, st1-st8, NJS-1H, D8804-7 and fs1fs2 mutants were both male and female sterile (Owen 1928; Hadley and Starnes 1964; Palmer 1974; Palmer and Kaul 1983; Skorupska and Palmer 1990; Ilarsian, et al. 1997; Palmer and Horner 2000; Kato and Palmer 2003; Speth, et al. 2015) (Johns and Palmer 1982; Zhao, et al. 1995; Li, et al. 2010). The ms1 was the first GMS line that showed male sterile and female fertile phenotype in soybean (Brim and Young 1971). In addition, ms2, ms4-ms7, ms12, MJ89-1, msMOS, msNJ, N7241S, Wh921, mst-M and ft were also belong to the male sterile and female fertile category (Singh and Jha 1978; Palmer 1979; Buss 1983; Graybosch, et al. 1984; Graybosch and Palmer 1985; Skorupska and Palmer 1989; Ma, et al. 1993; Jin, et al. 1997; Zhang, et al. 1999b; Palmer 2000; Tuanjie and Shouping 2005; Zhang 2019; Zhao, et al. 2019).
Although five PGMS and TGMS lines have been identified, so far, only MS3 (Glyma.02G107600), encoding a plant homeodomain (PHD) protein, has been identified (Hou, et al. 2022). The fertility of mutant ms3 can restore under long- day conditions, thus the mutant could be used to create a new, more stable photoperiod‐sensitive genic male sterility line for two‐line hybrid seed production in soybean. With the rapid development of BMS systems in rice and maize, more and more attempts have been taken in soybean. The 13 GMS lines (ms1, ms2, ms4-ms7, ms12, NJ89-1, msMOS, msNJ, N7241S, Wh921 and mst-M) displaying male sterile and female fertile phenotypes are suitable for exploiting this new technology in soybean. In order to make this design come true, plenty of works have been performed to explore the candidate sterile genes of above GMS mutants. MS4 (Glyma.02G243200) is the first GMS gene has been discovered in soybean by fine mapping, which encodes a MALE MEIOCYTE DEATH 1 (MMD1) protein (Thu, et al. 2019). The functional confirmation of MS4 in regulating male fertility was conducted by heterologous expression in Arabidopsis mmd1 mutant (Thu, et al. 2019). Subsequently, the function of MS12 (Glyma.10G117000) was also confirmed by QTL mapping and functional complementation of soybean gene in Arabidopsis cdc20.2 (cell division cycle 20.2) mutant (Zhang 2019). In 2021, both MS1 (Glyma.13G114200) and MS6 (Glyma.13G066600) have been identified by fine mapping (Fang, et al. 2021; Jiang, et al. 2021; Nadeem, et al. 2021; Yu, et al. 2021). MS6 encodes a Tapetal Development and Functional 1 (TDF1) protein, a R2R3 MYB transcription factor, and predominantly expressed in anther, where it regulated the formation of pollen grain (Yu, et al. 2021). Three studies have reported the mapping work of MS1 successively (Fang, et al. 2021; Jiang, et al. 2021; Nadeem, et al. 2021). MS1, encoding a NPK1-ACTIVATING KINESIN 2 (NACK2) protein, participates in regulating cell plate formation after cytokinesis by directly influencing phragmoplast expansion (Fang, et al. 2021). The identification and characterization of GMS genes will provide more choices for building the BMS systems for hybrid soybean production.
Challenges And Prospects In The Commercialization Of Hybrid Soybean
Although nearly 40 hybrid soybean varieties have been generated from the three-line hybrid system (cytoplasmic male sterility), the cultivation of hybrid soybean still have a long way to go. We believe that the three components are the keys to make hybrid soybean a commercial success:
1. Identify The Male Sterile Lines With High Out-crossing Rate
The outcrossing rate is the key determinant for hybrid seed production. The seed production has not been efficient and cost-effective for hybrid soybean. The main reason is that the mutations in causing the male sterility also very often have pleiotropic effects and lead to the defect in female function, which make the male sterile lines with low seed set. The identification of ms1 locus revealed that the gene was highly expressed in style and ovary and may also function in megagametogenesis or embryo development in soybean (Fang, et al. 2021). Studies had focused on the outcrossing rate on male sterile plants, the most promising record was the ms2 mutant, the outcrossing rate on male sterile plants was 74% of the self-pollinated plants (Carter, et al. 1986; Perez, et al. 2009). The feasible solution is to speed up cloning of the male sterile mutants that have good recorded with seed-set, and simultaneously generate new male sterile lines by genome editing to make the mutation only affect the male fertility and without any effect on female productivity and other growth habits.
2. Incorporate Genomic Selection To Precise Guidance On Hybridization Combination
Breeding 4.0 has been considered the next revolution of maize breeding (Wallace, et al. 2018). Even though the soybean breeding program is still at the Breeding 2.0 to 3.0 stages with molecular markers and genomic data to complement phenotypic data, the high-quality graph-based soybean pan-genome and the low cost of genome sequencing will turn promise into practice (Liu, Du, et al. 2020). The genotypes of soybean germplasm lines will be collected using high-throughput genotyping approaches such as next-generation sequencing (NGS) and SNP array platforms. The genetic variations among soybean germplasm of different origins/sources will make the selection of superior hybrid cost-effective.
3. Good Understanding Of The Molecular Mechanisms Of Anther Development In Soybean
Little is known about the biological processes and genes that regulate anther and pollen development in soybean. Like most (70%) angiosperms, soybean produces bicellular pollen. By contrast, rice and Arabidopsis both produce tricellular pollen, the biological significance of the evolution of these two types of pollen grains is still unclear (Williams, et al. 2014). Bicellular pollen undergoes mitotic division to form two sperm cells after germination; prior to anthesis, tricellular pollen forms a male germ unit (MGU) that develops rapidly, which may make tricellular pollen favored in angiosperms that demand rapid reproduction (Hackenberg and Twell 2019). So, the knowledge of extensive studies of anther and pollen formation in Arabidopsis and rice should not be simply transferred to soybean. The uncovering of anther specific genes, networks and hub genes in soybean anther development will provides important insights into the molecular events underlying soybean reproductive developmental processes, as well as valuable resources for the plant reproductive biology community in the areas of pollen evolution, pollination/fertilization, and hybrid breeding.
In summary, on-going and future research should consider the enhancement of hybrid seed production efficiency, and the long-term investment and commitment will definitely make the commercialization of hybrid soybean a reality.